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Cytochrome b5 reductases: Redox regulators of cell homeostasis

  • Robert Hall
    Affiliations
    Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Shuai Yuan
    Affiliations
    Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Katherine Wood
    Affiliations
    Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Mate Katona
    Affiliations
    Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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  • Adam C. Straub
    Correspondence
    For correspondence: Adam C. Straub
    Affiliations
    Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Center for Microvascular Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Open AccessPublished:October 28, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102654
      The cytochrome-b5 reductase (CYB5R) family of flavoproteins is known to regulate reduction-oxidation (redox) balance in cells. The five enzyme members are highly compartmentalized at the subcellular level and function as “redox switches” enabling the reduction of several substrates, such as heme and coenzyme Q. Critical insight into the physiological and pathophysiological significance of CYB5R enzymes has been gleaned from several human genetic variants that cause congenital disease and a broad spectrum of chronic human diseases. Among the CYB5R genetic variants, CYB5R3 is well-characterized and deficiency in expression and activity is associated with type II methemoglobinemia, cancer, neurodegenerative disorders, diabetes, and cardiovascular disease. Importantly, pharmacological and genetic-based strategies are underway to target CYB5R3 to circumvent disease onset and mitigate severity. Despite our knowledge of CYB5R3 in human health and disease, the other reductases in the CYB5R family have been understudied, providing an opportunity to unravel critical function(s) for these enzymes in physiology and disease. In this review, we aim to provide the broad scientific community an up-to-date overview of the molecular, cellular, physiological, and pathophysiological roles of CYB5R proteins.

      Keywords

      Abbreviations:

      AFR (ascorbate free radical), CoQ (coenzyme Q), CYB5R (cytochrome-b5 reductase), FAD (flavin adenine nucleotide), mARC (mitochondrial amidoxime reducing component), Mtln (mitoregulin), NO (nitric oxide), RCM (recessive congenital methemoglobinemia), sGC (soluble guanylate cyclase), VDAC1 (voltage-dependent anion-selective channel 1)
      The cytochrome-b5 reductase (CYB5R) family of enzymes, consisting of five members (
      • Siendones E.
      • SantaCruz-Calvo S.
      • Martín-Montalvo A.
      • Cascajo M.V.
      • Ariza J.
      • López-Lluch G.
      • et al.
      Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.
      ,
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ,
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ,
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ,
      • Fan J.
      • Du W.
      • Kim-Muller J.Y.
      • Son J.
      • Kuo T.
      • Larrea D.
      • et al.
      Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure.
      ), is a group of flavoprotein reductases that catalyze the transfer of electrons from NADH, generally through an electron carrier such as cytochrome b5 (CYB5), to the final substrate (
      • Siendones E.
      • SantaCruz-Calvo S.
      • Martín-Montalvo A.
      • Cascajo M.V.
      • Ariza J.
      • López-Lluch G.
      • et al.
      Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.
      ). CYB5R3, which is abundantly and ubiquitously expressed across cell types, has been studied extensively throughout the past half decade (
      • Siendones E.
      • SantaCruz-Calvo S.
      • Martín-Montalvo A.
      • Cascajo M.V.
      • Ariza J.
      • López-Lluch G.
      • et al.
      Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.
      ,
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ,
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ,
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ,
      • Fan J.
      • Du W.
      • Kim-Muller J.Y.
      • Son J.
      • Kuo T.
      • Larrea D.
      • et al.
      Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure.
      ,
      • Diaz-Ruiz A.
      • Lanasa M.
      • Garcia J.
      • Mora H.
      • Fan F.
      • Martin-Montalvo A.
      • et al.
      Overexpression of CYB5R3 and NQO1, two NAD+ -producing enzymes, mimics aspects of caloric restriction.
      ,
      • de Cabo R.
      • Siendones E.
      • Minor R.
      • Navas P.
      CYB5R3: a key player in aerobic metabolism and aging?.
      ,
      • Martin-Montalvo A.
      • Sun Y.
      • Diaz-Ruiz A.
      • Ali A.
      • Gutierrez V.
      • Palacios H.H.
      • et al.
      Cytochrome b5 reductase and the control of lipid metabolism and healthspan.
      ,
      • Wood K.C.
      • Durgin B.G.
      • Schmidt H.M.
      • Hahn S.A.
      • Baust J.J.
      • Bachman T.
      • et al.
      Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice.
      ). Structurally, CYB5R3 has been crystallized, revealing a “clam shell-like structure” with two critical structural domains: an NADH and [a] flavin adenine dinucleotide (FAD)-binding domain (Fig. 1). These two domains are held together by a linker region, which plays an important role in maintaining the two domains in the correct orientation and in close proximity to help facilitate electron transfer (
      • Nishida H.
      • Inaka K.
      • Miki K.
      Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases.
      ). CYB5R3 has been shown to reduce several critical substrates, such as heme and coenzyme Q (CoQ) (
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ,
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ,
      • Elahian F.
      • Sepehrizadeh Z.
      • Moghimi B.
      • Mirzaei S.A.
      Human cytochrome b5 reductase: structure, function, and potential applications.
      ). However, the other CYB5R family members have not been comprehensively studied. Therefore, much of our understanding of these proteins is based on our preexisting knowledge of CYB5R3 and the extensive sequence similarity between reductases within the CYB5R family (Fig. 2). The sequences of the five reductases are conserved, particularly within the FAD- and NADH-binding domains (Fig. 2), pointing to the importance of the interplay between these domains in the CYB5R family (
      • Wayne L.L.
      • Wallis J.G.
      • Kumar R.
      • Markham J.E.
      • Browse J.
      Cytochrome b5 reductase encoded by CBR1 is essential for a functional male gametophyte in Arabidopsis.
      ). The flavin-binding domain is necessary for CYB5R stability and function (
      • Marohnic C.C.
      • Bewley M.C.
      • Barber M.J.
      Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase.
      ,
      • Kimura S.
      • Nishida H.
      • Iyanagi T.
      Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis.
      ). Interestingly, among the five reductases, CYB5R1, CYB5R2, and CYB5R3 are the most structurally alike, sharing nearly indistinguishable motifs in both the FAD- and NADH-binding domains (Fig. 2). By contrast, CYB5R3 and CYB5R5 are the least analogous with a 27.87% sequence identity. Based on their primary sequences and predicted structures using Alpha fold (
      • Jumper J.
      • Evans R.
      • Pritzel A.
      • Green T.
      • Figurnov M.
      • Ronneberger O.
      • et al.
      Highly accurate protein structure prediction with AlphaFold.
      ), it is evident that CYB5R4 and CYB5R5 are the most structurally unique in the CYB5R family. Notably, CYB5R4 contains its own heme-binding domain (Fig. 2), akin to the cytochrome b5 carriers CYB5A and CYB5B, which is purportedly essential for electron transfer to target substrates (
      • Rodríguez-Marañón M.J.
      • Qiu F.
      • Stark R.E.
      • White S.P.
      • Zhang X.
      • Foundling S.I.
      • et al.
      13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.
      ,
      • Durley R.C.
      • Mathews F.S.
      Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution.
      ). Despite our extensive knowledge of CYB5R3, numerous questions pertaining to the other CYB5R family members remain unanswered. In this review, we lay out a detailed and compendious synopsis of CYB5R enzyme biology with the goal of highlighting salient contributions of these reductases and opportunities for future studies investigating their roles in cell signaling, physiology, and disease.
      Figure thumbnail gr1
      Figure 1Crystal structure and amino acid sequence of soluble rat CYB5R3 (PDB entry 1IB0). The side view (A) and top view (B) are depicted with FAD (orange) and NADH (blue) bound. The segments of the protein highlighted red illustrate clinically relevant mutations that have already been discovered in the literature. The position of these mutations in the CYB5R3 amino acid sequence is provided below (C). The orange and dark blue molecules depict bound FAD and NADH, respectively. The structure was constructed and visualized in the PyMOL software. FAD, flavin adenine nucleotide.
      Figure thumbnail gr2
      Figure 2Linear amino acid sequence (left) and crystal structure (right) of each CYB5R family member. Each structure depicts the purported membrane-bound isoform except for CYB5R4, which exists only as a soluble protein. Each structure was obtained from the AlphaFold protein structure database (
      • Jumper J.
      • Evans R.
      • Pritzel A.
      • Green T.
      • Figurnov M.
      • Ronneberger O.
      • et al.
      Highly accurate protein structure prediction with AlphaFold.
      ). The UniProt IDs are as follows: CYB5R1 – Q9UHQ9 (human); CYB5R2 – Q6BCY4 (human); CYB5R3 – P00387 (human); CYB5R4 – Q7L1T6 (human); CYB5R5 – Q6IPT4 (human). CYB5R, cytochrome-b5 reductase.

