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Mechanism of Fine-tuning pH Sensors in Proprotein Convertases

IDENTIFICATION OF A pH-SENSING HISTIDINE PAIR IN THE PROPEPTIDE OF PROPROTEIN CONVERTASE 1/3*
  • Danielle M. Williamson
    Affiliations
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239
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  • Johannes Elferich
    Affiliations
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239
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  • Ujwal Shinde
    Correspondence
    To whom correspondence should be addressed. Tel.: 503-494-8683; Fax: 503-494-8393.
    Affiliations
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Ruth L. Kirschstein National Research Service Award 5F30DK096752 (to D. M. W.). This work was also supported by National Science Foundation Grant MCB0746589 (to U. S.) and American Heart Association Predoctoral Training Grant 12PRE11470005 (to J. E.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:July 30, 2015DOI:https://doi.org/10.1074/jbc.M115.665430
      The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a “gatekeeper” that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.

      Introduction

      Eukaryotic cells have evolved an elegant series of membranous compartments to regulate protein synthesis, folding, activation, sorting, and export (
      • Soskine M.
      • Tawfik D.S.
      Mutational effects and the evolution of new protein functions.
      ,
      • Embley T.M.
      • Martin W.
      Eukaryotic evolution, changes and challenges.
      ). Beginning with their entry into the endoplasmic reticulum (ER)
      The abbreviations used are: ER
      endoplasmic reticulum
      PC
      proprotein convertase
      PROFUR
      furin propeptide
      MATFUR
      mature protease domain of furin
      PROPC1/3
      PC1/3 propeptide
      Abz
      aminobenzoic acid.
      as they exit the ribosome, proteins transit these secretory pathway compartments on their journey to their ultimate destination. Just as each group of compartments has mechanisms for maintaining precise pH and calcium balance (
      • Levin L.R.
      • Buck J.
      Physiological roles of acid-base sensors.
      ,
      • Srivastava J.
      • Barber D.L.
      • Jacobson M.P.
      Intracellular pH sensors: design principles and functional significance.
      ), each family of proteins has co-evolved means to sense the environmental changes to ensure optimal organismal homeostasis. How eukaryotic proteins have diverged from their prokaryotic ancestors to exploit the unique organellar environment of the secretory pathway for biological function remains a fundamental question in cell biology (
      • Shinde U.
      • Thomas G.
      Insights from bacterial subtilases into the mechanisms of intramolecular chaperone-mediated activation of furin.
      ).
      Proprotein convertases (PCs) are eukaryotic members of the ubiquitous superfamily of subtilases (
      • Thomas G.
      Furin at the cutting edge: from protein traffic to embryogenesis and disease.
      ). Comprising nine serine endoproteases (PC1/3, PC2, furin, PC4, PACE4, PC5/PC6, PC7/LPC/PC8, SKI/S1P, and NARC1/PCSK9), PCs are responsible for the conversion of a diverse range of precursor substrates to their active forms and thus play a central role in maintenance of physiologic homeostasis within cells and tissues (
      • Shinde U.
      • Thomas G.
      Insights from bacterial subtilases into the mechanisms of intramolecular chaperone-mediated activation of furin.
      ,
      • Seidah N.G.
      • Sadr M.S.
      • Chrétien M.
      • Mbikay M.
      The multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions.
      ). Furin, the most thoroughly characterized PC, is constitutively expressed in virtually all tissues and catalyzes the maturation of a diverse repertoire of hormones, enzymes, and receptor precursors within the secretory pathway (
      • Thomas G.
      Furin at the cutting edge: from protein traffic to embryogenesis and disease.
      ). Not surprisingly, misregulation of furin results in both hyper- and hypoactivity and has been associated with cancer invasiveness and metastasis (
      • Rashid S.
      • Tavori H.
      • Brown P.E.
      • Linton M.F.
      • He J.
      • Giunzioni I.
      • Fazio S.
      Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms.
      ,
      • Fu J.
      • Bassi D.E.
      • Zhang J.
      • Li T.
      • Nicolas E.
      • Klein-Szanto A.J.
      Transgenic overexpression of the proprotein convertase furin enhances skin tumor growth.
      ,
      • Arsenault D.
      • Lucien F.
      • Dubois C.M.
      Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.
      ,
      • Bassi D.E.
      • Zhang J.
      • Cenna J.
      • Litwin S.
      • Cukierman E.
      • Klein-Szanto A.J.
      Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.
      ,
      • Scamuffa N.
      • Sfaxi F.
      • Ma J.
      • Lalou C.
      • Seidah N.
      • Calvo F.
      • Khatib A.M.
      Prodomain of the proprotein convertase subtilisin/kexin Furin (ppFurin) protects from tumor progression and metastasis.
      ,
      • López de Cicco R.
      • Bassi D.E.
      • Zucker S.
      • Seidah N.G.
      • Klein-Szanto A.J.
      Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.
      ), susceptibility to viral and parasitic infection (
      • Pasquato A.
      • Ramos da Palma J.
      • Galan C.
      • Seidah N.G.
      • Kunz S.
      Viral envelope glycoprotein processing by proprotein convertases.
      ), and increased severity of cardiovascular disease (
      • Seidah N.G.
      • Awan Z.
      • Chrétien M.
      • Mbikay M.
      PCSK9: a key modulator of cardiovascular health.
      ,
      • Susan-Resiga D.
      • Essalmani R.
      • Hamelin J.
      • Asselin M.C.
      • Benjannet S.
      • Chamberland A.
      • Day R.
      • Szumska D.
      • Constam D.
      • Bhattacharya S.
      • Prat A.
      • Seidah N.G.
      Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10.
      ). Additionally, furin-deficient mice die at embryonic day 11 due to cardiac defects resulting from failed chorioallantoic fusion, axial rotation, and ventral closure (
      • Roebroek A.J.
      • Umans L.
      • Pauli I.G.
      • Robertson E.J.
      • van Leuven F.
      • Van de Ven W.J.
      • Constam D.B.
      Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.
      ). PC1/3 (also known as PC1 or PC3) along with PC2 is a neuroendocrine convertase responsible for the processing of several critical metabolic regulators, including insulin, glucagon, and proopiomelanocortin (
      • Zhu X.
      • Zhou A.
      • Dey A.
      • Norrbom C.
      • Carroll R.
      • Zhang C.
      • Laurent V.
      • Lindberg I.
      • Ugleholdt R.
      • Holst J.J.
      • Steiner D.F.
      Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects.
      ). Mice with knock-out of pcsk1 or pcsk2, the genes encoding PC1/3 and PC2 respectively, remain viable despite hormonal and/or neuroendocrine deficiencies (
      • Scamuffa N.
