Letters To The Editor
Cyclophilin B is really a major growth factor in breast milkCyclophilin B (CypB) was first described and its sequence was reported by Spik et al. in 1991 (1). These authors prepared and characterized recombinant CypB demonstrating that it had prolyl isomerase activity, which was inhibited by cyclosporin (1). At that time, no mitogenic activity was observed for CypB. This may be because of the absence of glycosylation, incorrect folding, denaturation, or to the cellular model used in these initial exploratory experiments (3T3). We wish to report that an earlier unpublished study (2) showed that CypB has growth factor activity on CCL-39 cells at around 30 ng/ml, similar to EGF, acidic FGF, and basic FGF.
SARS-CoV-2 Y453F is not the “cluster 5” variantI read with interest the recent publication by Bayarri-Olmos et al. (1) and would like to express some concerns regarding the study.
Reply to Douglass and Spiegel: A suite of mathematical solutions to describe ternary complex formation and their application to targeted protein degredation by heterobifunctional ligandsCalculating equilibrium concentration of a ternary complex for a given total ligand concentration (1, 2) and predicting the ternary complex concentration in equilibrium with a given free ligand concentration (3) are two totally different questions that lead to clearly distinct mathematical solutions. Even though an exact solution to one question can be an approximate answer to the other, the latter approach (3) is consistent with a long-established tradition of analyzing equilibrium behavior of binding reactions: the equilibrium dissociation constants, Kds, that are used in both approaches are defined by the free, not total, ligand concentration at equilibrium, and the universally adopted equation for a bimolecular binding reaction, B = Bmax ∗ [L]/([L] + Kd), is also a function of free ligand concentration at equilibrium.
Reply to Figueira et al.: Can NAD(P)+ transhydrogenase (NNT) mediate a physiologically meaningful increase in energy expenditure by mitochondria during H2O2 removal?In our recent publication (1), as well as an earlier publication from our lab (2), we demonstrate under specific experimental conditions designed to increase flux through redox buffering circuits in mitochondria, and thus NADPH demand, that at least a portion of the accompanying increase in JO2 (i.e., proton conductance) is directly and reproducibly attributed to nicotinamide nucleotide transhydrogenase (NNT; e.g., Fig. 3D in (1)). In their letter to the editor, Figueira et al. question whether flux through redox circuits linked to NNT can mediate a meaningful increase in energy expenditure.
Reply to Hayashi and Konishi: Noneffect of SARS-CoV-2 spike glycoprotein Y217N mutation on affinity between virus and ACE2We thank Hayashi and Konishi for their comments and interest in our article (1). We agree that by structural modeling analysis, angiotensin-converting enzyme 2 (ACE2 [Y217N]) mutant shows no changes in binding with receptor-binding domain (RBD) when compared with WT ACE2, which is depicted in Figure 4 in our article. We think the lower binding affinity of human ACE2 N217 with RBD was due to the following reasons. First, cell surface abundance of ACE2 Y217N was less compared with WT ACE2 (Fig. 5 in our article).
Noneffect of SARS-CoV-2 spike glycoprotein Y217N mutation on affinity between the virus and ACE2The interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. Zhang et al. (1) reportedly constructed human ACE2 (hACE2) with the Y217N mutation and found that this mutation completely blocked SARS-CoV-2 entry. Zhang et al. (1) performed an receptor binding domain (RBD) binding assay and found that WT hACE2 potently bound the RBD; however, hACE2 Y217N almost lost the ability to bind the RBD.
Glycolysis and gluconeogenesis: A teaching viewI read with interest the recent review “Tracking the carbons supplying gluconeogenesis” by Ankit M. Shah and Fredric E. Wondisford (1). The figures are clear, and they are a good teaching source. Nevertheless, I note some potential teaching issues as well as offer additional suggestions. It is well known and it is explained to our students that there are three reactions of glycolysis that are essentially irreversible: hexokinase, phosphofrutokinase-1, and pyruvate kinase. Thus, I would like to observe in relation to Figure 1 that
Reply to Hernández—Glycolysis and gluconeogenesis: A teaching viewWe thank Félix Hernández for his insightful and thoughtful comments as well as interest in our manuscript (1). Regarding suggestion 1, we omitted several glycolytic and gluconeogenic enzymes in the figure for simplicity. We specifically mentioned G6Pase and PEPCK as many investigators have studied these enzymes in particular when studying gluconeogenesis. Articles specifically referenced in our review article studied these two enzymes, and we wished to pictorially represent these enzymes. We further described each of these two enzymes’ specific functions in the text.