      CYB5R1

      To date, our knowledge on CYB5R1 function and physiology remains scant. A previous study reported CYB5R1 mRNA enrichment in human skeletal muscle and predicted protein localization to the mitochondria, plasma membrane, and endoplasmic reticulum (Table 1) (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ). CYB5R1 is a 34 kDa protein that harbors a 36-amino acid alpha-helical membrane anchor at the N-terminus, permitting CYB5R1 attachment to membranes. CYB5R1 structural analysis discloses conserved structural motifs in both the FAD- and NADH-binding domains, comparable to CYB5R3 (Fig. 2). These conserved structural motifs emerge from similar amino acid sequences, where CYB5R1 and CYB5R3 share a 63.54% sequence identity, the highest in the CYB5R family. CYB5R1 is also structurally homologous to CYB5R2, where there is a 58.82% sequence similarity between the two enzymes. Mass spectrometry analyses have identified numerous posttranslational modifications (PTMs) distinct to CYB5R1, most notably phosphorylation at site Y84 (
      • Mertins P.
      • Mani D.R.
      • Ruggles K.V.
      • Gillette M.A.
      • Clauser K.R.
      • Wang P.
      • et al.
      Proteogenomics connects somatic mutations to signalling in breast cancer.
      ). This residue is situated in the FAD-binding domain; thus, one might postulate that phosphorylation at Y84 may optimally govern electron transfer efficiency by the FAD molecule. Furthermore, K167, positioned within the linker region bridging the NADH and FAD domains, has been identified as an acetylation site (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). Located in the linker region, one might speculate that acetylation at K167 could induce a conformational change in both the NADH and FAD domains, thereby impacting CYB5R1 activity. To date, it is uncertain whether crosstalk between CYB5R1 phosphorylation and acetylation occurs. For instance, does phosphorylation at Y84 directly influence the addition or removal of the acetyl group at K167 and vice versa? Understanding the potential crosstalk between posttranslational modifications could shed light on how CYB5R1 activity is “turned on” or “turned off” under disparate pathophysiological conditions. Notably, it is uncertain whether these posttranslational modifications are linked with CYB5R1 activity or specific pathological conditions. Answering these questions could open doors for targeted therapeutic development.
      Table 1A comprehensive table indicating the subcellular location, cofactors, known roles within and outside of the cardiovascular (CV) system, and highest tissue expression for each of the five reductases in the CYB5R family
      ReductaseSubcellular locationCofactorsKnown role in CV systemKnown role outside of CV systemHuman tissue(s) with highest mRNA expressionReferences
      NADH-Cytochrome b5 reductase 1Mitochondria, extracellular space, plasma membraneNADH, FADUnknownInduction of lipid peroxidation and ferroptosis, lipid desaturationSkeletal muscle(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Yan B.
      • Ai Y.
      • Sun Q.
      • Ma Y.
      • Cao Y.
      • Wang J.
      • et al.
      Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1.
      ,
      • Oh Y.J.
      • Kim H.
      • Seo S.H.
      • Hwang B.G.
      • Chang Y.S.
      • Lee J.
      • et al.
      Cytochrome b5 reductase 1 triggers serial reactions that lead to iron uptake in plants.
      )
      NADH-Cytochrome b5 reductase 2Nucleus, cytosolNADH, FADUnknownProtection against prostate, nasopharynx, and colorectal cancerTestis, tibial nerve(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Franceschini N.
      • Fox E.
      • Zhang Z.
      • Edwards T.L.
      • Nalls M.A.
      • Sung Y.J.
      • et al.
      Genome-wide association analysis of blood-pressure traits in African-ancestry individuals reveals common associated genes in African and non-African populations.
      ,
      • Xiao X.
      • Zhao W.
      • Tian F.
      • Zhou X.
      • Zhang J.
      • Huang T.
      • et al.
      Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma.
      ,
      • Ming H.
      • Lan Y.
      • He F.
      • Xiao X.
      • Zhou X.
      • Zhang Z.
      • et al.
      Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.
      ,
      • Jo Y.S.
      • Kim M.S.
      • Yoo N.J.
      • Lee S.H.
      Intratumoral heterogeneity for inactivating frameshift mutation of CYB5R2 gene in colorectal cancers.
      ,
      • Devaney J.M.
      • Wang S.
      • Funda S.
      • Long J.
      • Taghipour D.J.
      • Tbaishat R.
      • et al.
      Identification of novel DNA-methylated genes that correlate with human prostate cancer and high-grade prostatic intraepithelial neoplasia.
      )
      NADH-Cytochrome b5 reductase 3Mitochondria, plasma membrane, ER, cytosolNADH, FADHeme reduction, lipid regulation, cholesterol biosynthesis, CoQ regulation, sGC regulation, protection against lipid peroxidationProtection of pancreatic beta cells against oxidative stress, drug metabolismArtery, aorta, adipocyte(
      • Siendones E.
      • SantaCruz-Calvo S.
      • Martín-Montalvo A.
      • Cascajo M.V.
      • Ariza J.
      • López-Lluch G.
      • et al.
      Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.
      ,
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ,
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ,
      • Fan J.
      • Du W.
      • Kim-Muller J.Y.
      • Son J.
      • Kuo T.
      • Larrea D.
      • et al.
      Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure.
      ,
      • de Cabo R.
      • Siendones E.
      • Minor R.
      • Navas P.
      CYB5R3: a key player in aerobic metabolism and aging?.
      ,
      • Martin-Montalvo A.
      • Sun Y.
      • Diaz-Ruiz A.
      • Ali A.
      • Gutierrez V.
      • Palacios H.H.
      • et al.
      Cytochrome b5 reductase and the control of lipid metabolism and healthspan.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ,
      • Villalba J.M.
      • Navarro F.
      • Gómez-Díaz C.
      • Arroyo A.
      • Bello R.I.
      • Navas P.
      Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane.
      ,
      • Rahaman M.M.
      • Nguyen A.T.
      • Miller M.P.
      • Hahn S.A.
      • Sparacino-Watkins C.
      • Jobbagy S.
      • et al.
      Cytochrome b5 reductase 3 modulates soluble guanylate cyclase redox state and cGMP signaling.
      ,
      • Navarro F.
      • Villalba J.M.
      • Crane F.L.
      • Mackellar W.C.
      • Navas P.
      A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane.
      )
      NADH-Cytochrome b5 reductase 4ER, cytosolNADPH, NADH, FAD, hemeUnknownProtection of pancreatic beta cells against oxidative stress, regulation of satiety and feeding behavior, fatty acid desaturation, iron homeostasisWhole blood cells (peripheral blood mononuclear cells, platelets, monocytes, T-lymphocytes, granulocytes)(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Zhu H.
      • Larade K.
      • Jackson T.A.
      • Xie J.
      • Ladoux A.
      • Acker H.
      • et al.
      NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.
      ,
      • Stroh M.A.
      • Winter M.K.
      • McCarson K.E.
      • Thyfault J.P.
      • Zhu H.
      NCB5OR deficiency in the cerebellum and midbrain leads to dehydration and alterations in thirst response, fasted feeding behavior, and voluntary exercise in mice.
      ,
      • Xie J.
      • Zhu H.
      • Larade K.
      • Ladoux A.
      • Seguritan A.
      • Chu M.
      • et al.
      Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.
      ,
      • Xu M.
      • Wang W.
      • Frontera J.R.
      • Neely M.C.
      • Lu J.
      • Aires D.
      • et al.
      Ncb5or deficiency increases fatty acid catabolism and oxidative stress.
      ,
      • Stroh M.A.
      • Winter M.K.
      • Swerdlow R.H.
      • McCarson K.E.
      • Zhu H.
      Loss of NCB5OR in the cerebellum disturbs iron pathways, potentiates behavioral abnormalities, and exacerbates harmaline-induced tremor in mice.
      )
      NADH-Cytochrome b5 reductase 5Nucleoplasm, ERNADH, FADUnknownMitigation of oxidative stress in colon polypsLow tissue specificity(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Wang J.
      • Wang T.
      • Bishop M.A.
      • Edwards J.F.
      • Yin H.
      • Dalton S.
      • et al.
      Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis.
      )
      A recent publication demonstrated that CYB5R1, localized to the ER membrane, functions in tandem with NADPH-cytochrome p450 reductase (POR) to catalyze lipid peroxidation and ferroptosis execution in HeLa cells (
      • Yan B.
      • Ai Y.
      • Sun Q.
      • Ma Y.
      • Cao Y.
      • Wang J.
      • et al.
      Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1.
      ). Ferroptosis is an iron-dependent form of cell death triggered by intracellular iron (Fe2+) accumulation and elevated hydrogen peroxide (H2O2) levels, which together lead to lipid peroxidation and execution of cell death (
      • Li J.
      • Cao F.
      • Yin H.-L.
      • Huang Z.-J.
      • Lin Z.-T.
      • Mao N.
      • et al.
      Ferroptosis: past, present and future.
      ). Cooperatively, these two enzymes are sufficient to induce lipid peroxidation and ferroptosis by reducing intracellular molecular oxygen, leading to hydrogen peroxide formation and subsequent membrane lipid oxidation via the Fenton reaction (
      • Yan B.
      • Ai Y.
      • Sun Q.
      • Ma Y.
      • Cao Y.
      • Wang J.
      • et al.
      Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1.
      ). The Fenton reaction is a reaction between free Fe2+ and H2O2 that yields a hydroxyl radical, a potent oxidant that attacks polyunsaturated fatty acids in membranes, ultimately leading to membrane rupture and cell death. Importantly, the authors noted that ferroptosis was mostly POR-dependent due to this enzymes capacity to generate a greater concentration of H2O2 (
      • Yan B.
      • Ai Y.
      • Sun Q.
      • Ma Y.
      • Cao Y.
      • Wang J.
      • et al.
      Membrane damage during ferroptosis is caused by oxidation of phospholipids catalyzed by the oxidoreductases POR and CYB5R1.
      ); thus, it appears CYB5R1 plays a lesser role in ferroptosis execution in HeLa cells. Interestingly, Woischke et al. (
      • Woischke C.
      • Blaj C.
      • Schmidt E.M.
      • Lamprecht S.
      • Engel J.
      • Hermeking H.
      • et al.
      CYB5R1 links epithelial-mesenchymal transition and poor prognosis in colorectal cancer.
      ) revealed a potential link between CYB5R1 overexpression and the risk of developing colorectal cancer, a seemingly contradictory finding to the ferroptosis study aforementioned. If CYB5R1 induces ferroptosis, one might expect that cancer cells would die; however, Woischke et al. assert that CYB5R1 is protective for colorectal cancer cells. Therefore, follow-up studies should be conducted to clear these discrepancies. As a whole, the connection between ferroptosis and cancer has been delineated in the literature (
      • Xia X.
      • Fan X.
      • Zhao M.
      • Zhu P.
      The relationship between ferroptosis and tumors: a novel landscape for therapeutic approach.
      ,
      • Wu S.
      • Li T.
      • Liu W.
      • Huang Y.
      Ferroptosis and cancer: complex relationship and potential application of exosomes.
      ), where ferroptosis-inducing drugs show promise in reducing tumor size and improving efficacy of chemotherapeutic drugs (
      • Wu Y.
      • Yu C.
      • Luo M.
      • Cen C.
      • Qiu J.
      • Zhang S.
      • et al.
      Ferroptosis in cancer treatment: another way to rome.
      ). Given CYB5R1’s reported role in ferroptosis and colorectal cancer, therapeutics targeting CYB5R1 could be beneficial in treating those suffering from certain cancers. The link between CYB5R1 and ferroptosis could also be important in various cell types, especially primary cells, and outside the realm of cancer research. This prospect should be investigated further to better understand the regulation of ferroptosis by CYB5R1.
      Beyond cancer, CYB5R1 transcript levels are significantly upregulated in retina samples collected from patients with diabetic retinopathy, a disease characterized by aberrations in the retinal microvasculature and blindness (
      • Govindarajan G.
      • Mathews S.
      • Srinivasan K.
      • Ramasamy K.
      • Periasamy S.
      Establishment of human retinal mitoscriptome gene expression signature for diabetic retinopathy using cadaver eyes.
      ). Importantly, similar changes in RNA were discerned in mice exhibiting diabetic retinopathy. The authors propose that CYB5R1 is localized to the mitochondria, where the enzyme likely plays a pivotal role in oxidative phosphorylation and ROS generation (
      • Peng L.
      • Ma W.
      • Xie Q.
      • Chen B.
      Identification and validation of hub genes for diabetic retinopathy.
      ). This suggests that CYB5R1 might be involved in mitochondrial oxidative stress pathways that factor into the development of diabetic retinopathy. By investigating a gene co-expression network in a human diabetic retinopathy dataset, the authors further surmise that CYB5R1 is acting on complex 1 of the electron transport chain (
      • Peng L.
      • Ma W.
      • Xie Q.
      • Chen B.
      Identification and validation of hub genes for diabetic retinopathy.
      ). Since CYB5R1 is an electron donor, it is possible that overexpression of CYB5R1 elicits electron leak during the CYB5R1 electron transfer reactions which results in the reduction of molecular oxygen to generate superoxide. These observations stipulate that models of diabetic retinopathy may be advantageous for understanding mechanisms by which CYB5R1 is involved in triggering oxidative stress in the retinal microcirculation.
      Beyond changes in expression levels of CYB5R1, it is unclear whether human genetic coding variants associate with human disease. Scanning of the human genome database revealed that an N44S mutation, positioned at the interface between the FAD domain and the membrane anchor, has a 44% allele frequency in East Asians and only 3% in populations with European ancestry (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ). Thus, future studies aimed at crystallizing both WT and CYB5R1 mutants could shed light on the structural and functional consequences of CYB5R1 mutations in human health and disease across ethnicities.

      CYB5R2

      In addition to CYB5R1, CYB5R2 has also been understudied. CYB5R2 mRNA has been predicted to be enriched in the testis and tibial nerve in humans (Table 1) (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ). CYB5R2 is a 31 kDa protein speculated to have nucleus and cytosolic confinement. When comparing the amino acid sequence of CYB5R2 to the other CYB5R family members, CYB5R2 has a 59.71% and 58.82% sequence similarity with CYB5R3 and CYB5R1, respectively. By contrast, CYB5R2 shares only a 28.35% and 28.82% sequence similarity with CYB5R4 and CYB5R5, respectively. CYB5R1, CYB5R2, and CYB5R3 all include similar structural motifs (Fig. 2)—namely, an FAD-binding domain consisting of six antiparallel β-sheets and one α-helix, an NADH domain comprised of five β-strands and four α-helices, a linker region consisting of three antiparallel β-sheets, and an alpha-helical membrane anchor (
      • Nishida H.
      • Inaka K.
      • Miki K.
      Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases.
      ). Whether CYB5R2 harbors a membrane anchor remains uncertain. CYB5R2 is phosphorylated at four amino acid sites (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). Two of them, S41 and T157, are situated in the FAD and NADH domains, respectively. On the other hand, two phosphorylation sites, T145 and N131, are situated within the linker region connecting the two domains. This suggests that the linker region is tightly regulated by phosphorylation to perpetuate interdomain interactions, stability, and biological activity. Future studies devoted to understanding the structural and functional implications of CYB5R2 modulation through phosphorylation and the crosstalk between different phosphorylation sites could provide invaluable insight into CYB5R2’s role in physiology and disease.
      The functional role of CYB5R2 in the cardiovascular system has not been investigated. However, one study conducted by Franceschini et al. (
      • Franceschini N.
      • Fox E.
      • Zhang Z.
      • Edwards T.L.
      • Nalls M.A.
      • Sung Y.J.
      • et al.
      Genome-wide association analysis of blood-pressure traits in African-ancestry individuals reveals common associated genes in African and non-African populations.
      ) identified a single nucleotide-polymorphism located 10 kilobases downstream of the CYB5R2 gene that associated with elevated blood pressure exclusively in those with African ancestry. Yet, whether CYB5R2 plays a role in blood pressure regulation has yet to be determined. Aside from this study, CYB5R2 has almost exclusively been investigated in the context of cancer. It was recently shown that CYB5R2 may act as a tumor suppressor gene in human nasopharyngeal cancer (
      • Xiao X.
      • Zhao W.
      • Tian F.
      • Zhou X.
      • Zhang J.
      • Huang T.
      • et al.
      Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma.
      ). This study revealed that CYB5R2 expression was reduced and the CYB5R2 promoter was hypermethylated in nasopharyngeal tumors. CYB5R2 promoter methylation associated with lymph node metastasis, suggesting that downregulation of CYB5R2 protein expression and methylation of its promoter in nasopharyngeal epithelium could potentially be used to forecast lymph node metastasis. Moreover, reconstitution of CYB5R2 in nasopharyngeal cancer cell lines suppressed cell proliferation and migration (
      • Xiao X.
      • Zhao W.
      • Tian F.
      • Zhou X.
      • Zhang J.
      • Huang T.
      • et al.
      Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma.
      ). Intriguingly, an unrelated study corroborated these findings. CYB5R2 was shown to upregulate genes that negatively impact angiogenesis in nasopharyngeal cancer cells and downregulate expression of vascular endothelial growth factor, thereby suppressing angiogenesis and tumor migration (
      • Ming H.
      • Lan Y.
      • He F.
      • Xiao X.
      • Zhou X.
      • Zhang Z.
      • et al.
      Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.
      ). These studies present unique findings that could lead to the development of targeted therapeutics, where enhancing CYB5R2 expression or activity could be a novel therapeutic approach for treating nasopharyngeal cancer.
      Jo et al. discovered a potential protective role for CYB5R2 in colorectal cancer by acting as a tumor suppressing gene. This group found that two separate colorectal cancer cell lines harbor a somatic frameshift mutation in the CYB5R2 gene, resulting in a truncated protein (
      • Jo Y.S.
      • Kim M.S.
      • Yoo N.J.
      • Lee S.H.
      Intratumoral heterogeneity for inactivating frameshift mutation of CYB5R2 gene in colorectal cancers.
      ). However, this investigation did not interrogate the clinical and histopathological parameters associated with colorectal tumors possessing the truncated CYB5R2 protein. These novel findings present a unique role of CYB5R2 in the pathogenesis of colorectal cancer and could lead to potential therapeutic interventions. However, further studies are needed to further illuminate the clinical implications of CYB5R2 frameshift mutations in colorectal cancer.
      Finally, CYB5R2 has been implicated in prostate cancer. Akin to the nasopharyngeal cancer study, CYB5R2 was found to be hypermethylated in prostate cancer in a tissue-specific manner, thereby facilitating prostate pathogenesis (
      • Devaney J.M.
      • Wang S.
      • Funda S.
      • Long J.
      • Taghipour D.J.
      • Tbaishat R.
      • et al.
      Identification of novel DNA-methylated genes that correlate with human prostate cancer and high-grade prostatic intraepithelial neoplasia.
      ). This implies that CYB5R2 may protect against prostate cancer. It would be curious to dissect the mechanisms responsible for the direct or indirect methylation of the CYB5R2 promoter that enable neutralization of the CYB5R2 gene. This study illustrates that epigenetic dysregulation of critical regulatory components, such as CYB5R2, can favor prostate carcinogenesis. These new findings demonstrate a potential role of CYB5R2 in mitigating the progression of prostate cancer, which could lead to the development of therapeutics that effectively target the expression or stability of CYB5R2.