      • Calvo F.
      • Chrétien M.
      • Seidah N.G.
      • Khatib A.M.
      Proprotein convertases: lessons from knockouts.
      ,
      • Stijnen P.
      • Tuand K.
      • Varga T.V.
      • Franks P.W.
      • Aertgeerts B.
      • Creemers J.W.
      The association of common variants in PCSK1 with obesity: a HuGE review and meta-analysis.
      ,
      • Benzinou M.
      • Creemers J.W.
      • Choquet H.
      • Lobbens S.
      • Dina C.
      • Durand E.
      • Guerardel A.
      • Boutin P.
      • Jouret B.
      • Heude B.
      • Balkau B.
      • Tichet J.
      • Marre M.
      • Potoczna N.
      • Horber F.
      • Le Stunff C.
      • Czernichow S.
      • Sandbaek A.
      • Lauritzen T.
      • Borch-Johnsen K.
      • Andersen G.
      • Kiess W.
      • Körner A.
      • Kovacs P.
      • Jacobson P.
      • Carlsson L.M.
      • Walley A.J.
      • Jørgensen T.
      • Hansen T.
      • Pedersen O.
      • Meyre D.
      • Froguel P.
      Common nonsynonymous variants in PCSK1 confer risk of obesity.
      ). Consistent with this, several studies characterizing mutations in PC1/3 have demonstrated an altered substrate processing that may underlie obesity, type II diabetes mellitus, and endocrine derangements (
      • Benzinou M.
      • Creemers J.W.
      • Choquet H.
      • Lobbens S.
      • Dina C.
      • Durand E.
      • Guerardel A.
      • Boutin P.
      • Jouret B.
      • Heude B.
      • Balkau B.
      • Tichet J.
      • Marre M.
      • Potoczna N.
      • Horber F.
      • Le Stunff C.
      • Czernichow S.
      • Sandbaek A.
      • Lauritzen T.
      • Borch-Johnsen K.
      • Andersen G.
      • Kiess W.
      • Körner A.
      • Kovacs P.
      • Jacobson P.
      • Carlsson L.M.
      • Walley A.J.
      • Jørgensen T.
      • Hansen T.
      • Pedersen O.
      • Meyre D.
      • Froguel P.
      Common nonsynonymous variants in PCSK1 confer risk of obesity.
      ,
      • Blanco E.H.
      • Peinado J.R.
      • Martín M.G.
      • Lindberg I.
      Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph.
      ,
      • Creemers J.W.
      • Choquet H.
      • Stijnen P.
      • Vatin V.
      • Pigeyre M.
      • Beckers S.
      • Meulemans S.
      • Than M.E.
      • Yengo L.
      • Tauber M.
      • Balkau B.
      • Elliott P.
      • Jarvelin M.R.
      • Van Hul W.
      • Van Gaal L.
      • Horber F.
      • Pattou F.
      • Froguel P.
      • Meyre D.
      Heterozygous mutations causing partial prohormone convertase 1 deficiency contribute to human obesity.
      ,
      • Prabhu Y.
      • Blanco E.H.
      • Liu M.
      • Peinado J.R.
      • Wheeler M.C.
      • Gekakis N.
      • Arvan P.
      • Lindberg I.
      Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.
      ,
      • Farooqi I.S.
      • Volders K.
      • Stanhope R.
      • Heuschkel R.
      • White A.
      • Lank E.
      • Keogh J.
      • O'Rahilly S.
      • Creemers J.W.
      Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in prohormone convertase 1/3.
      ). Further support for the correlation between polymorphisms in PC1/3 and metabolic disease is offered by reports of both individual patients (
      • Farooqi I.S.
      • Volders K.
      • Stanhope R.
      • Heuschkel R.
      • White A.
      • Lank E.
      • Keogh J.
      • O'Rahilly S.
      • Creemers J.W.
      Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in prohormone convertase 1/3.
      ,
      • Benjannet S.
      • Rondeau N.
      • Paquet L.
      • Boudreault A.
      • Lazure C.
      • Chrétien M.
      • Seidah N.G.
      Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2.
      ,
      • Jackson R.S.
      • Creemers J.W.
      • Farooqi I.S.
      • Raffin-Sanson M.L.
      • Varro A.
      • Dockray G.J.
      • Holst J.J.
      • Brubaker P.L.
      • Corvol P.
      • Polonsky K.S.
      • Ostrega D.
      • Becker K.L.
      • Bertagna X.
      • Hutton J.C.
      • White A.
      • Dattani M.T.
      • Hussain K.
      • Middleton S.J.
      • Nicole T.M.
      • Milla P.J.
      • Lindley K.J.
      • O'Rahilly S.
      Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency.
      ,
      • Jackson R.S.
      • Creemers J.W.
      • Ohagi S.
      • Raffin-Sanson M.L.
      • Sanders L.
      • Montague C.T.
      • Hutton J.C.
      • O'Rahilly S.
      Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.
      ,
      • Martín M.G.
      • Lindberg I.
      • Solorzano-Vargas R.S.
      • Wang J.
      • Avitzur Y.
      • Bandsma R.
      • Sokollik C.
      • Lawrence S.
      • Pickett L.A.
      • Chen Z.
      • Egritas O.
      • Dalgic B.
      • Albornoz V.
      • de Ridder L.
      • Hulst J.
      • Gok F.
      • Aydoğan A.
      • Al-Hussaini A.
      • Gok D.E.
      • Yourshaw M.
      • Wu S.V.
      • Cortina G.
      • Stanford S.
      • Georgia S.
      Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort.
      ) and epidemiologic studies (
      • Stijnen P.
      • Tuand K.
      • Varga T.V.
      • Franks P.W.
      • Aertgeerts B.
      • Creemers J.W.
      The association of common variants in PCSK1 with obesity: a HuGE review and meta-analysis.
      ).
      As unregulated protease activity can have devastating consequences on organismal homeostasis (
      • López-Otín C.
      • Bond J.S.
      Proteases: multifunctional enzymes in life and disease.
      ), PCs, like all subtilases, are synthesized as zymogens and are activated in a precise spatiotemporal fashion by intramolecular proteolysis (
      • Subbian E.
      • Yabuta Y.
      • Shinde U.P.
      Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin.
      ). A fundamental question has long been how the timing of activation is encoded and recognized by the protease. Our understanding of how this regulation is achieved is based on studies of profurin (
      • Dillon S.L.
      • Williamson D.M.
      • Elferich J.
      • Radler D.
      • Joshi R.
      • Thomas G.