Statistical and thermodynamic analysis of the binding of trans-activation response–binding proteins to HIV-1 TAR RNAIn a recent article (1), the authors examine the binding of lab-evolved trans-activation response (TAR)–binding proteins (TBPs) to HIV-1 TAR RNA. Here, we show our analysis of the thermodynamic data of the binding that identifies three quantitative features of the binding, which may provide further insight into the interactions.where ΔH° and ΔS° are experimentally measured values reported in the original paper (1). Based on the statistical parameters, probability density of the ΔG° can be generated using Equation 2:where σ = 1.9407 kJ/mol and ΔG°mean = –41.3 kJ/mol.
The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylationSzibor et al. (1) concluded that mitochondrial pyruvate oxidation is regulated primarily by cytosolic Ca2+ ([Ca2+]cyt) activation of the malate-aspartate shuttle, rather than by mitochondrial Ca2+ ([Ca2+]mit) activation of intramitochondrial dehydrogenases. Pyruvate dehydrogenase (PDH) activity largely reflects the ratio of active nonphosphorylated PDH to inactive phosphorylated PDH (PDHP) (2), but Szibor et al. (1) did not measure PDH/PDHP ratios. Moreover, their studies used unphysiological conditions with isolated mitochondria (saturating ADP); with synaptosomes, thymocytes, and fibroblasts (uncoupler and high pyruvate); and with perfused hearts (high pyruvate).
Reply to Rutter et al.: The roles of cytosolic and intramitochondrial Ca2+ and the mitochondrial Ca2+-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylationEach model used in the work referred to by Rutter et al. (1) addressed certain aspects of mitochondrial biology, and together, they fully support the conclusions made. Please note that we describe Ca2+-mediated regulation of oxidative phosphorylation (OXPHOS) fluxes (2, 3) and do not question Ca2+-responsiveness of pyruvate dehydrogenase en-zyme activity (4). To address concerns such as those raised by Rutter et al. (1), we studied glutamate/malate-dependent OXPHOS in the absence of exogenous pyruvate in mitochondria, omitted pyruvate from cell experiments, and implemented the working rat heart model perfused by Krebs–Henseleit (glucose) buffer.
Non-enzymatic action of expansinsFrom their simulations of endoglucanase Cel45A, Bharadwaj et al. (1) propose that structurally related expansins and MltA may cut glycan backbones without generating reducing ends. This is tenable for MltA, a peptidoglycan lytic transglycosylase whose action produces nonreducing 1,6-anhydro products, but is untenable for expansins.
Reply to Cosgrove: Non-enzymatic action of expansinsIn our computational study, we use molecular simulations to substantiate a hypothetical mechanism for glycosidic bond cleavage in the presence of a single catalytic acid at the active site of the mutant D10N HiCel45A. In addition to discussing this plausible mechanism from the context of structurally related MltA lytic transglycosylase and subfamily C GH45s, we also suggest the implications of the plausible mechanism for our current understanding of the action of expansins and lytic transglycosylases.
Arginine catabolism enzyme AgrE/ArgZ likely involves a cyanobacterial specific factorArginine is a proteinaceous amino acid that is also used in numerous metabolic processes, which is reflected in the presence of numerous different arginine utilization pathways in nature. In cyanobacteria, a novel enzyme showing arginine dihydrolase activity has recently been found and named ArgZ in Synechocystis (1) or AgrE in Anabaena (2). (AgrE stands for arginine-guanidine–removing enzyme.) Zhuang et al. (3) have reported the crystal structure of Synechocystis ArgZ, showing that amino acid residues 1–269 form an α/β propeller domain characteristic of guanidine group–modifying enzymes and describing the catalytic mechanism that removes two ammonia molecules and one CO2 molecule from arginine, producing ornithine.
Deciphering the roles of prominins in the visual systemThe mammalian Prom1 gene, which is critical for photoreceptor membrane biogenesis through its interaction with protocadherin 21 (1), has two zebrafish orthologs (prom1a and prom1b). In situ hybridization revealed co-expression of their transcripts in neural tissues and in the outer nuclear layer of the retina where perikarya of photoreceptors reside (2, 3). In contrast to mice, both orthologs were strongly and differentially expressed by retinal interneurons in the inner nuclear layer of the adult fish retina (2).
Reply to Corbeil et al.: Deletion of the transmembrane protein Prom1b in zebrafish disrupts outer-segment morphogenesis and causes photoreceptor degenerationWe thank Corbeil et al. (1) for their interest in our work.