      CYB5R3

      Structure and electron transfer reaction

      CYB5R3 has been extensively studied, concluding that this enzyme is vital for preserving cellular redox equilibrium. CYB5R3 is implicated in several redox reactions that affect lipid metabolism, cholesterol biosynthesis, drug metabolism, oxidative stress, and heme reduction (Table 1) (
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ). There are two isoforms of CYB5R3; a soluble, 31 kDa isoform located in the cytosol of erythrocytes (
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ) and a membrane-bound, 34 kDa isoform tethered to the ER, plasma membrane, and outer mitochondrial membrane via a myristoyl group (Fig. 3) in somatic cells (
      • Borgese N.
      • D'Arrigo A.
      • De Silvestris M.
      • Pietrini G.
      NADH-cytochrome b5 reductase and cytochrome b5 isoforms as models for the study of post-translational targeting to the endoplasmic reticulum.
      ,
      • Borgese N.
      • Aggujaro D.
      • Carrera P.
      • Pietrini G.
      • Bassetti M.
      A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes.
      ,
      • Kedar P.S.
      • Gupta V.
      • Warang P.
      • Chiddarwar A.
      • Madkaikar M.
      Novel mutation (R192C) in CYB5R3 gene causing NADH-cytochrome b5 reductase deficiency in eight Indian patients associated with autosomal recessive congenital methemoglobinemia type-I.
      ). The N-myristoylation of the membrane-bound isoform occurs on a glycine residue located at position 2 (
      • Borgese N.
      • Aggujaro D.
      • Carrera P.
      • Pietrini G.
      • Bassetti M.
      A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes.
      ). The catalytic domains of CYB5R3 are indistinguishable in the soluble and membrane isoforms (
      • Kurian J.R.
      • Bajad S.U.
      • Miller J.L.
      • Chin N.A.
      • Trepanier L.A.
      NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans.
      ), differing only in the N-terminus (
      • Kedar P.S.
      • Gupta V.
      • Warang P.
      • Chiddarwar A.
      • Madkaikar M.
      Novel mutation (R192C) in CYB5R3 gene causing NADH-cytochrome b5 reductase deficiency in eight Indian patients associated with autosomal recessive congenital methemoglobinemia type-I.
      ), which is spliced in the soluble isoform. Both isoforms of CYB5R3 contain two domains: an NADH and an FAD-binding domain. The FAD domain, consisting of six antiparallel β-sheets and one α-helix, seats a large cleft where the FAD prosthetic group is situated. The NADH domain, comprised of five β-strands and four α-helices, harbors a pocket in which NADH associates (
      • Nishida H.
      • Inaka K.
      • Miki K.
      Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases.
      ). These two domains are connected by a linker region which embodies three antiparallel β-sheets (
      • Nishida H.
      • Inaka K.
      • Miki K.
      Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases.
      ). Upon NADH binding to CYB5R3, which occurs in less than 2 milliseconds (
      • Gutiérrez-Merino C.
      • Martínez-Costa O.H.
      • Monsalve M.
      • Samhan-Arias A.K.
      Structural features of cytochrome b5-cytochrome b5 reductase complex formation and implications for the intramolecular dynamics of cytochrome b5 reductase.
      ), a conformational shift occurs in both the NADH and FAD domains that orients T66 closer to the N5 atom of FAD (
      • Yamada M.
      • Tamada T.
      • Takeda K.
      • Matsumoto F.
      • Ohno H.
      • Kosugi M.
      • et al.
      Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer.
      ). T66 is an important amino acid that facilitates efficient electron transfer from NADH to FAD by stabilizing the FAD moeity (
      • Kimura S.
      • Kawamura M.
      • Iyanagi T.
      Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer.
      ). Moreover, molecular dynamics simulations have revealed that R91 has a favorable electrostatic interaction with bound FAD, while K110 is a crucial bridging residue between FAD and NADH, enabling reducing equivalents to be passed to target substrates (
      • Govindarajan G.
      • Mathews S.
      • Srinivasan K.
      • Ramasamy K.
      • Periasamy S.
      Establishment of human retinal mitoscriptome gene expression signature for diabetic retinopathy using cadaver eyes.
      ). The domain rearrangement triggered by NADH binding creates a robust hydrogen-bonding network from the N5 of FAD to His49 and forms a stable stacking complex formed by the isoalloxazine ring of FAD and the nicotinamide ring of NAD+ (
      • Yamada M.
      • Tamada T.
      • Takeda K.
      • Matsumoto F.
      • Ohno H.
      • Kosugi M.
      • et al.
      Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer.
      ). This stacking complex upholds the planarity of the isoalloxazine ring and permits efficient electron transfer to the noncovalently bound FAD molecule (
      • Yamada M.
      • Tamada T.
      • Takeda K.
      • Matsumoto F.
      • Ohno H.
      • Kosugi M.
      • et al.
      Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer.
      ). Electrons transferred to FAD are subsequently passed to target substrates, where CYB5 is typically the first recipient of reducing equivalents (
      • Syed K.
      • Kattamuri C.
      • Thompson T.B.
      • Yadav J.S.
      Cytochrome b₅ reductase-cytochrome b₅ as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.
      ).
      Figure thumbnail gr3
      Figure 3Membrane-bound CYB5R3 is compartmentalized to the ER, OMM, and PM and catalyzes the transfer of electrons from NADH to target substrate. CYB5R3 reduces heme iron and oxidized CoQ in biological membranes to mitigate oxidative stress. In the ER, CYB5R3 reduces stearoyl-CoA desaturase-1 (SCD1) through transferring electrons to the electron mediator cytochrome b5 (isoform B). Reduction of SCD1 results in the desaturation of saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs). The N and G circles (left) reflect the N-myristoyl anchor and glycine residue by which N-myristoylation occurs, respectively.

      Prominent functions and regulation of CYB5R3

      CYB5R3 is considered a “master regulator” of redox balance in cells by catalyzing numerous reduction reactions. Membrane-bound CYB5R3 controls several biological reduction reactions, including CoQ reduction (
      • Villalba J.M.
      • Navarro F.
      • Gómez-Díaz C.
      • Arroyo A.
      • Bello R.I.
      • Navas P.
      Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane.
      ,
      • Villalba J.M.
      • Navas P.
      Plasma membrane redox system in the control of stress-induced apoptosis.
      ), heme reduction (
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ,
      • Straub A.C.
      • Lohman A.W.
      • Billaud M.
      • Johnstone S.R.
      • Dwyer S.T.
      • Lee M.Y.
      • et al.
      Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.
      ,
      • Amdahl M.B.
      • Sparacino-Watkins C.E.
      • Corti P.
      • Gladwin M.T.
      • Tejero J.
      Efficient reduction of vertebrate cytoglobins by the cytochrome b5/cytochrome b5 reductase/NADH system.
      ,
      • Rahaman M.M.
      • Nguyen A.T.
      • Miller M.P.
      • Hahn S.A.
      • Sparacino-Watkins C.
      • Jobbagy S.
      • et al.
      Cytochrome b5 reductase 3 modulates soluble guanylate cyclase redox state and cGMP signaling.
      ), and lipid elongation and desaturation (
      • Sacco J.C.
      • Trepanier L.A.
      Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.
      ,
      • Hildebrandt A.
      • Estabrook R.W.
      Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions.
      ) (Table 1). The most characterized is lipid metabolism in the liver, where CYB5R3 participates in fatty acid elongation and desaturation (Fig. 3) (
      • Martin-Montalvo A.
      • Sun Y.
      • Diaz-Ruiz A.
      • Ali A.
      • Gutierrez V.
      • Palacios H.H.
      • et al.
      Cytochrome b5 reductase and the control of lipid metabolism and healthspan.
      ). ER-localized CYB5R3 ideally positions the enzyme where fatty acid desaturation and elongation transpires, permitting the CYB5R3-dependent reduction of stearoyl-CoA desaturase-1 (Fig. 3). Mouse studies have shown that overexpressed CYB5R3 increased long chain unsaturated fatty acids, commensurate with a longer lifespan (
      • Martin-Montalvo A.
      • Sun Y.
      • Diaz-Ruiz A.
      • Ali A.
      • Gutierrez V.
      • Palacios H.H.
      • et al.
      Cytochrome b5 reductase and the control of lipid metabolism and healthspan.
      ). However, it is worth noting that no investigations have shown that endogenous CYB5R3 regulates fatty acid desaturation and elongation. Instead, these early studies only implicated CYB5R activity or peptide fragments with such an activity in lysates, usually the liver (
      • Oshino N.
      • Sato R.
      Stimulation by phenols of the reoxidation microsomal bound cytochrome b5 and its implication to fatty acid desaturation.
      ). The enzyme with CYB5R activity was later inferred to be on chromosome 22 where human CYB5R3 is located based on 2,6-dichlorophenolindophenol reduction activity (
      • Bull P.C.
      • Shephard E.A.
      • Povey S.
      • Santisteban I.
      • Phillips I.R.
      Cloning and chromosomal mapping of human cytochrome b5 reductase (DIA1).
      ). Despite our vast knowledge on CYB5R3, many questions endure regarding its redox regulatory roles during divergent physiological or pathological conditions.
      CYB5R3 is posttranslationally modified via phosphorylation, ubiquitination, and acetylation at several amino acid residues, the most abundant being phosphorylation at Y80 (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). Y80 is situated in the FAD domain near the FAD-binding site, implying that phosphorylation at this site might govern electron transfer efficacy to the FAD. It is possible that phosphorylation at Y80 mediates CYB5R3 activity by enhancing or reducing enzymatic activity. Yet, functional studies interrogating how Y80 phosphorylation impacts CYB5R3 activity and its relationship with disease have not been reported. Future studies dedicated to understanding PTMs at Y80 could provide valuable information into CYB5R3 regulation and its role in physiology and disease.