      • Shinde U.
      Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.
      ,
      • Elferich J.
      • Dillon S.
      • Shinde U.
      Proceeding of the 6th International Conference on Bioinformatics and Biomedical Engineering, Shanghai, China, May 17–20, 2012.
      ,
      • Elferich J.
      • Williamson D.M.
      • Krishnamoorthy B.
      • Shinde U.
      Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.
      ,
      • Williamson D.M.
      • Elferich J.
      • Ramakrishnan P.
      • Thomas G.
      • Shinde U.
      The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.
      ). This furin precursor contains an 83-residue N-terminal propeptide that is requisite for folding of its cognate catalytic domain in the ER (
      • Creemers J.W.
      • Vey M.
      • Schäfer W.
      • Ayoubi T.A.
      • Roebroek A.J.
      • Klenk H.D.
      • Garten W.
      • Van de Ven W.J.
      Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.
      ,
      • Anderson E.D.
      • Molloy S.S.
      • Jean F.
      • Fei H.
      • Shimamura S.
      • Thomas G.
      The ordered and compartment-specific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation.
      ,
      • Anderson E.D.
      • VanSlyke J.K.
      • Thulin C.D.
      • Jean F.
      • Thomas G.
      Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.
      ) after completion of which the propeptide gets cleaved but subsequently remains associated as an inhibitor until reaching the trans-Golgi network. The necessity of furin to reach the trans-Golgi network before becoming active (
      • Vey M.
      • Schäfer W.
      • Berghöfer S.
      • Klenk H.D.
      • Garten W.
      Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation.
      ) coupled with the understanding of the unique environments of each compartment of the secretory pathway (
      • Paroutis P.
      • Touret N.
      • Grinstein S.
      The pH of the secretory pathway: measurement, determinants, and regulation.
      ) argues for the presence of an encoded sensor that recognizes and responds to specific environmental signals (
      • Dillon S.L.
      • Williamson D.M.
      • Elferich J.
      • Radler D.
      • Joshi R.
      • Thomas G.
      • Shinde U.
      Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.
      ). In fact, studies demonstrate that it is the mildly acidic pH of the trans-Golgi network (pH ∼ 6.5) that triggers the release and degradation of the furin propeptide (PROFUR) and thus release of inhibition of the mature protease domain of furin (MATFUR) (
      • Anderson E.D.
      • Molloy S.S.
      • Jean F.
      • Fei H.
      • Shimamura S.
      • Thomas G.
      The ordered and compartment-specific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation.
      ,
      • Anderson E.D.
      • VanSlyke J.K.
      • Thulin C.D.
      • Jean F.
      • Thomas G.
      Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.
      ). Interestingly, although the PC1/3 precursor, pro-PC1/3, transits the secretory pathway in much the same way as the furin precursor, our previous work indicates that PC1/3 requires a lower pH for its activation (pH ∼ 5.5) and thus is likely activated in a later compartment (
      • Dillon S.L.
      • Williamson D.M.
      • Elferich J.
      • Radler D.
      • Joshi R.
      • Thomas G.
      • Shinde U.
      Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.
      ).
      Organellar pH can alter the protonation status of charged residues, thus altering the structure and stability of the protein both on a local and global scale (
      • Schönichen A.
      • Webb B.A.
      • Jacobson M.P.
      • Barber D.L.
      Considering protonation as a posttranslational modification regulating protein structure and function.
      ). Given that the pH range of interest in the case of the secretory pathway and PC activation falls within the physiologic range of ∼7.4 in the ER to ∼5.4 in the mature secretory granules, basic residues such as arginine (pKa ∼ 12.5) or acidic residues such as glutamate (pKa ∼ 4.2) are less likely to undergo altered protonation and be candidates for pH sensors; however, the imidazole ring of histidine has a pKa of ∼6.0, making it ideally situated to respond to pH changes in this range. Histidine is used as a pH sensor in a variety of biological molecules, including hemoglobin and class II MHC (
      • Zachos C.
      • Blanz J.
      • Saftig P.
      • Schwake M.
      A critical histidine residue within LIMP-2 mediates pH sensitive binding to its ligand β-glucocerebrosidase.
      ,
      • Baird F.E.
      • Pinilla-Tenas J.J.
      • Ogilvie W.L.
      • Ganapathy V.
      • Hundal H.S.
      • Taylor P.M.
      Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue.
      ,
      • Rötzschke O.
      • Lau J.M.
      • Hofstätter M.
      • Falk K.
      • Strominger J.L.
      A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules.
      ), and is enriched within the propeptides of eukaryotic proteases that transit the secretory pathway in contrast to both cytosolic proteases and prokaryotic orthologues (
      • Elferich J.
      • Dillon S.
      • Shinde U.
      Proceeding of the 6th International Conference on Bioinformatics and Biomedical Engineering, Shanghai, China, May 17–20, 2012.
      ,
      • Elferich J.
      • Williamson D.M.
      • Krishnamoorthy B.
      • Shinde U.
      Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.
      ). Indeed, we identified a histidine (His69) within PROFUR that regulates the compartment-specific activation of MATFUR (
      • Feliciangeli S.F.
      • Thomas L.
      • Scott G.K.
      • Subbian E.
      • Hung C.H.
      • Molloy S.S.
      • Jean F.
      • Shinde U.
      • Thomas G.
      Identification of a pH sensor in the furin propeptide that regulates enzyme activation.
      ). This histidine is in a loop adjacent to the secondary cleavage site nestled in a solvent-accessible pocket lined by hydrophobic residues. At the near-neutral pH of the ER, the deprotonated histidine acts as a hydrophobic residue, stabilizing the packing within this pocket and keeping the pH-sensitive loop protected against cleavage. Upon entry into the trans-Golgi network, the histidine is exposed to a ∼10-fold higher proton concentration and thus is protonated; as a result, the imidazole side chain becomes polar, disrupting the packing and driving a local conformational change that exposes the secondary cleavage site, allowing for rapid degradation and release of PROFUR from MATFUR (
      • Williamson D.M.
      • Elferich J.
      • Ramakrishnan P.
      • Thomas G.
      • Shinde U.
      The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.
      ).
      Propeptide domains alone encode sufficient information for regulating the organelle-specific pH-dependent activation of cognate protease domains (
      • Dillon S.L.
      • Williamson D.M.
      • Elferich J.
      • Radler D.
      • Joshi R.
      • Thomas G.
      • Shinde U.
      Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.