Phb1:Phb2 heterodimers in the mitochondria—beyond functional interdependenceLi et al. (1) showed that in vivo deletion of prohibitin-2 (Phb2) in hepatocytes (Hep-Phb2−/−) leads to impaired gluconeogenesis, reduced food intake, severe hypoglycemia, and, subsequently, poor survival. Phb2 and its homologous protein Phb1 form heterodimers in the mitochondria and are functionally interdependent (1–3). Consequently, the knockdown of either member leads to a parallel loss of the other member (1–4). Thus, it remains unclear whether or not Phb1 and Phb2 have protein-specific functions in the mitochondria.
Reply to Mishra: Prohibitin heterodimers—a complex time dependence for carcinogenesisDr. Mishra (1) points to an interesting aspect of Phb1/Phb2 interdependence and tumor formation. Constitutive liver-specific Phb1-KO mice develop hepatocellular carcinoma (2). Through a putative mirror effect, Dr. Mishra extrapolates on the phenotype of our Hep-Phb2−/− mice (3) that should supposedly be similar to liver-specific Phb1-KO (2), according to Phb1/Phb2 interdependence. While both models are liver-specific, our Phb2-KO is time-specific (Phb2fl/fl;Alb-Cre-ERT2), not constitutive. Specifically, we induced the KO in adult mice (8 weeks old), avoiding the absence of liver prohibitins at the embryonic and developmental stages.
Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifactEyre et al. (1) recently claimed that the histidine-involved collagen cross-links, histidinohydroxylysinonorleucine (HHL)2 (2) and dehydrohistidinohydroxymerodesmosine (HHMD) (3), are both laboratory artifacts. We have several concerns about this study.
Reply to Yamauchi et al.: Analyses of lysine aldehyde cross-linking in collagen reveal that the mature cross-link histidinohydroxylysinonorleucine is an artifactYamauchi et al. (1) question the data in our recent paper (2) as support for our conclusions. We disagree and maintain that histidinohydroxylysinonorleucine (HHL)2 (3, 4) does not exist as a natural product in collagen.
On “Isomerization as the secret Achilles’ heel of long-lived proteins”This summary (1) calls to our attention the importance of often-overlooked stochastic processes that occur as proteins age—especially long-lived proteins. The demonstration by Lyon et al. (2) of the failed chaperone function of α-crystallins and its relationship to cataract and potentially other protein precipitation diseases is elegant. They should, however, acknowledge that Groenen et al. (3) and Voorter et al. (4) published on this topic decades ago. Goenen showed how simple water-based hydrolysis after racemization can lead to the extensive cleavages that are observed in the aged lens.
APPealing for a role in cellular iron effluxDlouhy et al. (1) recently refined how β-amyloid precursor protein (APP)2 participates in iron (Fe) efflux and hinted that APP may be unnecessary for ferroportin-supported Fe efflux. APP and Fe efflux have long been our interest, given recent work on the APP mRNA 5′-UTR and how regulation of APP translation operates through an interleukin-1 acute box, an iron response element (IRE), where iron-responsive protein 1 (IRP1) binds, and a target sequence for microRNA-346 (2). Untangling the roles of IRE, IRP1, interleukin-1, and miR-346 at the APP 5′-UTR is critical in Alzheimer’s disease (AD).
Reply to Lahiri et al.: APPealing for a role in cellular iron effluxLahiri et al. (1) expressed concern that by not detecting FRET between ferroportin-CFP and APP-YFP,2 we “hinted that APP is unnecessary for ferroportin-supported Fe efflux.” There is no question that APP is unnecessary for ferroportin-dependent Fe efflux, as illustrated by the work by MacKenzie and co-workers (2), who, using expression of Fpn in Xenopus oocytes, have provided details of ferroportin function. Also, we have shown that whereas knockdown of the essential ferroxidase hephaestin reduces iron efflux in primary hippocampal neurons, knockdown of APP does not (3).
Essentiality of n-6 fatty acidsIt was with great interest I read the recent JBC Reflections by William L. Smith (1). However, I was a bit provoked by his statement in the abstract that “Their essentiality (i.e. n-6 fatty acids) is largely due to their function as prostaglandin precursors.” This is not correct.
Reply to Hansen: Essentiality of n-3 fatty acidsProfessor Hansen makes an important point that arose from my imprecise use of the word “largely” in the sentence to which he refers (1). Regrettably, I failed to note the significance of linoleic acid itself functioning in the maintenance of the water barrier in skin. In young rats, the scaly skin syndrome of omega-6 essential fatty acid (EFA) deficiency appears about 2 months after placing animals on EFA-deficient diets (2). Hansen (1) references the brilliant biochemical studies in this area by Brash and co-workers.