      CYB5 as an essential substrate

      One of the most crucial protein partner substrates of CYB5R3 is CYB5. The CYB5 family plays a key role in mediating the electron transfer reaction carried out by CYB5R3 (Table 2). CYB5 is a small, ubiquitously expressed heme protein found in plants, animals, and fungi, functioning as an electron transporter in a plethora of reactions (
      • Zheng H.
      • Li X.
      • Shi L.
      • Jing Y.
      • Song Q.
      • Chen Y.
      • et al.
      Genome-wide identification and analysis of the cytochrome B5 protein family in Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).
      ). Studies in plants, yeast, and mammals have demonstrated that CYB5 can also accept electrons from NADPH:cytochrome P450 reductase (
      • Wayne L.L.
      • Wallis J.G.
      • Kumar R.
      • Markham J.E.
      • Browse J.
      Cytochrome b5 reductase encoded by CBR1 is essential for a functional male gametophyte in Arabidopsis.
      ). In vertebrates, there are two isoforms of CYB5: CYB5A, anchored to the endoplasmic reticulum membrane, and CYB5B, anchored to the outer mitochondrial membrane (
      • Deng B.
      • Parthasarathy S.
      • Wang W.
      • Gibney B.R.
      • Battaile K.P.
      • Lovell S.
      • et al.
      Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
      ). Both isoforms contain a heme-binding domain with near-identical folds, comprised of six alpha-helices and five beta-sheets (
      • Rodríguez-Marañón M.J.
      • Qiu F.
      • Stark R.E.
      • White S.P.
      • Zhang X.
      • Foundling S.I.
      • et al.
      13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.
      ,
      • Durley R.C.
      • Mathews F.S.
      Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution.
      ). CYB5 and CYB5R3 are ubiquitously expressed proteins generally involved in NADH-dependent electron transport, where CYB5R3 transfers two reducing equivalents from NADH to FAD situated in the FAD-binding domain, then ultimately to CYB5 (
      • Syed K.
      • Kattamuri C.
      • Thompson T.B.
      • Yadav J.S.
      Cytochrome b₅ reductase-cytochrome b₅ as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2.
      ). A study with purified enzyme showed that CYB5R3 binds to CYB5 with a Km of 20 μM and reaches a Vmax of 272 μmol min−1 mg−1 when NADH is used as an electron donor (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ). One study revealed, through site-directed mutagenesis, that electrostatic interactions between lysine residues in CYB5R3 and the carboxyl groups in CYB5 maintains the two proteins in a tight complex for electron transfer (
      • Shirabe K.
      • Nagai T.
      • Yubisui T.
      • Takeshita M.
      Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 studied by site-directed mutagenesis.
      ). Critically, the reduction of FAD is the rate-limiting step in the electron transfer from CYB5R3 to CYB5 (
      • Kimura S.
      • Kawamura M.
      • Iyanagi T.
      Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer.
      ). CYB5 acts as an electron transfer mediator during CYB5R3-catalyzed reactions as shown through P450 monooxygenation (
      • Hildebrandt A.
      • Estabrook R.W.
      Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactions.
      ), fatty acid desaturation and elongation (
      • Shimakata T.
      • Mihara K.
      • Sato R.
      Reconstitution of hepatic microsomal stearoyl-coenzyme A desaturase system from solubilized components.
      ,
      • Keyes S.R.
      • Alfano J.A.
      • Jansson I.
      • Cinti D.L.
      Rat liver microsomal elongation of fatty acids. Possible involvement of cytochrome b5.
      ), myoglobin reduction (
      • Liu X.
      • Tong J.
      • Zweier J.R.
      • Follmer D.
      • Hemann C.
      • Ismail R.S.
      • et al.
      Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.
      ), cytoglobin reduction (
      • Amdahl M.B.
      • Sparacino-Watkins C.E.
      • Corti P.
      • Gladwin M.T.
      • Tejero J.
      Efficient reduction of vertebrate cytoglobins by the cytochrome b5/cytochrome b5 reductase/NADH system.
      ,
      • Liu X.
      • Tong J.
      • Zweier J.R.
      • Follmer D.
      • Hemann C.
      • Ismail R.S.
      • et al.
      Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.
      ,
      • Liu X.
      • El-Mahdy M.A.
      • Boslett J.
      • Varadharaj S.
      • Hemann C.
      • Abdelghany T.M.
      • et al.
      Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.
      ), and hemoglobin reduction (
      • Zweier J.L.
      • Ilangovan G.
      Regulation of nitric oxide metabolism and vascular tone by cytoglobin.
      ). However, CYB5R3 has been shown to function without CYB5, as shown with electron transfer to CoQ to stabilize ascorbate (
      • Villalba J.M.
      • Navarro F.
      • Gómez-Díaz C.
      • Arroyo A.
      • Bello R.I.
      • Navas P.
      Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane.
      ,
      • Navarro F.
      • Villalba J.M.
      • Crane F.L.
      • Mackellar W.C.
      • Navas P.
      A phospholipid-dependent NADH-coenzyme Q reductase from liver plasma membrane.
      ,
      • Villalba J.M.
      • Navarro F.
      • Córdoba F.
      • Serrano A.
      • Arroyo A.
      • Crane F.L.
      • et al.
      Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.
      ), the recycling of plasma membrane vitamin E (
      • Kagan V.E.
      • Arroyo A.
      • Tyurin V.A.
      • Tyurina Y.Y.
      • Villalba J.M.
      • Navas P.
      Plasma membrane NADH-coenzyme Q0 reductase generates semiquinone radicals and recycles vitamin E homologue in a superoxide-dependent reaction.
      ), and the protection against ceramide-induced apoptosis (
      • Navas P.
      • Fernandez-Ayala D.M.
      • Martin S.F.
      • Lopez-Lluch G.
      • De Caboa R.
      • Rodriguez-Aguilera J.C.
      • et al.
      Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells.
      ).
      Table 2Known associated partners and substrates of human CYB5R3
      Associated partners and substratesPrimary subcellular location(s)Tissue location(s)Functional roleReferences
      Cytochrome b5 (isoforms A and B)Mitochondria, cytosolUbiquitousElectron acceptor and carrier(
      • Zheng H.
      • Li X.
      • Shi L.
      • Jing Y.
      • Song Q.
      • Chen Y.
      • et al.
      Genome-wide identification and analysis of the cytochrome B5 protein family in Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).
      ,
      • Deng B.
      • Parthasarathy S.
      • Wang W.
      • Gibney B.R.
      • Battaile K.P.
      • Lovell S.
      • et al.
      Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
      )
      Soluble guanylate cyclaseCytosolPrimarily in heart tissue, particularly vascular smooth muscleElectron acceptor leading to heme iron reduction, catalyzes reduction of GTP to cGMP(
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Rahaman M.M.
      • Nguyen A.T.
      • Miller M.P.
      • Hahn S.A.
      • Sparacino-Watkins C.
      • Jobbagy S.
      • et al.
      Cytochrome b5 reductase 3 modulates soluble guanylate cyclase redox state and cGMP signaling.
      ,
      • Kang Y.
      • Liu R.
      • Wu J.-X.
      • Chen L.
      Structural insights into the mechanism of human soluble guanylate cyclase.
      )
      Mitochondrial amidoxime reducing component (mARC)MitochondriaUbiquitousElectron transfer reaction, catalyzes the reduction of N-oxygenated molecules, drug metabolism(
      • Wahl B.
      • Reichmann D.
      • Niks D.
      • Krompholz N.
      • Havemeyer A.
      • Clement B.
      • et al.
      Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes.
      ,
      • Jakobs H.H.
      • Mikula M.
      • Havemeyer A.
      • Strzalkowska A.
      • Borowa-Chmielak M.
      • Dzwonek A.
      • et al.
      The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice.
      ,
      • Plitzko B.
      • Ott G.
      • Reichmann D.
      • Henderson C.J.
      • Wolf C.R.
      • Mendel R.
      • et al.
      The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells.
      ,
      • Krompholz N.
      • Krischkowski C.
      • Reichmann D.
      • Garbe-Schönberg D.
      • Mendel R.-R.
      • Bittner F.
      • et al.
      The mitochondrial amidoxime reducing component (mARC) is involved in detoxification of N-hydroxylated base analogues.
      )
      Coenzyme QMitochondriaUbiquitousElectron-transferring membrane protein complex in the mitochondrial respiratory chain, protection against lipid peroxidation(
      • Turunen M.
      • Olsson J.
      • Dallner G.
      Metabolism and function of coenzyme Q.
      ,
      • Yuan S.
      • Schmidt H.M.
      • Wood K.C.
      • Straub A.C.
      CoenzymeQ in cellular redox regulation and clinical heart failure.
      )
      HemoglobinCytosol, extracellularUbiquitous, but most highly expressed in the bloodElectron acceptor leading to heme iron reduction, oxygen transport to peripheral tissues(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Trent J.T.
      • Hargrove M.S.
      A ubiquitously expressed human hexacoordinate hemoglobin.
      ,
      • Marengo-Rowe A.J.
      Structure-function relations of human hemoglobins.
      )
      MyoglobinCytosol, extracellularPrimarily cardiac and skeletal muscleStorage and transport of oxygen from the cell membrane to the mitochondria, nitric oxide regulation(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Liu X.
      • Tong J.
      • Zweier J.R.
      • Follmer D.
      • Hemann C.
      • Ismail R.S.
      • et al.
      Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.
      ,
      • Wittenberg J.B.
      Myoglobin-facilitated oxygen diffusion: role of myoglobin in oxygen entry into muscle.
      )
      MitoregulinMitochondriaPrimarily cardiac and skeletal muscle, adipose tissueRegulates mitochondrial complex assembly and respiration rate, controls mitochondrial ROS levels, maintains cellular lipid composition(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Stein C.S.
      • Jadiya P.
      • Zhang X.
      • McLendon J.M.
      • Abouassaly G.M.
      • Witmer N.H.
      • et al.
      Mitoregulin: a lncRNA-encoded microprotein that supports mitochondrial supercomplexes and respiratory efficiency.
      )
      FoxO1Mitochondria, nucleus, cytosolPrimarily skeletal muscleInsulin signaling, regulation of metabolic homeostasis in response to oxidative stress(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.-A.
      • Matsuhisa M.
      • Hori M.
      • et al.
      The forkhead transcription factor Foxo1 bridges the JNK pathway and the transcription factor PDX-1 through its intracellular translocation.
      ,
      • Kitamura Y.I.
      • Kitamura T.
      • Kruse J.-P.
      • Raum J.C.
      • Stein R.
      • Gu W.
      • et al.
      FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction.
      ,
      • Kim-Muller J.Y.
      • Kim Y.J.R.
      • Fan J.
      • Zhao S.
      • Banks A.S.
      • Prentki M.
      • et al.
      FoxO1 deacetylation decreases fatty acid oxidation in β-cells and sustains insulin secretion in diabetes.
      ,
      • Lu H.
      • Huang H.
      FOXO1: a potential target for human diseases.
      )
      Molecular oxygenUbiquitousUbiquitousOxidative phosphorylation(
      • Wilson D.F.
      • Harrison D.K.
      • Vinogradov S.A.
      Oxygen, pH, and mitochondrial oxidative phosphorylation.
      ,
      • Wilson D.F.
      Oxidative phosphorylation: regulation and role in cellular and tissue metabolism.
      )
      VDAC1MitochondriaPrimarily skeletal muscleFacilitates the transport of metabolites and ions across the outer mitochondrial membrane(
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Camara A.K.S.
      • Zhou Y.
      • Wen P.-C.
      • Tajkhorshid E.
      • Kwok W.-M.
      Mitochondrial VDAC1: a key gatekeeper as potential therapeutic target.
      ,
      • Nikiforova A.B.
      • Saris N.-E.L.
      • Kruglov A.G.
      External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?.
      ,
      • Shimada H.
      • Hirai K.-I.
      • Simamura E.
      • Hatta T.
      • Iwakiri H.
      • Mizuki K.
      • et al.
      Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase.
      )
      NOX4Plasma Membrane, nucleus, mitochondria, ERPrimarily in the kidney, arteryOxygen sensor, catalyzes the reduction of molecular oxygen to ROS(
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ,
      • Nisimoto Y.
      • Diebold B.A.
      • Cosentino-Gomes D.
      • Lambeth J.D.
      Nox4: a hydrogen peroxide-generating oxygen sensor.
      ,
      • Sedeek M.
      • Nasrallah R.
      • Touyz R.M.
      • Hébert R.L.
      NADPH oxidases, reactive oxygen species, and the kidney: friend and foe.
      ,
      • Brandes R.P.
      • Weissmann N.
      • Schröder K.
      Nox family NADPH oxidases: molecular mechanisms of activation.
      )
      AscorbateUbiquitousUbiquitousPotent antioxidant(
      • Bakalova R.
      • Zhelev Z.
      • Miller T.
      • Aoki I.
      • Higashi T.
      Vitamin C versus cancer: ascorbic acid radical and impairment of mitochondrial respiration?.
      ,
      • Beyer R.E.
      The role of ascorbate in antioxidant protection of biomembranes: interaction with vitamin E and coenzyme Q.
      )
      CytoglobinCytosolUbiquitousFacilitates oxygen transport, protection against oxidative stress, NO scavenging(
      • Amdahl M.B.
      • Sparacino-Watkins C.E.
      • Corti P.
      • Gladwin M.T.
      • Tejero J.
      Efficient reduction of vertebrate cytoglobins by the cytochrome b5/cytochrome b5 reductase/NADH system.
      ,
      • Liu X.
      • Tong J.
      • Zweier J.R.
      • Follmer D.
      • Hemann C.
      • Ismail R.S.
      • et al.
      Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.
      ,
      • Liu X.
      • El-Mahdy M.A.
      • Boslett J.
      • Varadharaj S.
      • Hemann C.
      • Abdelghany T.M.
      • et al.
      Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.
      ,
      • Zweier J.L.
      • Ilangovan G.
      Regulation of nitric oxide metabolism and vascular tone by cytoglobin.
      ,
      • Amdahl M.B.
      • DeMartino A.W.
      • Tejero J.
      • Gladwin M.T.
      Cytoglobin at the crossroads of vascular remodeling.
      )
      The main subcellular and tissue location, as well as the functional role, of each substrate/partner are shown.