      ). Thus, when the propeptides of furin and PC1/3 are swapped, the pH-dependent protease activation is transferred in a propeptide-dictated manner both in vitro and in cells. Interestingly, the histidine pH sensor identified in PROFUR is absolutely conserved within the propeptides of all PCs, including PROPC1/3. Despite their structural similarity and the presence of the conserved histidine throughout the family of PCs, it remains unclear how the pH sensitivity of other PCs is encoded.
      In this study, we asked whether the pH-sensing histidine is conserved in PROPC1/3 and why does MATPC1/3 not become active at the same pH as furin? We present biochemical and structural data that suggest that the conserved histidine residue plays a critical role in pH-dependent activation similar to what has been established in furin; however, an additional histidine residue modulates this pH sensitivity such that a more acidic environment is required for PC1/3 activation.

      Discussion

      Organellar pH is a critical regulator of a wide range of intracellular events, including zymogen activation, signaling cascades, ion channel activity, and receptor-ligand interactions. Each of these events must be tightly regulated both temporally and spatially to maintain organismal homeostasis, and many eukaryotic proteins have evolved to exploit environmental pH as a cue to regulate their activation via alterations in the protonation status of titratable amino acid side chains. Not surprisingly then, protonation with increasing organellar acidification is the major biochemical cue that regulates the final activation step of PCs. We are only just beginning to understand how the differing pH sensitivity of individual PCs is encoded and how the addition of a single proton can drive the biochemical and biophysical changes required to initiate protease activation. We have earlier demonstrated that protonation of His69 can disrupt a hydrophobic pocket within PROFUR to drive local unfolding of the secondary cleavage site loop to facilitate PROFUR proteolysis that results in furin activation (
      • Williamson D.M.
      • Elferich J.
      • Ramakrishnan P.
      • Thomas G.
      • Shinde U.
      The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.
      ,
      • Feliciangeli S.F.
      • Thomas L.
      • Scott G.K.
      • Subbian E.
      • Hung C.H.
      • Molloy S.S.
      • Jean F.
      • Shinde U.
      • Thomas G.
      Identification of a pH sensor in the furin propeptide that regulates enzyme activation.
      ). Although His69 is conserved within all PCs, individual PCs nonetheless undergo activation at different pH values with actual proton concentrations that vary over 10-fold, suggesting that additional complexities remain to be determined.
      Our results demonstrate that although histidine residues are mostly conserved within orthologues of PC1/3 and furin individually they show significant differences between the two families with only the primary pH sensor residue (His69 in PROFUR and His72 in PROPC1/3) absolutely conserved throughout the alignment (Fig. 2). The remaining histidines, His67, His75, and His85, in PROPC1/3 are located within proximity of the cleavage site loop. His67 (which corresponds to His66 in furin) is situated at the interface with the protease domain and has been demonstrated to have no influence on pH sensing in furin (
      • Feliciangeli S.F.
      • Thomas L.
      • Scott G.K.
      • Subbian E.
      • Hung C.H.
      • Molloy S.S.
      • Jean F.
      • Shinde U.
      • Thomas G.
      Identification of a pH sensor in the furin propeptide that regulates enzyme activation.
      ). Furthermore, preliminary studies using leucine and arginine substitutions at positions 67 and 85 in PC1/3 suggest that these variants do not affect the secondary structure of the isolated propeptide domain or their IC50 values (data not shown). It is noteworthy that (i) the counterpart to His69, which is solvent-accessible in furin, appears more buried within PC1/3; (ii) the imidazole side chain of His75 is solvent-exposed and does not have an analogous counterpart in furin; and (iii) His75 precedes a proline residue in PC1/3. Experimental studies have established that when a histidine side chain precedes a proline residue the proline isomerization rates increase up to 10-fold when the imidazole side chain is protonated relative to the deprotonated state (
      • Reimer U.
      • Scherer G.
      • Drewello M.
      • Kruber S.
      • Schutkowski M.
      • Fischer G.
      Side-chain effects on peptidyl-prolyl cis/trans isomerisation.
      ,
      • Texter F.L.
      • Spencer D.B.
      • Rosenstein R.
      • Matthews C.R.
      Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.
      ). In this study, we therefore analyzed the role of the conserved histidine in PC1/3 (His72) as well as that of a second histidine (His75), which we propose acts as a “gatekeeper” that regulates solvent accessibility and thus protonation of the primary pH sensing histidine.
      To decipher the role of the two histidine residues in regulating the pH-dependent activation of PC1/3, we used constructs with a C-terminal KDEL sequence that retains the noncovalently bound inhibition complex within the ER; in this case, pro-PC1/3 has undergone the pH-independent primary processing subsequent to folding, but the propeptide remains associated, acting as an inhibitor of protease activity. Our results indicated that mutation of either His72 or His75 to a nonprotonatable mimic was sufficient to block the second processing event and thus activation of PC1/3 at all pH values assayed, indicating that the protonation of both histidine residues is necessary and that neither alone are sufficient to drive pH-dependent activation of PC1/3. It is noteworthy that when replaced with an arginine, a surrogate that mimics a constitutively protonated state, the H72R-PROPC1/3 did not promote activation, whereas the H75R-PROPC1/3 underwent marginal activation at pH 7.4. Although activation at pH 6.4 and 5.4 increased for both H72R-PROPC1/3 and H75R-PROPC1/3, only the double variant (H72R/H75R-PROPC1/3) mediated robust activation at all pH values. On this basis, we propose that both His72 and His75 appear to work cooperatively to mediate the pH-dependent activation of PC1/3 (Fig. 3).
      The biophysical analysis of the isolated propeptides further supported the idea that both histidine residues were important in the mechanism of activation for this protease. Here, the pH-dependent structural transitions seen in the various mutant propeptides are particularly insightful. The H72L-PROPC1/3, H75L-PROPC1/3, and H72L/H75L-PROPC1/3 variants behaved similarly to WT PROPC1/3 between pH 6.0 and 8.0. However, at pH less than 6.0, the WT PROPC1/3 began to unfold with a calculated pKa of ∼5.2 when the data were fit to a two-proton model (Fig. 5A). Under these conditions, all histidine to leucine PROPC1/3 variants were significantly more stable than the WT PROPC1/3, and although the H75L-PROPC1/3 lost about 50% of its ellipticity at 222 nm when compared with WT PROPC1/3, H72L-PROPC1/3 and H72L/H75L-PROPC1/3 variants underwent only marginal unfolding (∼20%) under identical conditions (Fig. 5B). Although these results indicate that a single substitution of histidine with nonprotonatable leucine residues at position 72 or 75 blunts the ability of PROPC1/3 variant to respond to more acidic pH, the subtle differences in the behavior of the individual variants allows one to begin teasing apart the differing roles of these residues.
