Propeptides of Proteases Evolved Sensors to Exploit
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PROPEPTIDES OF PROTEASES EVOLVED SENSORS TO EXPLOIT ORGANELLAR PH Johannes Elferich, Danielle M. Williamson, Bala Krishnamoorthy¶, and Ujwal Shinde* Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR, 97239, USA ¶ Department of Mathematics, Washington State University Pullman, WA, 99164, USA *Corresponding Author: Ujwal Shinde, [email protected] Phone (503)-494-8683 Facsimile: (503)-494-8393 Running title: Protease evolution to exploit organelle pH SUMMARY: Eukaryotic cells maintain strict control over protein secretion, in part by utilizing the pH-gradient maintained within their secretory pathway. How eukaryotic proteins evolved from prokaryotic orthologs to exploit the pH-gradient for biological function remains a fundamental question in cell biology. We have previously demonstrated that protein domains located within precursor proteins, propeptides, encode histidine-driven pH-sensors to regulate organelle-specific activation of the eukaryotic proteases, furin and proprotein convertase-1/3. Using bioinformatics, we analyzed over 10,000 unique proteases within evolutionarily unrelated families, and established that eukaryotic propeptides are enriched in histidines when compared to prokaryotic orthologs. On this basis, we propose that eukaryotic proteins evolved to contain histidines within cognate propeptides to exploit the tightly controlled pH-gradient of the secretory pathway, thereby directing activation within specific organelles. Enrichment of histidine in propeptides may therefore be used to predict the presence of pH sensors in other proteases or even protease substrates. HIGHLIGHTS: • Histidine residues in propeptides act as pH-sensors in furin, a eukaryotic protease • Histidine is enriched in eukaryotic, but not prokaryotic, subtilase propeptides • Histidine enrichment is found in protein families unrelated to subtilases • We propose histidine enrichment as an evolutionary mechanism to sense organellar pH INTRODUCTION: Eukaryotes are descendants of distinct prokaryotic cells that united symbiotically to subsequently evolve complex cellular compartment called organelles (Embley and Martin, 2006). Although both prokaryotes and eukaryotes are able to secrete proteins, only eukaryotes employ multi-compartmental secretory and endocytotic pathways. These pathways maintain a precise pH-gradient that acidifies from the endoplasmic reticulum (pH~7.2) to secretory vesicles (pH~5.5). This gradient provides the unique environmental conditions essential for the optimal structure and function of proteins within distinct biochemical pathways (Casey et al., 2010). Since many secreted eukaryotic proteins have prokaryotic orthologs, how and when eukaryotic proteins evolved the ability to regulate their activity within different organelles is a central question germane to our understanding of protein trafficking. Comparing secreted eukaryotic proteins with their bacterial orthologs may potentially provide information about mechanisms by which protein activity is regulated during trafficking through the secretory pathway. Proteases hydrolyze peptide bonds and likely arose early during evolution as simple catabolic catalysts that generated amino acid residues in primitive organisms (Lopez-Otin and Bond, 2008). Due to their ubiquitous distribution within prokaryotes, eukaryotes, and archea, the three domains of life, proteases are well suited for analysis of selective pressures that drove adaptation of eukaryotic proteins to the complex organelle trafficking system. Since uncontrolled proteolysis has catastrophic consequences, cells appear to have evolved two distinct mechanisms that maintain protease activities under exquisite spatiotemporal control (Lopez- Otin and Bond, 2008). The first mechanism involves co-evolution of specific endogenous inhibitors, typically within compartments distinct from those containing active enzymes. The second mechanism involves proteases being synthesized as inactive precursors called zymogens, which become active by limited intra- or intermolecular proteolysis. In some cases the two regulatory mechanisms are combined; N-terminal propeptides co-evolved to facilitate folding of cognate catalytic domains and act as potent inhibitors after cleavage from the catalytic domain (Shinde and Inouye, 1993; Shinde and Thomas, 2011). Subtilases – a ubiquitous super-family of serine proteases – represents an ideal group of homologs to analyze protein adaptation to eukaryotic organelles, since they exist in all three domains of life. Bacterial subtilisin and mammalian proprotein convertase (PC) sub-families constitute the most extensively studied enzymes (Shinde and Thomas, 2011). Despite evolutionary