      The roles of CYB5R3 in mediating redox balance and oxidative stress

      A multitude of studies have identified key substrates of CYB5R3 that play a role in mediating nitric oxide (NO) and ROS signaling. One example is soluble guanylate cyclase (sGC) in vascular smooth muscle cells (Table 2). sGC is activated upon binding of NO, catalyzing the formation of cyclic GMP, leading to blood vessel dilation. The major prerequisite for NO-induced sGC activation is reduced heme iron (Fe2+) in the sGC β H-NOX domain (
      • Rahaman M.M.
      • Nguyen A.T.
      • Miller M.P.
      • Hahn S.A.
      • Sparacino-Watkins C.
      • Jobbagy S.
      • et al.
      Cytochrome b5 reductase 3 modulates soluble guanylate cyclase redox state and cGMP signaling.
      ). In the presence of oxidants, sGC can become oxidized and insensitive to NO, a state that contributes to a myriad of diseases (
      • Stasch J.-P.
      • Schmidt P.M.
      • Nedvetsky P.I.
      • Nedvetskaya T.Y.
      • Arun Kumar H.S.
      • Meurer S.
      • et al.
      Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.
      ,
      • Stasch J.-P.
      • Schmidt P.
      • Alonso-Alija C.
      • Apeler H.
      • Dembowsky K.
      • Haerter M.
      • et al.
      NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle.
      ,
      • Geschka S.
      • Kretschmer A.
      • Sharkovska Y.
      • Evgenov O.V.
      • Lawrenz B.
      • Hucke A.
      • et al.
      Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats.
      ). sGC heme iron is maintained in its Fe2+ state via direct interaction with CYB5R3, thereby regulating cGMP signaling needed for downstream activation of protein kinase G–dependent signaling and blood vessel dilation (
      • Rahaman M.M.
      • Nguyen A.T.
      • Miller M.P.
      • Hahn S.A.
      • Sparacino-Watkins C.
      • Jobbagy S.
      • et al.
      Cytochrome b5 reductase 3 modulates soluble guanylate cyclase redox state and cGMP signaling.
      ). Studies with purified enzyme demonstrated that sGC heme iron is reduced by CYB5R3 with a rate constant of 1.56 × 104 M−1 min−1 (
      • Peng L.
      • Ma W.
      • Xie Q.
      • Chen B.
      Identification and validation of hub genes for diabetic retinopathy.
      ). Two studies demonstrated that mice with CYB5R3 deficiency in vascular smooth muscle cells exhibited increased mean arterial systemic pressure as a consequence of impaired sGC reduction in angiotensin II–induced hypertension and in sickle cell disease (
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ,
      • Wood K.C.
      • Durgin B.G.
      • Schmidt H.M.
      • Hahn S.A.
      • Baust J.J.
      • Bachman T.
      • et al.
      Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice.
      ). These studies demonstrated that targeting the CYB5R3-sGC axis could alleviate the poor outcomes associated with cardiovascular diseases. CYB5R3 has also been shown to cooperate with NADPH oxidase 4 (NOX4), an NADPH oxidase that reduces molecular oxygen to generate predominately hydrogen peroxide in vascular endothelial cells on the outer mitochondrial membrane (Table 2) (
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ). It has been demonstrated that CYB5R3 bolsters NOX4-derived hydrogen peroxide production at the outer mitochondrial membrane (
      • Nisimoto Y.
      • Diebold B.A.
      • Cosentino-Gomes D.
      • Lambeth J.D.
      Nox4: a hydrogen peroxide-generating oxygen sensor.
      ) and is optimal when coupled with CoQ (
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ). Importantly, CYB5R3’s regulation of NOX4-dependent hydrogen peroxide production reduces vascular wall inflammation and tempers inflammatory signaling. This newfound molecular interaction provides key insight into possible therapeutic options for clinical management of inflammatory diseases, potentially in patients who possess loss-of-function mutations in the CYB5R3 gene (
      • Yuan S.
      • Hahn S.A.
      • Miller M.P.
      • Sanker S.
      • Calderon M.J.
      • Sullivan M.
      • et al.
      Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.
      ).
      It is widely accepted that CYB5R3 plays an important role in antioxidant stress responses and can be viewed as a “resilience enzyme” that protects cells from stress. Under stress conditions, CYB5R3 maintains membrane embedded α-tocopherol and ascorbate, potent membrane antioxidants in living cells, in their reduced state (Fig. 3 and Table 2) (
      • De Cabo R.
      • Cabello R.
      • Rios M.
      • López-Lluch G.
      • Ingram D.K.
      • Lane M.A.
      • et al.
      Calorie restriction attenuates age-related alterations in the plasma membrane antioxidant system in rat liver.
      ,
      • de Cabo R.
      • Burgess J.R.
      • Navas P.
      Adaptations to oxidative stress induced by vitamin E deficiency in rat liver.
      ,
      • Bello R.I.
      • Alcaín F.J.
      • Gómez-Díaz C.
      • López-Lluch G.
      • Navas P.
      • Villalba J.M.
      Hydrogen peroxide- and cell-density-regulated expression of NADH-cytochrome b5 reductase in HeLa cells.
      ). This is achieved through the CYB5R3-catalyzed reduction of CoQ in biological membranes (Fig. 3). CoQ is a molecule present in all cells and membranes, where it functions not only as an important electron carrier in the mitochondrial respiratory chain (
      • Turunen M.
      • Olsson J.
      • Dallner G.
      Metabolism and function of coenzyme Q.
      ) but also as a potent antioxidant that can independently quench ROS or through reducing α-tocopherol and ascorbate free radical (AFR) (Table 2) (
      • Hernández-Camacho J.D.
      • Bernier M.
      • López-Lluch G.
      • Navas P.
      Coenzyme Q10 supplementation in aging and disease.
      ). As such, CYB5R3 reduction of CoQ is essential to the membrane antioxidant pathway (
      • Siendones E.
      • SantaCruz-Calvo S.
      • Martín-Montalvo A.
      • Cascajo M.V.
      • Ariza J.
      • López-Lluch G.
      • et al.
      Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Tissue-based map of the human proteome.
      ,
      • Hyun D.-H.
      • Emerson S.S.
      • Jo D.-G.
      • Mattson M.P.
      • de Cabo R.
      Calorie restriction up-regulates the plasma membrane redox system in brain cells and suppresses oxidative stress during aging.
      ) for protection against lipid peroxidation, a process in which oxidants attack polyunsaturated fatty acid phospholipids leading to the degradation and subsequent perturbation of cell membranes (
      • Ayala A.
      • Muñoz M.F.
      • Argüelles S.
      Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal.
      ). Thus, maintaining CoQ and α-tocopherol in their reduced states is essential for antioxidant protection. The conversion of AFR to ascorbate, a potent antioxidant (
      • Shirabe K.
      • Landi M.T.
      • Takeshita M.
      • Uziel G.
      • Fedrizzi E.
      • Borgese N.
      A novel point mutation in a 3’ splice site of the NADH-cytochrome b5 reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia.
      ), is also pivotal in protecting cells against lipid peroxidation (
      • Ramana K.V.
      • Srivastava S.
      • Singhal S.S.
      Lipid peroxidation products in human health and disease.
      ), a reaction carried out by CYB5R3 as previously described. Although not a direct interaction, CYB5R3 and voltage-dependent anion-selective channel 1 (VDAC1); an outer mitochondrial membrane protein responsible for the passage of metabolites, ions, and nucleotides into the mitochondria (
      • Camara A.K.S.
      • Zhou Y.
      • Wen P.-C.
      • Tajkhorshid E.
      • Kwok W.-M.
      Mitochondrial VDAC1: a key gatekeeper as potential therapeutic target.
      ); have been shown to work in tandem as a “redox-cycling system” to mediate AFR transport (Table 2) (
      • Bakalova R.
      • Zhelev Z.
      • Miller T.
      • Aoki I.
      • Higashi T.
      Vitamin C versus cancer: ascorbic acid radical and impairment of mitochondrial respiration?.
      ). In addition to oxidative stress suppression, the reduction of AFR to ascorbate restores the cellular ascorbate pool and maintains the cellular NAD+/NADH ratio (
      • Bakalova R.
      • Zhelev Z.
      • Miller T.
      • Aoki I.
      • Higashi T.
      Vitamin C versus cancer: ascorbic acid radical and impairment of mitochondrial respiration?.
      ), both of which are critical for governing the intracellular redox state and metabolic processes. VDAC1 controls membrane integrity while also governing the flow of AFR into the mitochondrial matrix (
      • Nikiforova A.B.
      • Saris N.-E.L.
      • Kruglov A.G.
      External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?.
      ,
      • Shimada H.
      • Hirai K.-I.
      • Simamura E.
      • Hatta T.
      • Iwakiri H.
      • Mizuki K.
      • et al.
      Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase.
      ). Given the indirect nature of CYB5R3-VDAC1 interactions, further studies must be performed to characterize alternative signaling mediators and pathways that could be involved in the “redox-cycling system” to mitigate excessive oxidative stress.

      Globins as primary substrates for CYB5R3

      Among CYB5R3’s most important substrates in human physiology are globins, a family of heme-containing globular proteins necessary for oxygen transport to tissues (Table 2). The soluble form of CYB5R3 is responsible for the reduction of methemoglobin to hemoglobin in erythrocytes, permitting adequate oxygen binding and delivery to downstream tissues (
      • Passon P.G.
      • Reed D.W.
      • Hultquist D.E.
      Soluble cytochrome b5 from human erythrocytes.
      ,
      • Hultquist D.E.
      • Passon P.G.
      Catalysis of methaemoglobin reduction by erythrocyte cytochrome B5 and cytochrome B5 reductase.
      ). In this reaction, CYB5R3 reduces the ferric iron within methemoglobin to convert it to ferrous iron (
      • Elahian F.
      • Sepehrizadeh Z.
      • Moghimi B.
      • Mirzaei S.A.
      Human cytochrome b5 reductase: structure, function, and potential applications.
      ). The reduction of FAD is a rate-limiting step in this electron transfer reaction (
      • Elahian F.
      • Sepehrizadeh Z.
      • Moghimi B.
      • Mirzaei S.A.
      Human cytochrome b5 reductase: structure, function, and potential applications.
      ,
      • Kimura S.
      • Kawamura M.
      • Iyanagi T.
      Role of Thr(66) in porcine NADH-cytochrome b5 reductase in catalysis and control of the rate-limiting step in electron transfer.
      ). Importantly, the reduction of methemoglobin is not exclusive to the soluble isoform of CYB5R3. Straub et al. demonstrated a novel paradigm between membrane-bound CYB5R3 and alpha globin expressed in arterial endothelial cells. At the myoendothelial junction, the heme iron of alpha globin is redox-regulated by CYB5R3 to control NO diffusion and vascular tone (
      • Straub A.C.
      • Lohman A.W.
      • Billaud M.
      • Johnstone S.R.
      • Dwyer S.T.
      • Lee M.Y.
      • et al.
      Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.
      ). This study highlights that CYB5R3 plays a significant role in mediating globin redox state not only in erythrocytes but also in arterial endothelial cells. Additionally, CYB5R3 also directly reduces myoglobin, a globin hemoprotein that possesses a reactive heme iron for binding oxygen and subsequent transport from the plasma membrane to the mitochondria in muscle fibers (
      • Wittenberg J.B.
      Myoglobin-facilitated oxygen diffusion: role of myoglobin in oxygen entry into muscle.
      ). When the iron is oxidized to its ferric form (metmyoglobin), oxygen binding is mitigated and oxidative phosphorylation is hampered (
      • Arihara K.
      • Cassens R.G.
      • Greaser M.L.
      • Luchansky J.B.
      • Mozdziak P.E.
      Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle.
      ). CYB5R3 is responsible for reducing metmyoglobin iron to its ferrous state, which maintains the physiological role of myoglobin in muscle tissue (
      • Arihara K.
      • Cassens R.G.
      • Greaser M.L.
      • Luchansky J.B.
      • Mozdziak P.E.
      Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle.
      ). Lastly, cytoglobin can also be reduced by CYB5R3. Cytoglobin is similar to hemoglobin and myoglobin containing a hexacoordinate heme that facilitates oxygen transport and protects against oxidative stress (
      • Trent J.T.
      • Hargrove M.S.
      A ubiquitously expressed human hexacoordinate hemoglobin.
      ). The reduction reaction involves CYB5B as an intermediate substrate of CYB5R3 in the transfer of electrons to cytoglobin (
      • Amdahl M.B.
      • DeMartino A.W.
      • Tejero J.
      • Gladwin M.T.
      Cytoglobin at the crossroads of vascular remodeling.
      ). The existence of this reaction has been illustrated in vascular smooth muscle cells, with cytoglobin playing a key role in regulating blood pressure and vascular tone via NO-scavenging mechanisms (
      • Amdahl M.B.
      • Sparacino-Watkins C.E.
      • Corti P.
      • Gladwin M.T.
      • Tejero J.
      Efficient reduction of vertebrate cytoglobins by the cytochrome b5/cytochrome b5 reductase/NADH system.
      ,
      • Liu X.
      • El-Mahdy M.A.
      • Boslett J.
      • Varadharaj S.
      • Hemann C.
      • Abdelghany T.M.
      • et al.
      Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.
      ). Interestingly, the reduction of purified, human cytoglobin occurs at an order of magnitude faster than other heme-containing globins (
      • Amdahl M.B.
      • Sparacino-Watkins C.E.
      • Corti P.
      • Gladwin M.T.
      • Tejero J.
      Efficient reduction of vertebrate cytoglobins by the cytochrome b5/cytochrome b5 reductase/NADH system.
      ,
      • Liu X.
      • El-Mahdy M.A.
      • Boslett J.
      • Varadharaj S.
      • Hemann C.
      • Abdelghany T.M.
      • et al.
      Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.
      ,
      • Zweier J.L.
      • Ilangovan G.
      Regulation of nitric oxide metabolism and vascular tone by cytoglobin.
      ). Although this interaction between CYB5R3 and globins has been demonstrated indirectly, the direct interaction has not been shown experimentally. In fact, loss of CYB5R3 in smooth muscle cells in vivo did not impact sodium nitroprusside–stimulated vasodilation, suggesting that there is likely another reductase controlling cytoglobin-mediated NO scavenging in the compensation for the loss of CYB5R3 (
      • Durgin B.G.
      • Hahn S.A.
      • Schmidt H.M.
      • Miller M.P.
      • Hafeez N.
      • Mathar I.
      • et al.
      Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.
      ). Currently, it is unclear whether the reduction of all globins by CYB5R3 requires CYB5 as an electron intermediate. It is worth investigating whether hemoglobin and myoglobin, like cytoglobin, also require CYB5 as the intermediate electron carrier in vitro and in vivo. Additionally, it remains to be determined whether other reductases in the CYB5R family directly reduce hemoglobin or require CYB5 and how this may be different based on tissue and cell type. Future studies aimed at investigating these possibilities are warranted to better understand how the CYB5R family of enzymes might govern globin reduction.