      First, given that the single H72L variant was sufficient to attenuate the pH-dependent structural changes within the propeptide (Fig. 5B) and block pH-dependent activation (Fig. 3A), we propose that the protonation status of His72 is a major driver of the requisite unfolding of the cleavage loop of PROPC1/3. Second, the observation that the H75L underwent partial unfolding, albeit to a lesser degree than the WT PROPC1/3, indicates that although protonation of His72 is necessary it is not sufficient, and thus the combinatorial effect of protonation of both residues appears to be necessary for activation of PC1/3. As a final note, the small change in secondary structural content in the double H72R/H75R-PROPC1/3 variant was likely due to the influence of other titratable groups, which at low pH values may enhance the local changes required for propeptide processing and release, and thus further studies are needed to more fully investigate this possibility.
      By introducing an arginine in place of the histidines of interest, we were able to assess the effect of a positive charge at these positions within the propeptides. Again, the differences between the variants shed light on the differing role that each of these histidines play and lends further support to the notion that there is a synergistic effect between His72 and His75 that allows for the precise spatiotemporal regulation of the loosening of secondary structure required for PROPC1/3 processing. Even at pH 8, H72R-PROPC1/3 displayed markedly less secondary structure than the WT-PROPC1/3, reflecting its role as a major driver of destabilization. There was an additional loss of α helicity as pH dropped below ∼6 either due to the aggregate effect of protonation of His75 or the influence of other side chains. The behavior of H75R-PROPC1/3 is again of particular interest: although somewhat destabilized relative to WT-PROPC1/3, it was nonetheless well structured at pH 8 when compared with H72R-PROPC1/3 and H72R/H75R-PROPC1/3. As pH was lowered, H75R-PROPC1/3 immediately began to lose secondary structure but plateaued at pH 6. This again highlights our assertion that the positive charge at either of these positions singly is not sufficient to account for the activation behavior of PC1/3 but rather that cooperative action between early protonation of His75 followed by later protonation of His72 is required.
      A final piece of insight is offered by the ability of the propeptides to inhibit the protease domain (Fig. 6). As would be expected, IC50 values (at pH 6.8) for the leucine variant propeptides roughly approximated those of the wild type, whereas those for the H75R and H72R/H75R variant were ∼15-fold higher, indicating that they are notably weaker inhibitors. At first, it may seem surprising that the H72R variant has an IC50 that is comparable with that of the wild type; however, we propose that this reflects a critical role of the histidine at position 75: in addition to modulating the solvent accessibility of the hydrophobic pocket it overlays, protonation of His75 may be involved in a destabilization of the propeptide-protease complex. As we are yet unable to produce the mature protease in sufficient quantities, we are unable to more directly determine kon/koff and more thoroughly characterize the interaction between the protease and its cognate propeptide.
      Taking the above as a whole, we can then propose a mechanism by which PC1/3 is activated (Fig. 7). We believe that the two histidine residues within the cleavage loop are in proximity of one another and require sequential protonation to allow for activation. His75, which sits at the top of the hydrophobic pocket, is likely protonated first and begins the local unfolding of the cleavage loop. It is tempting to speculate that protonation of His75 facilitates local unfolding by enhancing the kinetics of cis-trans isomerization of Pro76 as demonstrated by systematic studies in a pentapeptide series, Ac-Ala-Xaa-Pro-Ala-Lys-NH2 (
      • Reimer U.
      • Scherer G.
      • Drewello M.
      • Kruber S.
      • Schutkowski M.
      • Fischer G.
      Side-chain effects on peptidyl-prolyl cis/trans isomerisation.
      ). These studies, which examined the influence of each of the 20 amino acids at position Xaa on the energetics of proline isomerization using NMR spectroscopy, demonstrated that the rates of proline isomerization are enhanced severalfold only when the side chains of tyrosine and histidine residues are protonated. The occurrence of histidine residues preceding a proline is higher than the overall frequency of the individual amino acids would predict (
      • Reimer U.
      • el Mokdad N.
      • Schutkowski M.
      • Fischer G.
      Intramolecular assistance of cis/trans isomerization of the histidine-proline moiety.
      ). Thus we propose that the protonation of His75 enhances local unfolding of the loop, which allows His72 to become more solvent-accessible and simultaneously disrupt the interface between the propeptide and protease. Once solvent-accessible, His72 can then be protonated, thus delivering the second and final blow that drives the processing and dissociation of the propeptide, thus releasing inhibition from the protease. In this model, His75 acts as a gatekeeper residue, blocking access to the hydrophobic pocket in its deprotonated state, a restriction that can be removed via protonation. The model for two protonation events is also supported by CD data on the WT-PROPC1/3. Notably, when we fit the CD data from pH-dependent unfolding experiments to a modified form of the Henderson-Hasselbalch equation (
      • Tanford C.
      • De P.K.
      The unfolding of β-lactoglobulin at pH 3 by urea, formamide, and other organic substances.
      ), we found that a model of two titratable groups better fit the conformational changes in PROPC1/3 in response to changing pH than one that only allowed for a single protonation (Fig. 5A). We also speculate that the close proximity of His72 and His75 may also be critical to understanding the mechanism of activation and reason that PC1/3 is only activated in acidic environs. When both residues are unprotonated, hydrophobic packing of the core of the propeptide is the major stabilizing force similar to the furin propeptide. One must ask then is it simply a greater degree of stability in the structure of PROPC1/3 that requires the stronger disrupting force of two positive charges to drive its unfolding, or does the protonation of one residue influence the protonation of the second? Concomitant work in our laboratory has been focused on defining pKa values of the histidines within PROFUR and PROPC1/3. Although the pKa values of His67, His75, and His85 are all ∼6.0, the pKa of His72 is acid-shifted with a value of ∼5.5 (
      • Elferich J.
      • Williamson D.M.
      • David L.L.
      • Shinde U.
      Determination of histidine pKa values in the propeptides of furin and PC1/3 using histidine hydrogen-deuterium exchange mass spectrometry.
      ), which similar to the pH of activation of PC1/3. Therefore, we believe that the spatial arrangement and interaction of His72 and His75 can explain why PC1/3 is activated by a 10-fold higher proton concentration than its paralogue furin.
      Figure thumbnail gr7
      FIGURE 7Model of histidine protonation in PROPC1/3. Two-dimensional schematic representations of the NMR solution structures of the PC1/3 propeptide (Protein Data Bank code 1KN6) displaying histidines side chains of interest are shown. Left, both histidines are deprotonated, maintaining packing of core and protecting cleavage site. Center, His75 is protonated, causing partial unfolding of the cleavage loop and exposing His72. Right, both histidines are protonated, causing cleavage loop to unfold completely and exposing cleavage site to allow processing.
