Identification of a pH sensor in the furin propeptide that regulates enzyme activation.

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-Arg107/ 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 69HRGVTKR75/, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. 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 His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 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.

D-MEKK1, the Drosophila orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediate the activation of D-JNK by cadmium and arsenite in Schneider cells.

BACKGROUND: The family of c-Jun NH2-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). RESULTS: Using Drosophila melanogaster S2 cells, we demonstrate here that cadmium and arsenite activate Drosophila JNK (D-JNK) via oxidative stress as well, thus providing a simpler model system to study JNK signaling. To elucidate the signaling pathways that lead to activation of D-JNK in response to cadmium or arsenite, we employed RNA interference (RNAi) to knock down thirteen upstream regulators of D-JNK, either singly or in combinations of up to seven at a time. CONCLUSION: D-MEKK1, the fly orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediates the activation of D-JNK by cadmium and arsenite.

Aberrant folding of a mutant Stat5b causes growth hormone insensitivity and proteasomal dysfunction.

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 Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P 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 Stat5bA630P was <15% of Stat5bWT (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 Stat5bA630P 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 Stat5bA630P could produce additional defects through inhibition of proteasome function.

Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin.

Several secreted proteases are synthesized with N-terminal propeptides that function as intramolecular chaperones (IMCs) and direct the folding of proteases to their native functional states. Using subtilisin E as our model system, we had earlier established that (i) release and degradation of the IMC from its complex with the protease upon completion of folding is the rate-determining step to protease maturation and, (ii) IMC of SbtE is an extremely charged, intrinsically unstructured polypeptide that adopts an alpha-beta structure only in the presence of the protease. Here, we explore the mechanism of IMC release and the intricate relationship between IMC structure and protease activation. We establish that the release of the first IMC from its protease domain is a non-deterministic event that subsequently triggers an activation cascade through trans-proteolysis. By in silico simulation of the protease maturation pathway through application of stochastic algorithms, we further analyze the sub-stages of the release step. Our work shows that modulating the structure of the IMC domain through external solvent conditions can vary both the time and randomness of protease activation. This behavior of the protease can be correlated to varying the release-rebinding equilibrium of IMC, through simulation. Thus, a delicate balance underlies IMC structure, release, and protease activation. Proteases are ubiquitous enzymes crucial for fundamental cellular processes and require deterministic activation mechanisms. Our work on SbtE establishes that through selection of an intrinsically unstructured IMC domain, nature appears to have selected for a viable deterministic handle that controls a fundamentally random event. While this outlines an important mechanism for regulation of protease activation, it also provides a unique approach to maintain industrially viable subtilisins in extremely stable states that can be activated at will.

Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases.

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.

Folding pathway mediated by an intramolecular chaperone. A functional peptide chaperone designed using sequence databases.

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 wild-type 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 alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. 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.

Folding pathway mediated by an intramolecular chaperone: dissecting conformational changes coincident with autoprocessing and the role of Ca(2+) in subtilisin maturation.

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.