      Mitochondrial-associated partners of CYB5R3

      Membrane-bound CYB5R3 can localize to the outer mitochondrial membrane. As such, several mitochondrial-associated partners of CYB5R3 have been discovered, such as mitochondrial amidoxime reducing component (mARC) and mitoregulin (Mtln). Studies show that CYB5R3 interacts with mARC, a mammalian molybdenum-containing enzyme that exists in two isoforms, mARC1 and mARC2 (
      • Wahl B.
      • Reichmann D.
      • Niks D.
      • Krompholz N.
      • Havemeyer A.
      • Clement B.
      • et al.
      Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes.
      ), and catalyzes the reduction of N-oxygenated and N-hydroxylated structures, respectively (Table 2) (
      • Jakobs H.H.
      • Mikula M.
      • Havemeyer A.
      • Strzalkowska A.
      • Borowa-Chmielak M.
      • Dzwonek A.
      • et al.
      The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice.
      ). However, cell culture studies show that electron transfer by mARC requires its strong interaction with CYB5B alone or both CYB5B and CYB5R3 (
      • Plitzko B.
      • Ott G.
      • Reichmann D.
      • Henderson C.J.
      • Wolf C.R.
      • Mendel R.
      • et al.
      The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells.
      ,
      • Krompholz N.
      • Krischkowski C.
      • Reichmann D.
      • Garbe-Schönberg D.
      • Mendel R.-R.
      • Bittner F.
      • et al.
      The mitochondrial amidoxime reducing component (mARC) is involved in detoxification of N-hydroxylated base analogues.
      ). The synergy of mARC and CYB5R3, and in some instances also CYB5, is essential for regulating N-reductive drug metabolism in human cells (
      • Plitzko B.
      • Havemeyer A.
      • Bork B.
      • Bittner F.
      • Mendel R.
      • Clement B.
      Defining the role of the NADH-cytochrome-b5 reductase 3 in the mitochondrial amidoxime reducing component enzyme system.
      ). It is unclear whether the other CYB5R family members play a role in this N-reductive system. CYB5R3 also interacts with the 56 amino acid-long peptide Mtln (Table 2). Mtln is encoded by the gene LINC00116 and is localized to the mitochondria. Mtln is also important for mitochondrial respiratory complex I activity (
      • Chugunova A.
      • Loseva E.
      • Mazin P.
      • Mitina A.
      • Navalayeu T.
      • Bilan D.
      • et al.
      LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism.
      ), decreasing mitochondrial ROS (
      • Stein C.S.
      • Jadiya P.
      • Zhang X.
      • McLendon J.M.
      • Abouassaly G.M.
      • Witmer N.H.
      • et al.
      Mitoregulin: a lncRNA-encoded microprotein that supports mitochondrial supercomplexes and respiratory efficiency.
      ), and forming mitochondrial super complexes (
      • Stein C.S.
      • Jadiya P.
      • Zhang X.
      • McLendon J.M.
      • Abouassaly G.M.
      • Witmer N.H.
      • et al.
      Mitoregulin: a lncRNA-encoded microprotein that supports mitochondrial supercomplexes and respiratory efficiency.
      ). CYB5R3 interacts with Mtln at the mitochondrial membrane, where it likely maintains lipid homeostasis, metabolism, and integrity of the mitochondrial membrane, given its role in fatty acid desaturation and cholesterol biosynthesis (
      • Keyes S.R.
      • Alfano J.A.
      • Jansson I.
      • Cinti D.L.
      Rat liver microsomal elongation of fatty acids. Possible involvement of cytochrome b5.
      ,
      • Chugunova A.
      • Loseva E.
      • Mazin P.
      • Mitina A.
      • Navalayeu T.
      • Bilan D.
      • et al.
      LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism.
      ). It was speculated that Mtln acts to stabilize CYB5R3 and protects it from partial or complete proteolysis (
      • Chugunova A.
      • Loseva E.
      • Mazin P.
      • Mitina A.
      • Navalayeu T.
      • Bilan D.
      • et al.
      LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism.
      ) but it has only been found to bolster CYB5R3 activity related to lipid metabolism through an unknown mechanism. It is worth mentioning that the authors do not distinguish between a direct or indirect interaction. The conclusion that CYB5R3 interacts with Mtln was based largely on copurification studies. To support their findings, additional cell-based and functional assays assessing interactions, mitochondrial efficiency, and membrane composition should be performed to ascertain whether the two enzymes interact at the mitochondrial membrane.

      CYB5R3’s role in cardiovascular disease

      CYB5R3 deficiency is linked to cardiovascular disease. Among the most well-characterized diseases caused by CYB5R3 deficiency is recessive congenital methemoglobinemia (RCM), a hereditary disease where the oxygen carrying capacity of hemoglobin is compromised in erythrocytes (Fig. 4) (
      • Ewenczyk C.
      • Leroux A.
      • Roubergue A.
      • Laugel V.
      • Afenjar A.
      • Saudubray J.M.
      • et al.
      Recessive hereditary methaemoglobinaemia, type II: delineation of the clinical spectrum.
      ). Nearly 60 different human genetic variants for CYB5R3 have been reported with evidence of a substantial role in the pathogenesis of RCM (
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ) (Fig. 1). Loss-of-function mutations in CYB5R3 increase erythrocytic methemoglobin levels, limiting oxygen binding and delivery to tissues (
      • Marengo-Rowe A.J.
      Structure-function relations of human hemoglobins.
      ). RCM exists in two forms: type 1 and type 2. Type 1 is caused primarily by missense mutations and that gives rise to an enzymatically active but unstable CYB5R3 protein. Type 1 manifests as cyanosis (
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ), an abnormal discoloration of the skin caused by high levels of deoxygenated ferric hemoglobin (
      • McMullen S.M.
      • Patrick W.
      Cyanosis.
      ). Patients with type 1 RCM, however, present with normal life expectancy and no neurological symptoms (
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ). In contrast, type 2 RCM exhibits serious consequences. Caused by full stop or deletions that enzymatically inactivate CYB5R3, patients present with severe cyanosis and neurological deterioration, progressive microencephaly, and growth retardation (
      • Nicolas-Jilwan M.
      Recessive congenital methemoglobinemia type II: hypoplastic basal ganglia in two siblings with a novel mutation of the cytochrome b5 reductase gene.
      ). Interestingly, full stops or deletions are commonly located in the FAD-binding sites of CYB5R3 (
      • Gupta V.
      • Kulkarni A.
      • Warang P.
      • Devendra R.
      • Chiddarwar A.
      • Kedar P.
      Mutation update: variants of the CYB5R3 gene in recessive congenital methemoglobinemia.
      ). An additional study conducted by Carew et al. (
      • Carew N.T.
      • Schmidt H.M.
      • Yuan S.
      • Galley J.C.
      • Hall R.
      • Altmann H.M.
      • et al.
      Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.
      ) demonstrated that the cardiomyocyte-specific deletion of CYB5R3 in male mice causes cardiac hypertrophy and sudden cardiac death. These phenotypic differences are accompanied by elevated oxidative stress, decreased CoQ levels, and hemoprotein dysregulation in mouse CYB5R3-cardiomyocyte–specific knockout hearts (
      • Carew N.T.
      • Schmidt H.M.
      • Yuan S.
      • Galley J.C.
      • Hall R.
      • Altmann H.M.
      • et al.
      Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.
      ). From a translational point of view, Carew et al. (
      • Carew N.T.
      • Schmidt H.M.
      • Yuan S.
      • Galley J.C.
      • Hall R.
      • Altmann H.M.
      • et al.
      Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.
      ) revealed that a high-frequency missense genetic variant of CYB5R3, T117S, is associated with decreased event-free survival in those with African ancestry suffering from heart failure with reduced ejection fraction. It was shown that the membrane-bound T117S variant exhibits 50% reduced enzymatic activity when compared to WT CYB5R3. Together, this study demonstrates that CYB5R3 is critical for cardiomyocyte function and that the T117S CYB5R3 variant could be utilized as a genetic biomarker for persons of African ancestry that may be susceptible to an increased risk of death from heart failure with reduced ejection fraction.
      Figure thumbnail gr4
      Figure 4The known implications of CYB5R3 in human health and disease.

      CYB5R3 and neurodegeneration

      CYB5R3 has also been implicated in neurodegenerative disorders, such as Alzheimer’s disease (Fig. 4). Mitochondrial dysfunction is an established feature of Alzheimer’s disease (
      • Swerdlow R.H.
      Mitochondria and mitochondrial cascades in Alzheimer’s disease.
      ,
      • Wang X.
      • Wang W.
      • Li L.
      • Perry G.
      • Lee H.
      • Zhu X.
      Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease.
      ) and in the frequently utilized 5xFAD mouse model, a model that has a total of five Alzheimer’s disease–linked mutations: the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP and the M146L and L286V mutations in PSEN1 (
      • Jankowsky J.L.
      • Zheng H.
      Practical considerations for choosing a mouse model of Alzheimer’s disease.
      ). 5xFAD mice exhibited reduced CYB5R3 levels in cerebrospinal fluid (
      • Wang H.
      • Dey K.K.
      • Chen P.-C.
      • Li Y.
      • Niu M.
      • Cho J.-H.
      • et al.
      Integrated analysis of ultra-deep proteomes in cortex, cerebrospinal fluid and serum reveals a mitochondrial signature in Alzheimer’s disease.
      ). The authors assumed that decreased CYB5R3 levels in the cerebrospinal fluid is commensurate with a decrease in mitochondrial number, but they did not consider that CYB5R3 also localizes to the ER and plasma membrane, in addition to the mitochondria. It is possible that Alzheimer’s disease etiology involves a causal effect for loss of CYB5R3 in mitochondrial dysfunction; however, more studies in the neural system must be performed to establish such a relationship.

      CYB5R3 and diabetes

      Evidence has shown that CYB5R3 also plays a prominent role in pancreatic beta cell function (Fig. 4). Type II diabetes is associated with pancreatic beta cell failure, resulting in insulin resistance and inadequate glucose sensing. As such, pancreatic beta cells are unable to maintain insulin production, leading to a reduction in beta cell mass and function (
      • Halban P.A.
      • Polonsky K.S.
      • Bowden D.W.
      • Hawkins M.A.
      • Ling C.
      • Mather K.J.
      • et al.
      β-Cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment.
      ,
      • Accili D.
      • Talchai S.C.
      • Kim-Muller J.Y.
      • Cinti F.
      • Ishida E.
      • Ordelheide A.M.
      • et al.
      When β-cells fail: lessons from dedifferentiation.
      ). Several pathways have been identified in beta cell failure; of particular importance is the protective response orchestrated by the transcription factor FoxO1 (
      • Kawamori D.
      • Kaneto H.
      • Nakatani Y.
      • Matsuoka T.-A.
      • Matsuhisa M.
      • Hori M.
      • et al.
      The forkhead transcription factor Foxo1 bridges the JNK pathway and the transcription factor PDX-1 through its intracellular translocation.
      ,
      • Kitamura Y.I.
      • Kitamura T.
      • Kruse J.-P.
      • Raum J.C.
      • Stein R.
      • Gu W.
      • et al.
      FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction.
      ). Failure of FoxO1 to induce this stress response leads to mitochondrial dysfunction (
      • Kim-Muller J.Y.
      • Kim Y.J.R.
      • Fan J.
      • Zhao S.
      • Banks A.S.
      • Prentki M.
      • et al.
      FoxO1 deacetylation decreases fatty acid oxidation in β-cells and sustains insulin secretion in diabetes.
      ). It was discovered that CYB5R3, the main CYB5R isoform expressed in pancreatic beta cells in humans (
      • Baron M.
      • Veres A.
      • Wolock S.L.
      • Faust A.L.
      • Gaujoux R.
      • Vetere A.
      • et al.
      A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure.
      ), is a target of FoxO1. It is possible that when FoxO1 targeting of CYB5R3 is dysregulated, beta cell mitochondrial electron transport chain functions will be underprotected and mitochondrial ROS production excessive (Table 2) (
      • Fan J.
      • Du W.
      • Kim-Muller J.Y.
      • Son J.
      • Kuo T.
      • Larrea D.
      • et al.
      Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure.
      ). This could lead to overt oxidative stress that is deleterious for pancreatic beta cell function.