      This work offers significant insight into the question of how each member of the PC family encodes unique organelle-specific information about activation and processing. We believe that although the primary pH sensor (His69 in furin and His72 in PC1/3) has been evolutionarily conserved throughout PCs, concomitant with histidine enrichment has been a tuning of the pH sensitivity of these proteases. As seen in the multiple sequence alignment, only the position of the primary pH sensor has been absolutely conserved; thus domain-specific conservation as opposed to position-specific conservation seems to be crucial to encoding pH sensitivity in the propeptides of proteases in this family. Looking more broadly at secreted proteases across eukaryotes and prokaryotes, we had reported previously that there is an enrichment of histidine residues in the propeptides of secreted eukaryotic proteases that is not seen in their prokaryotic paralogues. Again, we see that although there is a bias for more histidines within this particular domain there is no appreciable bias for specific positions (
      • Elferich J.
      • Dillon S.
      • Shinde U.
      Proceeding of the 6th International Conference on Bioinformatics and Biomedical Engineering, Shanghai, China, May 17–20, 2012.
      ). These observations highlight the importance of spatial juxtapositioning of titratable groups in regions of proteins to tune their pH sensitivity, an evolutionary adaptation that was likely concomitant with the emergence of compartmentalization and specialization allowed by the secretory pathway within eukaryotic cells.
      The issue of “mistuning” via mutation is also interesting to consider. The most obvious case to consider would be the loss of the conserved pH sensor, yielding either an inactivatable or constitutively active protease depending on the type of mutation. More interesting, however, would be the mutation of one of the surrounding residues and its effect on the local environs and protonatability of not only the primary pH sensor but also the gatekeeper or tuning residues. Evidence for the plausibility of this notion is lent by several single nucleotide polymorphisms in PROFUR and PROPC1/3. Preliminary characterization of one of these polymorphisms, ΔR80Q (
      • Pickett L.A.
      • Yourshaw M.
      • Albornoz V.
      • Chen Z.
      • Solorzano-Vargas R.S.
      • Nelson S.F.
      • Martín M.G.
      • Lindberg I.
      Functional consequences of a novel variant of PCSK1.
      ), indicates that changes in the propeptide can affect the biosynthesis, secretion, and catalytic activity similar to other previously described SNPs in the catalytic domain, and thus it is hypothesized that it may contribute to cancer (including head and neck carcinoma and lung adenocarcinoma (
      • Forbes S.A.
      • Beare D.
      • Gunasekaran P.
      • Leung K.
      • Bindal N.
      • Boutselakis H.
      • Ding M.
      • Bamford S.
      • Cole C.
      • Ward S.
      • Kok C.Y.
      • Jia M.
      • De T.
      • Teague J.W.
      • Stratton M.R.
      • McDermott U.
      • Campbell P.J.
      COSMIC: exploring the world's knowledge of somatic mutations in human cancer.
      )) or metabolic disease. Further characterization of these polymorphisms would be insightful not only in understanding the basis on which they are able to cause disease but also in the broader sense of understanding what role propeptides have in dictating proteolytic activity and processing. Similarly, environmental homeostasis is required for optimal proteolytic function; perturbation of cellular pH through disease (
      • Weisz O.A.
      Organelle acidification and disease.
      ) may cause activation of a PC prematurely or prevent activation all together. The role of the pH gradient in the secretory pathway in orchestrating proteolytic processing of substrates cannot be understated. Thus as we continue to understand the interplay among the proprotein convertases, proton concentration, and pH sensors, we hope to gain better insight into how things go wrong in the disease state and how we can better approach solutions to restore homeostasis.

      Author Contributions

      D. M. W. made substantial contributions to conception, design, and acquisition of data. J. E. helped with the analysis. U. S. helped draft and revise the article and helped with the analysis of data. All authors reviewed the results and approved the final version of the manuscript.

      References

        • Soskine M.
        • Tawfik D.S.
        Mutational effects and the evolution of new protein functions.
        Nat. Rev. Genet. 2010; 11: 572-582
        • Embley T.M.
        • Martin W.
        Eukaryotic evolution, changes and challenges.
        Nature. 2006; 440: 623-630
        • Levin L.R.
        • Buck J.
        Physiological roles of acid-base sensors.
        Annu. Rev. Physiol. 2015; 77: 347-362
        • Srivastava J.
        • Barber D.L.
        • Jacobson M.P.
        Intracellular pH sensors: design principles and functional significance.
        Physiology. 2007; 22: 30-39
        • Shinde U.
        • Thomas G.
        Insights from bacterial subtilases into the mechanisms of intramolecular chaperone-mediated activation of furin.
        Methods Mol. Biol. 2011; 768: 59-106
        • Thomas G.
        Furin at the cutting edge: from protein traffic to embryogenesis and disease.
        Nat. Rev. Mol. Cell Biol. 2002; 3: 753-766
        • Seidah N.G.
        • Sadr M.S.
        • Chrétien M.
        • Mbikay M.
        The multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions.
        J. Biol. Chem. 2013; 288: 21473-21481
        • Rashid S.
        • Tavori H.
        • Brown P.E.
        • Linton M.F.
        • He J.
        • Giunzioni I.
        • Fazio S.
        Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms.
        Circulation. 2014; 130: 431-441
        • Fu J.
        • Bassi D.E.
        • Zhang J.
        • Li T.
        • Nicolas E.
        • Klein-Szanto A.J.
        Transgenic overexpression of the proprotein convertase furin enhances skin tumor growth.
        Neoplasia. 2012; 14: 271-282
        • Arsenault D.
        • Lucien F.
        • Dubois C.M.
        Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.
        J. Cell Physiol. 2012; 227: 789-800
        • Bassi D.E.
        • Zhang J.
        • Cenna J.
        • Litwin S.
        • Cukierman E.
        • Klein-Szanto A.J.
        Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.
        Neoplasia. 2010; 12: 516-526
        • Scamuffa N.
        • Sfaxi F.
        • Ma J.
        • Lalou C.
        • Seidah N.
        • Calvo F.
        • Khatib A.M.
        Prodomain of the proprotein convertase subtilisin/kexin Furin (ppFurin) protects from tumor progression and metastasis.
        Carcinogenesis. 2014; 35: 528-536
        • López de Cicco R.
        • Bassi D.E.
        • Zucker S.
        • Seidah N.G.
        • Klein-Szanto A.J.
        Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.