divergence, proteins in these subfamilies display common folds with conserved catalytic triads. Subtilases are almost always expressed as zymogens, with amino and occasionally carboxy propeptide extensions. They are classified into two sub-families; Extracellular Serine Proteases (ESP) and Intracellular Serine Proteases (ISP) (Subbian et al., 2004). ESPs have 80-100 residue long propeptides that catalyze folding and act as inhibitors after cleavage, while ISPs have shorter propeptides that only act as inhibitors in the zymogen. Catalytic domains and propeptides of mammalian PCs are closely related to protease domains of ESPs and not ISPs (Shinde and Thomas, 2011). Similar to bacterial ESPs, propeptides of PCs assist folding and require two ordered steps of proteolytic cleavage for activation. The two proteolytic cleavages are precisely controlled within different organelles. The first cleavage occurs rapidly after protein folding in the endoplasmic reticulum and results in a non-covalent complex between the propeptide and the catalytic domain. Activation requires an additional cleavage within the propeptide; in the case of furin this cleavage occurs only after the protein is trafficked to a different organelle, the trans-golgi-network (TGN) (Anderson et al., 2002). Other PCs are activated in a similar manner, but within different compartments (Seidah and Prat, 2012). Experiments in vitro show that the pH of the TGN is sufficient to trigger the second activating cleavage of furin (Anderson et al., 1997) and that a histidine residue in the propeptide acts as a pH-sensor (Feliciangeli et al., 2006). We recently showed that propeptides of PCs mediate the pH of activation, as swapping propeptides between PCs reassigned the pH of activation (Dillon et al., 2012). We therefore hypothesized that propeptides of eukaryotic subtilases evolved to sense organelle pH in order to direct activation. Such a broad hypothesis is difficult to test experimentally, as it would require biochemical studies on a large number of proteins. We overcame this problem by predicting properties of protein sequences based on our hypothesis, and testing these against sequence databases using statistical methods. Histidine is the only residue with an intrinsic pKa near the physiological range (~6.5) and therefore likely involved in pH-sensing mechanisms. In this paper we show that enrichment of histidines in propeptides correlates with the requirement to sense pH for activation within the subtilase family. Furthermore, we demonstrate similar enrichment in other protease families, indicating that enrichment of histidines in propeptides is a common mechanism to regulate activity in the secretory pathway. RESULTS: Propeptide sequences of subtilases are more divergent than cognate catalytic domains: To identify conserved sequence elements unique to either prokaryotic or eukaryotic subtilases, we performed an evolutionary conservation analysis using the ConSurf server (Ashkenazy et al., 2010). The analysis of prokaryotic subtilisin and eukaryotic proprotein convertase families was initiated using sequences of Subtilisin E and Proprotein Convertase 1/3 (PC1/3), respectively. The resulting conservation scores were mapped on the crystal structure of the propepide:Subtilisin E complex (PDB: 1SCJ) and on a homology model of the propeptide:PC1 complex (based on PDB: 1P8J and 1KN6), respectively (Figure 1). Catalytic domains of eukaryotic and prokaryotic subtilases depict a highly conserved core. On the contrary, propeptides demonstrate less sequence conservation, with the dibasic cleavage motif at the C- terminus of eukaryotic propeptides representing the only conserved region. Since histidine 69 was demonstrated to function as a pH sensor in furin (Feliciangeli et al., 2006), and given that propeptides of furin and PC1/3 alone are sufficient to impart organelle- specific pH-dependent activation of cognate catalytic domains (Dillon et al., 2012), we analyzed whether histidine residues demonstrate any sequence conservation within propeptides. Although we could not identify absolutely conserved histidine residues in propeptides of eukaryotic subtilases, several positions in our alignment contain a histidine residue in a substantial fraction of sequences, especially at the position corresponding to histidine 69 in furin (53.3% of sequences). In contrast, prokaryotic subtilases, which do not traverse the secretory pathway, appear to encode less histidines within their propeptides. However, when catalytic sequences are compared, we find strictly conserved histidine residues within prokaryotic and eukaryotic sequences, and studies indicate that they play essential roles in catalysis or protein stability (Carter and Wells, 1987). Hence, biased enrichment for histidine residues appears