      CYB5R3 and cancer

      CYB5R3 has also been implicated in various cancers over the past decade (Fig. 4) (
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ,
      • Rajcevic U.
      • Petersen K.
      • Knol J.C.
      • Loos M.
      • Bougnaud S.
      • Klychnikov O.
      • et al.
      iTRAQ-based proteomics profiling reveals increased metabolic activity and cellular cross-talk in angiogenic compared with invasive glioblastoma phenotype.
      ,
      • Lund R.R.
      • Leth-Larsen R.
      • Caterino T.D.
      • Terp M.G.
      • Nissen J.
      • Lænkholm A.-V.
      • et al.
      NADH-cytochrome b5 reductase 3 promotes colonization and metastasis formation and is a prognostic marker of disease-free and overall survival in estrogen receptor-negative breast cancer.
      ). Several studies have highlighted CYB5R3 overexpression in cancer cells to protect against oxidative stress and apoptosis (
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ,
      • Rajcevic U.
      • Petersen K.
      • Knol J.C.
      • Loos M.
      • Bougnaud S.
      • Klychnikov O.
      • et al.
      iTRAQ-based proteomics profiling reveals increased metabolic activity and cellular cross-talk in angiogenic compared with invasive glioblastoma phenotype.
      ,
      • Lund R.R.
      • Leth-Larsen R.
      • Caterino T.D.
      • Terp M.G.
      • Nissen J.
      • Lænkholm A.-V.
      • et al.
      NADH-cytochrome b5 reductase 3 promotes colonization and metastasis formation and is a prognostic marker of disease-free and overall survival in estrogen receptor-negative breast cancer.
      ). Several research groups demonstrated that CYB5R3 overexpression and polymorphisms increase the risk of breast cancer in women, especially women of African ancestry (
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ,
      • Lund R.R.
      • Leth-Larsen R.
      • Caterino T.D.
      • Terp M.G.
      • Nissen J.
      • Lænkholm A.-V.
      • et al.
      NADH-cytochrome b5 reductase 3 promotes colonization and metastasis formation and is a prognostic marker of disease-free and overall survival in estrogen receptor-negative breast cancer.
      ). The risk of CYB5R3 polymorphism-associated breast cancer is further exacerbated in females who smoke cigarettes. Since CYB5R3 plays a role in drug metabolism (
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ), it is possible that the loss-of-function polymorphisms in CYB5R3 result in accumulated carcinogen and cellular damage (
      • Blanke K.L.
      • Sacco J.C.
      • Millikan R.C.
      • Olshan A.F.
      • Luo J.
      • Trepanier L.A.
      Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.
      ). Moreover, CYB5R3 overexpression is described in cancer cells of the lung. Genetic knockdown of CYB5R3 in lung cancer cells revealed slow proliferation and metastasis but did not affect cancer cell survival, pointing to a potential link between CYB5R3 and lung cancer (
      • Lund R.R.
      • Leth-Larsen R.
      • Caterino T.D.
      • Terp M.G.
      • Nissen J.
      • Lænkholm A.-V.
      • et al.
      NADH-cytochrome b5 reductase 3 promotes colonization and metastasis formation and is a prognostic marker of disease-free and overall survival in estrogen receptor-negative breast cancer.
      ). It is worth noting, however, that this study also reported a contradictory finding with CYB5R3 overexpression contributing to increased tumor size. Interestingly, the same study showed that CYB5R3 deficiency was deleterious in breast cancer, as evidenced by increased tumor colonization and metastasis. This could be due to differential cell lines utilized in these studies. These findings might also suggest that CYB5R3 has a differential role among tissues and, therefore, the progression of different types of cancers.

      CYB5R3 as a therapeutic target: Recent advances

      Given the implications of CYB5R3 in human physiology and disease, efforts have been dedicated to devising CYB5R3-targeted therapeutics. One study demonstrated that treating HEK293 and rat renal endothelial cells with propylthiouracil derivatives ZINC05626394 (IC50 = 10.81 μM) and ZINC39395747 (IC50 = 9.14 μM) inhibit CYB5R3 activity by roughly 75%. In addition, acutely administered ZINC39395747 increased NO bioavailability in renal vascular cells, augmented renal blood flow, and reduced systemic blood pressure in hypertensive rats (
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ). However, it is not entirely clear whether these inhibitors block other NADH reductases in vivo, such as the other CYB5R family members. More selective CYB5R3 inhibitors could be a promising treatment for acutely modulating blood pressure (
      • Rahaman M.M.
      • Reinders F.G.
      • Koes D.
      • Nguyen A.T.
      • Mutchler S.M.
      • Sparacino-Watkins C.
      • et al.
      Structure guided chemical modifications of propylthiouracil reveal novel small molecule inhibitors of cytochrome b5 reductase 3 that increase nitric oxide bioavailability.
      ).
      As previously described, CYB5R3 plays an essential role in cellular redox and metabolic homeostasis, a hallmark of longevity (
      • Caldeira da Silva C.C.
      • Cerqueira F.M.
      • Barbosa L.F.
      • Medeiros M.H.G.
      • Kowaltowski A.J.
      Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity.
      ,
      • López-Otín C.
      • Blasco M.A.
      • Partridge L.
      • Serrano M.
      • Kroemer G.
      The hallmarks of aging.
      ). As such, several pharmacological and genetic-based approaches have been developed to target CYB5R3 in hopes of extending lifespan and delaying age-related diseases associated with metabolic and redox imbalances, such as Alzheimer’s and Parkinson’s disease (
      • Martin-Montalvo A.
      • Sun Y.
      • Diaz-Ruiz A.
      • Ali A.
      • Gutierrez V.
      • Palacios H.H.
      • et al.
      Cytochrome b5 reductase and the control of lipid metabolism and healthspan.
      ,
      • Rizvi S.I.
      • Pandey K.B.
      Activation of the erythrocyte plasma membrane redox system by resveratrol: a possible mechanism for antioxidant properties.
      ,
      • Lee J.
      • Park A.H.
      • Lee S.-H.
      • Lee S.-H.
      • Kim J.-H.
      • Yang S.-J.
      • et al.
      Beta-lapachone, a modulator of NAD metabolism, prevents health declines in aged mice.
      ,
      • Calabrese V.
      • Cornelius C.
      • Dinkova-Kostova A.T.
      • Calabrese E.J.
      • Mattson M.P.
      Cellular stress responses, the hormesis paradigm, and vitagenes: novel targets for therapeutic intervention in neurodegenerative disorders.
      ). Overexpression of CYB5R3 in mouse models leads to extended lifespan, bolstered physical performance, ameliorated chronic inflammation, and protection against carcinogenesis (
      • Diaz-Ruiz A.
      • Lanasa M.
      • Garcia J.
      • Mora H.
      • Fan F.
      • Martin-Montalvo A.
      • et al.
      Overexpression of CYB5R3 and NQO1, two NAD+ -producing enzymes, mimics aspects of caloric restriction.
      ). These findings are commensurate with CYB5R3’s role in generating intracellular NAD+ for utilization by sirtuins, NAD+-dependent histone deacetylases that are essential for DNA repair, controlling inflammation, and antioxidant defenses (
      • Diaz-Ruiz A.
      • Lanasa M.
      • Garcia J.
      • Mora H.
      • Fan F.
      • Martin-Montalvo A.
      • et al.
      Overexpression of CYB5R3 and NQO1, two NAD+ -producing enzymes, mimics aspects of caloric restriction.
      ,
      • Shen A.
      • Kim H.-J.
      • Oh G.-S.
      • Lee S.-B.
      • Lee S.H.
      • Pandit A.
      • et al.
      NAD+ augmentation ameliorates acute pancreatitis through regulation of inflammasome signalling.
      ,
      • Ross D.
      • Siegel D.
      Functions of NQO1 in cellular protection and coq10 metabolism and its potential role as a redox sensitive molecular switch.
      ,
      • Kupis W.
      • Pałyga J.
      • Tomal E.
      • Niewiadomska E.
      The role of sirtuins in cellular homeostasis.
      ). The NAD+/NADH ratio is regulated, in part, by CYB5R3 and is vital for cellular homeostasis, where too low of a ratio is associated with higher sensitivity to oxidative stress (
      • Siendones E.
      • Ballesteros M.
      • Navas P.
      Cellular and molecular mechanisms of recessive hereditary methaemoglobinaemia type II.
      ). Given CYB5R3’s role in age-related processes, drugs aimed at boosting CYB5R3 activity chronically and modulating the NAD+/NADH ratio in the cytosol and mitochondria (
      • de Cabo R.
      • Siendones E.
      • Minor R.
      • Navas P.
      CYB5R3: a key player in aerobic metabolism and aging?.
      ) could be promising for treating age-related metabolic diseases (
      • Verdin E.
      NAD+ in aging, metabolism, and neurodegeneration.
      ,
      • Imai S.-I.
      A possibility of nutriceuticals as an anti-aging intervention: activation of sirtuins by promoting mammalian NAD biosynthesis.
      ,
      • Herranz D.
      • Muñoz-Martin M.
      • Cañamero M.
      • Mulero F.
      • Martinez-Pastor B.
      • Fernandez-Capetillo O.
      • et al.
      Sirt1 improves healthy ageing and protects from metabolic syndrome-associated cancer.
      ) and maintaining cellular redox balance to prevent disease onset or severity.