        Cancer Res. 2005; 65: 4162-4171
        • Pasquato A.
        • Ramos da Palma J.
        • Galan C.
        • Seidah N.G.
        • Kunz S.
        Viral envelope glycoprotein processing by proprotein convertases.
        Antiviral Res. 2013; 99: 49-60
        • Seidah N.G.
        • Awan Z.
        • Chrétien M.
        • Mbikay M.
        PCSK9: a key modulator of cardiovascular health.
        Circ. Res. 2014; 114: 1022-1036
        • Susan-Resiga D.
        • Essalmani R.
        • Hamelin J.
        • Asselin M.C.
        • Benjannet S.
        • Chamberland A.
        • Day R.
        • Szumska D.
        • Constam D.
        • Bhattacharya S.
        • Prat A.
        • Seidah N.G.
        Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10.
        J. Biol. Chem. 2011; 286: 22785-22794
        • Roebroek A.J.
        • Umans L.
        • Pauli I.G.
        • Robertson E.J.
        • van Leuven F.
        • Van de Ven W.J.
        • Constam D.B.
        Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.
        Development. 1998; 125: 4863-4876
        • Zhu X.
        • Zhou A.
        • Dey A.
        • Norrbom C.
        • Carroll R.
        • Zhang C.
        • Laurent V.
        • Lindberg I.
        • Ugleholdt R.
        • Holst J.J.
        • Steiner D.F.
        Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10293-10298
        • Scamuffa N.
        • Calvo F.
        • Chrétien M.
        • Seidah N.G.
        • Khatib A.M.
        Proprotein convertases: lessons from knockouts.
        FASEB J. 2006; 20: 1954-1963
        • Stijnen P.
        • Tuand K.
        • Varga T.V.
        • Franks P.W.
        • Aertgeerts B.
        • Creemers J.W.
        The association of common variants in PCSK1 with obesity: a HuGE review and meta-analysis.
        Am. J. Epidemiol. 2014; 180: 1051-1065
        • Benzinou M.
        • Creemers J.W.
        • Choquet H.
        • Lobbens S.
        • Dina C.
        • Durand E.
        • Guerardel A.
        • Boutin P.
        • Jouret B.
        • Heude B.
        • Balkau B.
        • Tichet J.
        • Marre M.
        • Potoczna N.
        • Horber F.
        • Le Stunff C.
        • Czernichow S.
        • Sandbaek A.
        • Lauritzen T.
        • Borch-Johnsen K.
        • Andersen G.
        • Kiess W.
        • Körner A.
        • Kovacs P.
        • Jacobson P.
        • Carlsson L.M.
        • Walley A.J.
        • Jørgensen T.
        • Hansen T.
        • Pedersen O.
        • Meyre D.
        • Froguel P.
        Common nonsynonymous variants in PCSK1 confer risk of obesity.
        Nat. Genet. 2008; 40: 943-945
        • Blanco E.H.
        • Peinado J.R.
        • Martín M.G.
        • Lindberg I.
        Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph.
        Endocrinology. 2014; 155: 3434-3447
        • Creemers J.W.
        • Choquet H.
        • Stijnen P.
        • Vatin V.
        • Pigeyre M.
        • Beckers S.
        • Meulemans S.
        • Than M.E.
        • Yengo L.
        • Tauber M.
        • Balkau B.
        • Elliott P.
        • Jarvelin M.R.
        • Van Hul W.
        • Van Gaal L.
        • Horber F.
        • Pattou F.
        • Froguel P.
        • Meyre D.
        Heterozygous mutations causing partial prohormone convertase 1 deficiency contribute to human obesity.
        Diabetes. 2012; 61: 383-390
        • Prabhu Y.
        • Blanco E.H.
        • Liu M.
        • Peinado J.R.
        • Wheeler M.C.
        • Gekakis N.
        • Arvan P.
        • Lindberg I.
        Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.
        Endocrinology. 2014; 155: 2391-2401
        • Farooqi I.S.
        • Volders K.
        • Stanhope R.
        • Heuschkel R.
        • White A.
        • Lank E.
        • Keogh J.
        • O'Rahilly S.
        • Creemers J.W.
        Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in prohormone convertase 1/3.
        J. Clin. Endocrinol. Metab. 2007; 92: 3369-3373
        • Benjannet S.
        • Rondeau N.
        • Paquet L.
        • Boudreault A.
        • Lazure C.
        • Chrétien M.
        • Seidah N.G.
        Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2.
        Biochem. J. 1993; 294: 735-743
        • Jackson R.S.
        • Creemers J.W.
        • Farooqi I.S.
        • Raffin-Sanson M.L.
        • Varro A.
        • Dockray G.J.
        • Holst J.J.
        • Brubaker P.L.
        • Corvol P.
        • Polonsky K.S.
        • Ostrega D.
        • Becker K.L.
        • Bertagna X.
        • Hutton J.C.
        • White A.
        • Dattani M.T.
        • Hussain K.
        • Middleton S.J.
        • Nicole T.M.
        • Milla P.J.
        • Lindley K.J.
        • O'Rahilly S.
        Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency.
        J. Clin. Investig. 2003; 112: 1550-1560
        • Jackson R.S.
        • Creemers J.W.
        • Ohagi S.
        • Raffin-Sanson M.L.
        • Sanders L.
        • Montague C.T.
        • Hutton J.C.
        • O'Rahilly S.
        Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.
        Nat. Genet. 1997; 16: 303-306
        • Martín M.G.
        • Lindberg I.
        • Solorzano-Vargas R.S.
        • Wang J.
        • Avitzur Y.
        • Bandsma R.
        • Sokollik C.
        • Lawrence S.
        • Pickett L.A.
        • Chen Z.
        • Egritas O.
        • Dalgic B.
        • Albornoz V.
        • de Ridder L.
        • Hulst J.
        • Gok F.
        • Aydoğan A.
        • Al-Hussaini A.
        • Gok D.E.
        • Yourshaw M.
        • Wu S.V.
        • Cortina G.
        • Stanford S.
        • Georgia S.
        Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort.
        Gastroenterology. 2013; 145: 138-148
        • López-Otín C.
        • Bond J.S.
        Proteases: multifunctional enzymes in life and disease.
        J. Biol. Chem. 2008; 283: 30433-30437
        • Subbian E.
        • Yabuta Y.
        • Shinde U.P.
        Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin.
        J. Mol. Biol. 2005; 347: 367-383
        • Dillon S.L.
        • Williamson D.M.
        • Elferich J.
        • Radler D.
        • Joshi R.
        • Thomas G.
        • Shinde U.
        Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.
