The folding and activation of furin occur through two pH-and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg 107 2 generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69 HRGVTKR 75 2, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His 69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His 69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg 107 . Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2-and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His 69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg 75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.The pH gradient formed by the various membranous compartments comprising the secretory and endocytic pathways has essential and manifold roles ranging from the regulation of protein traffic to the control of protein conformation and enzyme activities, including the processing of prohormones and proproteins by proprotein convertases (PCs). 2 The PCs, a family of calcium-dependent serine endoproteases, cleave proproteins and prohormones at doublets or clusters of basic amino acids thus generating mature bioactive proteins as well as hormonal processing intermediates that require additional post-translational modifications to gain full bioactivity (1, 2). The proteolytic maturation of prohormones by the neuroendocrine-specific PC1/3 and PC2 requires the acidic pH of maturing secretory granules (3-5), whereas the proteolytic activation of proprotein substrates by the ubiquitously expressed PC furin in the trans-Golgi network (TGN)/endosomal system is compartment-and hence pH-specific (2, 6). This compartmentspecific processing by furin is mediated in part by the pH-dependent changes in the conformation of furin substrates as well as the utilization of pH-sensitive furin cleavage sites. Furin efficiently cleaves a number of proprotein substrates at the consensus site -Arg-X-Lys/Arg-Arg-2 (2). Kinetic studies show that the P1 and P4 Arg residues are required for the efficient processing of furin substrates, whereas the P2 Arg has a modulatory role (7,8). However, at acidic pH the absence of a P2 or P4 Arg can be compensated for by the presence of positively charged residues at P6 or possibly adjacen...

No abstract

Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca(2+) binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca(2+) ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca(2+) ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links alpha-helix-2 and beta-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca(2+) binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.

Subtilisin E (SbtE) is a member of the ubiquitous superfamily of serine proteases called subtilases and serves as a model for understanding propeptide-mediated protein folding mechanisms. Unlike most proteins that adopt thermodynamically stable conformations, the native state of SbtE is trapped into a kinetically stable conformation. While kinetic stability offers distinct functional advantages to the native state, the constraints that dictate the selection between kinetic and thermodynamic folding and stability remain unknown. Using highly conserved subtilases, we demonstrate that adaptive evolution of sequence dictates selection of folding pathways. Intracellular and extracellular serine proteases (ISPs and ESPs, respectively) constitute two subfamilies within the family of subtilases that have highly conserved sequences, structures, and catalytic activities. Our studies on the folding pathways of subtilisin E (SbtE), an ESP, and its homologue intracellular serine protease 1 (ISP1), an ISP, show that although topology, contact order, and hydrophobicity that drive protein folding reactions are conserved, ISP1 and SbtE fold through significantly different pathways and kinetics. While SbtE absolutely requires the propeptide to fold into a kinetically trapped conformer, ISP1 folds to a thermodynamically stable state more than 1 million times faster and independent of a propeptide. Furthermore, kinetics establish that ISP1 and SbtE fold through different intermediate states. An evolutionary analysis of folding constraints in subtilases suggests that observed differences in folding pathways may be mediated through positive selection of specific residues that map mostly onto the protein surface. Together, our results demonstrate that closely related subtilases can fold through distinct pathways and mechanisms, and suggest that fine sequence details can dictate the choice between kinetic and thermodynamic folding and stability.

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5b A630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were ϳ3-fold higher than Stat5b A630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5b A630P was <15% of Stat5b WT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5b A630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5b A630P could produce additional defects through inhibition of proteasome function.Sequence-specific transcription factors are modular proteins containing distinct domains that mediate their actions, including the ability to move among different subcellular compartments, to bind to DNA in chromatin in the nucleus, and to interact with transcriptional co-activators, co-repressors, and other regulatory molecules (1). The Stat 2 family consists of seven related proteins (Stats 1, 2, 3, 4, 5a, 5b, and 6) that are responsible for many of the transcriptional effects of cytokines, growth factors, and hormones (2, 3). Stat proteins are found in latent form in the cytoplasm of unstimulated cells. They are activated upon ligand binding to its receptor by a series of steps consisting of receptorinitiated tyrosine phosphorylation, dimerization, transport into the nucleus, binding to DNA response elements on target genes, and recruitment of a complex of co-activator proteins that stimulate transcription (2, 3).Growth hormone (GH) plays a central role in regulating somatic growth and intermediary metabolism in many vertebrate species, including humans (4). Upon binding to its transmembrane receptor, GH triggers the activation of the receptor-associated tyrosine-protein kinase Jak2, which in addition to recruiting a variety of other signaling molecules leads to the activation of Stats 1, 3, 5a, and 5b (4, 5). Many ...

Background: The family of c-Jun NH 2 -terminal kinases (JNK) plays important roles in embryonic development and in cellular responses to stress. Toxic metals and their compounds are potent activators of JNK in mammalian cells. The mechanism of mammalian JNK activation by cadmium and sodium arsenite involves toxicant-induced oxidative stress. The study of mammalian signaling pathways to JNK is complicated by the significant degree of redundancy among upstream JNK regulators, especially at the level of JNK kinase kinases (JNKKK).

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or ''intramolecular chaperones'' to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wildtype propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined ␣؊␤ conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT⅐subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.Subtilisin E is an alkaline serine protease isolated from Bacillus subtilis (1). In vivo this protein is produced as pre-prosubtilisin (2), wherein the pre-region (signal peptide) facilitates protein secretion and the pro-region (propeptide) functions as an intramolecular chaperone that guides correct folding of subtilisin (2-6). Upon completion of folding, the propeptide is autoproteolytically removed (7). This is necessary because the propeptide is a potent inhibitor of subtilisin activity (4).Propeptide-mediated folding mechanisms exist in several unrelated protease families. However, there is no sequence conservation in propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a common mechanism of action (9). Subtilisin (2-6) and ␣-lytic protease (10 -13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models for the subtilase family that spans prokaryotes and eukaryotes. Amino acid sequence analysis of this family establishes that alth...

Efficient and precise assembly of polypeptides into native functional states is critical for normal cellular processes. Understanding how a specific structure is encoded in the polypeptide sequence and what drives the structural progression to the native state is essential to deciphering the folding problem. Several prokaryotic and eukaryotic proteins require their propeptide-domains to function as dedicated intramolecular chaperones (IMCs). In this manuscript, we investigate the elementary steps in the IMC mediated maturation of Subtilisin E, a bacterial serine protease, and a prototype for the eukaryotic proprotein convertases (PCs). Through detailed analyses, we have attempted to define the unimolecular folding energy landscape for SbtE to understand how the stabilization of folding intermediates influences the maturation process, an aspect that is difficult to study in eukaryotic PCs. Our studies demonstrate that a rapid hydrophobic collapse precedes acquisition of tertiary structure during the folding of Pro-SbtE and results in formation of a moltenglobule like intermediate. Induction of structure within the IMC stabilizes both the molten globule-like folding intermediate and the native state, and appears to expedite initial stages of folding, purely through thermodynamic stabilization of the folded state. While the induced structure does not affect the activation energies in the unimolecular folding reaction, it is detrimental to the autoproteolytic cleavage of the precursor and subsequent release and degradation of the inhibitory IMC-domain since both these stages require some degree of unfolding. Completion of Pro-SbtE maturation results in the formation of a kinetically trapped and extremely stable native state. Hence, our results suggest that the SbtE IMC appears to have evolved to be intrinsically unstructured and to bind with its cognate protease with a specific affinity that is critical for biological regulation. E. Subbian et al. 74