      CYB5R4

      Aside from CYB5R3, CYB5R4, also known as Ncb5or, is the most extensively studied in the CYB5R family. CYB5R4 is a 59 kDa flavohemoprotein that is ubiquitously expressed only in animal tissues and is the largest and most structurally unique in the CYB5R family, consisting of three distinct domains. CYB5R4 is the only CYB5R family member to contain a cytochrome b5 domain with coordinated heme that is lodged between two alpha-helices (
      • Deng B.
      • Parthasarathy S.
      • Wang W.
      • Gibney B.R.
      • Battaile K.P.
      • Lovell S.
      • et al.
      Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
      ). The N-terminal b5 domain is linked to the C-terminal b5R domain via a CS (CHORD-SGT1) domain, comprised of roughly 90 amino acid residues and nine β-sheets (
      • Deng B.
      • Parthasarathy S.
      • Wang W.
      • Gibney B.R.
      • Battaile K.P.
      • Lovell S.
      • et al.
      Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
      ). The CS domain of CYB5R4 differs from its structural homologs, featuring an additional β-sheet structure involving residues G256 and P267, forming two strands (β8 and β9) separated by a five-residue loop that orient in an antiparallel fashion (
      • Benson D.R.
      • Lovell S.
      • Mehzabeen N.
      • Galeva N.
      • Cooper A.
      • Gao P.
      • et al.
      Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.
      ). Beyond P267, a classic type I β-turn from R268 and T271 forms a linkage to the b5R domain (
      • Benson D.R.
      • Lovell S.
      • Mehzabeen N.
      • Galeva N.
      • Cooper A.
      • Gao P.
      • et al.
      Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.
      ). Interestingly, CS domains exist in diverse proteins and are commonly involved in protein–protein interactions, contributing to the potential diverse functions of CYB5R4 (
      • Garcia-Ranea J.A.
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      p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families.
      ). Relative to CYB5R3, the B5R domain of CYB5R4 contains notable gaps and insertions, further illustrating the unique structural character of CYB5R4 (
      • Zhu H.
      • Qiu H.
      • Yoon H.W.
      • Huang S.
      • Bunn H.F.
      Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
      ). The cytochrome b5R domain binds FAD and NAD(P)H prosthetic groups, both serving as important enzymatic cofactors in the electron transfer reaction (
      • Karplus P.A.
      • Daniels M.J.
      • Herriott J.R.
      Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family.
      ). The unique ability of CYB5R4 to utilize both NADH and NADPH is supported by a recent structural study on its FAD domain (
      • Benson D.R.
      • Lovell S.
      • Mehzabeen N.
      • Galeva N.
      • Cooper A.
      • Gao P.
      • et al.
      Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.
      ).
      Unlike the other CYB5R family members, CYB5R4 is a soluble protein that localizes on the ER membrane (
      • Zhu H.
      • Larade K.
      • Jackson T.A.
      • Xie J.
      • Ladoux A.
      • Acker H.
      • et al.
      NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.
      ). However, it should be noted that a more recent study failed to validate CYB5R4’s ER localization (
      • Zámbó V.
      • Tóth M.
      • Schlachter K.
      • Szelényi P.
      • Sarnyai F.
      • Lotz G.
      • et al.
      Cytosolic localization of NADH cytochrome b₅ oxidoreductase (Ncb5or).
      ). The authors demonstrated that CYB5R4 is positioned in the cytosol and they speculated that CYB5R4 is not an integral protein anchored to the ER membrane but can be transiently recruited from the cytosol for fatty acid desaturation (
      • Zámbó V.
      • Tóth M.
      • Schlachter K.
      • Szelényi P.
      • Sarnyai F.
      • Lotz G.
      • et al.
      Cytosolic localization of NADH cytochrome b₅ oxidoreductase (Ncb5or).
      ). The condition and mechanism for CYB5R4’s recruitment to the ER remains to be investigated (
      • Zámbó V.
      • Tóth M.
      • Schlachter K.
      • Szelényi P.
      • Sarnyai F.
      • Lotz G.
      • et al.
      Cytosolic localization of NADH cytochrome b₅ oxidoreductase (Ncb5or).
      ). Given the multidomain structure of CYB5R4, full-length CYB5R4 has been resistant to crystallization; however, high-resolution crystal structures of the individual domains have been discovered (
      • Deng B.
      • Parthasarathy S.
      • Wang W.
      • Gibney B.R.
      • Battaile K.P.
      • Lovell S.
      • et al.
      Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.
      ). Future studies aimed at characterizing CYB5R4 structure are warranted to verify the absence of a membrane anchor domain. This would provide insight as to how CYB5R4 localization differs based on cell type and how CYB5R4 function changes as a consequence.
      CYB5R4 is ubiquitinated and phosphorylated at numerous amino acid residues, the most abundant being phosphorylated at S471 and S476 (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). These two residues are situated in the NADH domain near the NADH-binding site, suggesting that the phosphorylation of these residues could impact either NADH binding or the efficiency of electron transfer from NADH to FAD. CYB5R4 has also been shown to be ubiquitinated at K277 and K442, two important modifications that could impact CYB5R4 stability and functional activity (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ). One might speculate that under certain physiological conditions or stressors, ubiquitination at K277 and K442 could serve to target CYB5R4 to the proteasome for degradation. This could be due to either excess CYB5R4 that is unnecessary for the cell or mutated CYB5R4 that is deleterious for normal physiological processes. It is also possible that ubiquitination at these sites could alter protein localization or interacting partners. Because the function of these identified PTMs remains uncertain, future research dedicated to understanding PTMs of CYB5R4 is needed to understand the details in CYB5R4 regulation at a posttranslational level and its contribution to disease.
      Kinetic measurements have demonstrated that human CYB5R4 can reduce numerous substrates in vivo, such as cytochrome c, methemoglobin, molecular oxygen, and ferricyanide (
      • Zhu H.
      • Qiu H.
      • Yoon H.W.
      • Huang S.
      • Bunn H.F.
      Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
      ,
      • Zhu H.
      • Larade K.
      • Jackson T.A.
      • Xie J.
      • Ladoux A.
      • Acker H.
      • et al.
      NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum.
      ). CYB5R4 also reduces its own heme moiety through the consumption of NAD(P)H, where the FAD group bound at the reductase domain is necessary for mediating electron transfer from NAD(P)H to the heme moiety (
      • Zhu H.
      • Qiu H.
      • Yoon H.W.
      • Huang S.
      • Bunn H.F.
      Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
      ). Electrons are subsequently transferred to oxygen resulting in the generation of superoxide that can be dismutated to hydrogen peroxide.
      Several studies have suggested that the loss of CYB5R4 results in diabetes mellitus as evidenced by mitochondrial dysfunction, disrupted ion channel signaling and iron homeostasis, and the progressive loss of white adipose tissue in the liver (
      • Zámbó V.
      • Tóth M.
      • Schlachter K.
      • Szelényi P.
      • Sarnyai F.
      • Lotz G.
      • et al.
      Cytosolic localization of NADH cytochrome b₅ oxidoreductase (Ncb5or).
      ,
      • Stroh M.A.
      • Winter M.K.
      • McCarson K.E.
      • Thyfault J.P.
      • Zhu H.
      NCB5OR deficiency in the cerebellum and midbrain leads to dehydration and alterations in thirst response, fasted feeding behavior, and voluntary exercise in mice.
      ). Notably, knockout of CYB5R4 caused early-onset diabetes in mice, irrespective of peripheral insulin sensitivity (
      • Xie J.
      • Zhu H.
      • Larade K.
      • Ladoux A.
      • Seguritan A.
      • Chu M.
      • et al.
      Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.
      ). CYB5R4 likely plays an important role in protecting pancreatic beta cells against oxidative stress by preventing the accumulation of ROS, similar to CYB5R3 (
      • Xie J.
      • Zhu H.
      • Larade K.
      • Ladoux A.
      • Seguritan A.
      • Chu M.
      • et al.
      Absence of a reductase, NCB5OR, causes insulin-deficient diabetes.
      ). These unique findings identify a unique enzyme in CYB5R4 that could be targeted therapeutically for those suffering from diabetes mellitus. While the studies on CYB5R4 are certainly the most abundant in the CYB5R family, aside from CYB5R3, our mechanistic understanding of this enzyme is still incomplete. Therefore, interrogating the mechanisms involved in CYB5R4-mediated pancreatic beta cell protection must be performed to facilitate the development of high-quality therapies for diabetes mellitus. Moreover, one publication investigated the role of ER-associated CYB5R4 in mouse liver. They created a liver-specific CYB5R4 KO and found that free fatty acids, lipid catabolism, and oxidative stress are enriched in hepatocytes, characterized by increased mitochondrial content, PCG1 alpha expression, fatty acid oxidation rates, and oxidized glutathione content (
      • Xu M.
      • Wang W.
      • Frontera J.R.
      • Neely M.C.
      • Lu J.
      • Aires D.
      • et al.
      Ncb5or deficiency increases fatty acid catabolism and oxidative stress.
      ). In addition, CYB5R4 liver knockouts exhibited heightened lipotoxicity. These are novel findings and suggest that CYB5R4 may be a unique therapeutic target for those suffering with diabetes mellitus. Studies aimed at elucidating the mechanisms by which CYB5R4 might be mediating the pathogenesis of diabetes, and the assessment of potential interacting partners in vitro and in vivo would further facilitate the development of new therapeutic avenues. Finally, CYB5R4 deficiency has been characterized in the brain, where it was found that the conditional deletion of CYB5R4 in the mouse cerebellum and midbrain results in altered iron homeostasis and locomotor activity and potentiates behavioral abnormalities (
      • Stroh M.A.
      • Winter M.K.
      • McCarson K.E.
      • Thyfault J.P.
      • Zhu H.
      NCB5OR deficiency in the cerebellum and midbrain leads to dehydration and alterations in thirst response, fasted feeding behavior, and voluntary exercise in mice.
      ,
      • Stroh M.A.
      • Winter M.K.
      • Swerdlow R.H.
      • McCarson K.E.
      • Zhu H.
      Loss of NCB5OR in the cerebellum disturbs iron pathways, potentiates behavioral abnormalities, and exacerbates harmaline-induced tremor in mice.
      ). Deletion of CYB5R4 resulted in altered drinking and feeding behavior, neuroendocrine thirst regulation, and energy expenditure (
      • Stroh M.A.
      • Winter M.K.
      • McCarson K.E.
      • Thyfault J.P.
      • Zhu H.
      NCB5OR deficiency in the cerebellum and midbrain leads to dehydration and alterations in thirst response, fasted feeding behavior, and voluntary exercise in mice.
      ). Therefore, CYB5R4 could be playing a role in modulating the integrity of cerebellar regulation of satiety cues and voluntary exercise (
      • Stroh M.A.
      • Winter M.K.
      • McCarson K.E.
      • Thyfault J.P.
      • Zhu H.
      NCB5OR deficiency in the cerebellum and midbrain leads to dehydration and alterations in thirst response, fasted feeding behavior, and voluntary exercise in mice.
      ). The findings of this study combined with efforts to more rigorously understand the mechanisms by which CYB5R4 mediates cerebellar and midbrain processes could fuel pharmacological developments in this up-and-coming field of CYB5R4 research.

      CYB5R5

      CYB5R5, also known as CYB5RL, is unequivocally the least studied in the CYB5R family. CYB5R5 is a 36 kDa flavoprotein that is the least expressed of the CYB5R family members (Table 1). While the predicted structure of CYB5R5 (Fig. 2) does not share the same structural motifs conserved across CYB5R1, CYB5R2, and CYB5R3, it is purported that CYB5R5 also utilizes both NADH and FAD as cofactors (Table 1). It is also uncertain whether CYB5R5 harbors a membrane anchor (Fig. 2). CYB5R5 is the least similar to CYB5R3, sharing a 27.87% sequence identity, and is the most similar to CYB5R4, sharing a 30.25% sequence identity. CYB5R5 has a very low tissue specificity and is therefore not enriched in any human tissue. CYB5R5 is predicted to be localized to the nucleoplasm and the ER but has not been proven experimentally. CYB5R5 is ubiquitinated and phosphorylated at K55 and T76, respectively (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: mutations, PTMs and recalibrations.
      ,
      • Mertins P.
      • Yang F.
      • Liu T.
      • Mani D.R.
      • Petyuk V.A.
      • Gillette M.A.
      • et al.
      Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.
      ). These two residues are situated within the purported membrane anchor region near the beginning of the FAD domain. One might postulate that phosphorylation at T76 could potentially disrupt CYB5R5 membrane targeting and localization, which could inevitably interfere with normal CYB5R5 function. It is conceivable that ubiquitination at K55 could impact protein localization and protein–protein interactions and could also serve as a regulatory mechanism to stimulate the degradation of CYB5R5 in cases where CYB5R5 is not needed for a particular function or when CYB5R5 is nonfunctional. Future studies aimed to understand these PTM’s of CYB5R5 could not only help discern the crosstalk between these two sites but also provide crucial details as to how CYB5R5 activity is regulated at the protein level.
      A study from Wang et al. uncovered a novel germline truncation mutation at R51 in CYB5R5, R51X, enriched in colon polyps in canines. With this mutation, CYB5R5 function is compromised, accelerating the generation of ROS and oxidative stress in the colon (
      • Wang J.
      • Wang T.
      • Bishop M.A.
      • Edwards J.F.
      • Yin H.
      • Dalton S.
      • et al.
      Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis.
      ). In response, Bacteroides uniformis, an anaerobic bacterium that resides in the colon, expresses thioredoxin and nitroreductase, which together act as a bacterial redox system to mitigate oxidative stress induced by the R51X CYB5R5 mutation and the more aerobic environment of the jejunum (
      • Wang J.
      • Wang T.
      • Bishop M.A.
      • Edwards J.F.
      • Yin H.
      • Dalton S.
      • et al.
      Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis.
      ). In turn, cell death is ameliorated, leading to the uncontrolled proliferation of cancer cells in the colon and the onset of extreme polyposis. Despite these findings, it is critical that future functional studies are conducted to validate these findings in canines and possibly extend similar studies to humans. Nevertheless, this study presents a significant first step into understanding the role of CYB5R5 in human health and disease, presenting a potential therapeutic target for colon cancer.

      Conclusions and future directions

      A multitude of unanswered questions remain surrounding the biological roles of the CYB5R family of proteins. These include the following: (1) do the different CYB5R isoforms cooperate with one another or are their biological roles functionally separate and how do their roles differ based on tissue or cell type, (2) how are CYB5R proteins regulated at the transcriptional and posttranslational level and how does this govern enzymatic function, (3) are there genetic variants of the CYB5R isoforms that can be identified to predict high risk patients for different diseases, and (4) how can we leverage this information to design targeted CYB5R therapeutics? Collectively, these preexisting CYB5R studies have led to valuable insight emphasizing the importance of CYB5R enzymes in human physiology and disease. As further studies begin to uncover novel roles of CYB5R enzymes, emphasis should be placed on developing innovative strategies to therapeutically target CYB5R proteins to treat a plethora of diseases.

      Conflict of interest

      Dr Straub is a consultant and stockholder for Creegh Pharmaceuticals. Dr Straub received research funds from Bayer Pharmaceuticals. All other authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank the University of Pittsburgh and the ARCS foundation for their continued support.

      Author contributions

      R. H., S. Y., K. W., M. K., and A. C. S. conceptualization; R. H., S. Y., K. W., M. K., and A. C. S. writing–original draft.

      Funding and additional information

      Financial support for this work was provided by the National Institutes of Health grants: R35 HL161177 (A. C. S.), R01 HL 149825 (A. C. S.), R01 HL 153532 (A. C. S.), American Heart Association grants: Established Investigator Award 19EIA34770095 (A. C. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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