        J. Mol. Biol. 2012; 423: 47-62
        • Elferich J.
        • Dillon S.
        • Shinde U.
        Proceeding of the 6th International Conference on Bioinformatics and Biomedical Engineering, Shanghai, China, May 17–20, 2012.
        (Abstract Number 71241) IEEE, New York2012
        • Elferich J.
        • Williamson D.M.
        • Krishnamoorthy B.
        • Shinde U.
        Propeptides of eukaryotic proteases encode histidines to exploit organelle pH for regulation.
        FASEB J. 2013; 27: 2939-2945
        • Williamson D.M.
        • Elferich J.
        • Ramakrishnan P.
        • Thomas G.
        • Shinde U.
        The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.
        J. Biol. Chem. 2013; 288: 19154-19165
        • Creemers J.W.
        • Vey M.
        • Schäfer W.
        • Ayoubi T.A.
        • Roebroek A.J.
        • Klenk H.D.
        • Garten W.
        • Van de Ven W.J.
        Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.
        J. Biol. Chem. 1995; 270: 2695-2702
        • Anderson E.D.
        • Molloy S.S.
        • Jean F.
        • Fei H.
        • Shimamura S.
        • Thomas G.
        The ordered and compartment-specific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation.
        J. Biol. Chem. 2002; 277: 12879-12890
        • Anderson E.D.
        • VanSlyke J.K.
        • Thulin C.D.
        • Jean F.
        • Thomas G.
        Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.
        EMBO J. 1997; 16: 1508-1518
        • Vey M.
        • Schäfer W.
        • Berghöfer S.
        • Klenk H.D.
        • Garten W.
        Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation.
        J. Cell Biol. 1994; 127: 1829-1842
        • Paroutis P.
        • Touret N.
        • Grinstein S.
        The pH of the secretory pathway: measurement, determinants, and regulation.
        Physiology. 2004; 19: 207-215
        • Schönichen A.
        • Webb B.A.
        • Jacobson M.P.
        • Barber D.L.
        Considering protonation as a posttranslational modification regulating protein structure and function.
        Annu. Rev. Biophys. 2013; 42: 289-314
        • Zachos C.
        • Blanz J.
        • Saftig P.
        • Schwake M.
        A critical histidine residue within LIMP-2 mediates pH sensitive binding to its ligand β-glucocerebrosidase.
        Traffic. 2012; 13: 1113-1123
        • Baird F.E.
        • Pinilla-Tenas J.J.
        • Ogilvie W.L.
        • Ganapathy V.
        • Hundal H.S.
        • Taylor P.M.
        Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue.
        Biochem. J. 2006; 397: 369-375
        • Rötzschke O.
        • Lau J.M.
        • Hofstätter M.
        • Falk K.
        • Strominger J.L.
        A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16946-16950
        • Feliciangeli S.F.
        • Thomas L.
        • Scott G.K.
        • Subbian E.
        • Hung C.H.
        • Molloy S.S.
        • Jean F.
        • Shinde U.
        • Thomas G.
        Identification of a pH sensor in the furin propeptide that regulates enzyme activation.
        J. Biol. Chem. 2006; 281: 16108-16116
        • Yabuta Y.
        • Subbian E.
        • Oiry C.
        • Shinde U.
        Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases.
        J. Biol. Chem. 2003; 278: 15246-15251
        • Subbian E.
        • Yabuta Y.
        • Shinde U.
        Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases.
        Biochemistry. 2004; 43: 14348-14360
        • Subbian E.
        • Williamson D.M.
        • Shinde U.
        Protein folding mediated by an intramolecular chaperone: the energy landscape for unimolecular pro-subtilisin E maturation.
        Adv. Biosci. Biotechnol. 2015; 6: 73-88
        • Tanford C.
        • De P.K.
        The unfolding of β-lactoglobulin at pH 3 by urea, formamide, and other organic substances.
        J. Biol. Chem. 1961; 236: 1711-1715
        • Tangrea M.A.
        • Bryan P.N.
        • Sari N.
        • Orban J.
        Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus.
        J. Mol. Biol. 2002; 320: 801-812
        • Tangrea M.A.
        • Alexander P.
        • Bryan P.N.
        • Eisenstein E.
        • Toedt J.
        • Orban J.
        Stability and global fold of the mouse prohormone convertase 1 pro-domain.
        Biochemistry. 2001; 40: 5488-5495
        • Zandberg W.F.
        • Benjannet S.
        • Hamelin J.
        • Pinto B.M.
        • Seidah N.G.
        N-Glycosylation controls trafficking, zymogen activation and substrate processing of proprotein convertases PC1/3 and subtilisin kexin isozyme-1.
        Glycobiology. 2011; 21: 1290-1300
        • Reimer U.
        • Scherer G.
        • Drewello M.
        • Kruber S.
        • Schutkowski M.
        • Fischer G.
        Side-chain effects on peptidyl-prolyl cis/trans isomerisation.
        J. Mol. Biol. 1998; 279: 449-460
        • Texter F.L.
        • Spencer D.B.
        • Rosenstein R.
        • Matthews C.R.
        Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.
        Biochemistry. 1992; 31: 5687-5691
        • Reimer U.
        • el Mokdad N.
        • Schutkowski M.
        • Fischer G.
        Intramolecular assistance of cis/trans isomerization of the histidine-proline moiety.
        Biochemistry. 1997; 36: 13802-13808
        • Elferich J.
        • Williamson D.M.
        • David L.L.
        • Shinde U.
        Determination of histidine pKa values in the propeptides of furin and PC1/3 using histidine hydrogen-deuterium exchange mass spectrometry.
        Anal. Chem. 2015; 87: 7909-7917
        • Pickett L.A.
        • Yourshaw M.
        • Albornoz V.
        • Chen Z.
        • Solorzano-Vargas R.S.
        • Nelson S.F.
        • Martín M.G.
        • Lindberg I.
        Functional consequences of a novel variant of PCSK1.
        PLoS One. 2013; 8e55065
        • Forbes S.A.
        • Beare D.
        • Gunasekaran P.
        • Leung K.
        • Bindal N.
        • Boutselakis H.
        • Ding M.
        • Bamford S.
        • Cole C.
        • Ward S.
        • Kok C.Y.
        • Jia M.
        • De T.
        • Teague J.W.
        • Stratton M.R.
        • McDermott U.
        • Campbell P.J.
        COSMIC: exploring the world's knowledge of somatic mutations in human cancer.
        Nucleic Acids Res. 2015; 43: D805-D811
        • Weisz O.A.
        Organelle acidification and disease.
        Traffic. 2003; 4: 57-64