The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.     

Cold-adapted maturation of thermophilic WF146 protease by mimicking the propeptide binding interactions of psychrophilic subtilisin S41

Thermophilic WF146 protease matures efficiently at 60 degrees C, but quite slowly at low temperatures. In this report, seven amino acid residues involved in interactions between the mature domain and the propeptide of the enzyme were substituted by corresponding residues of psychrophilic subtilisin S41 to generate mutant Mut7 (S105G/G107D/Y117E/S136N/V143G/K144E/D145S). Mut3 (S105G/G107D/Y117E) and Mut4 (S136N/V143G/K144E/D145S) were also constructed. Transferring structural features from S41 endowed Mut7 with a remarkably increased maturation rate, as well as an improved caseinolytic activity at 25 degrees C. Moreover, Mut3 and Mut4 each obtained one of the above endowments. Further studies suggest that low-temperature activity and maturation rate are not necessarily linked, and uncoupling structural elements modulating the two properties may be advantageous to cold adaptation.     

Backbone NMR assignments of DFP-inhibited mature subtilisin E

Here we report the backbone chemical shifts of the DFP-inhibited mature subtilisin E, which was uniformly labeled by ¹³C, ¹⁵N with a supplement of excess calcium.     

The precursor to B-type natriuretic peptide is an O-linked glycoprotein

Human pro-B-type natriuretic peptide (proBNP), the precursor for B-type natriuretic peptide (BNP), was expressed in Chinese hamster ovary cells (CHO) and compared by Western blot analysis to BNP cross-reacting material immunoprecipitated from the plasma of heart failure patients. Both recombinant and native forms co-migrated as a diffuse band centered around 25 kDa and were reduced to a 12 kDa species by treatment with a mixture of O-link deglycosylation enzymes. The 108-amino acid CHO-expressed protein was examined by tryptic mapping and LC-MS and found to be an O-linked glycoprotein. Determination of the sites of O-glycosyl addition by blank cycle sequencing of tryptic and Glu-C (Staphylococcus aureus V8 protease) peptides showed that there are seven sites of glycosylation confined to a 36-amino acid residue stretch within the center of the propeptide region. This data is consistent with previous observations of higher molecular weight isoforms of BNP.     

Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor

The gene of Serratia proteamaculans proteinase, protealysin, was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a precursor of 341 amino acids (AAs) with a significant homology to thermolysin-like proteinases (TLPs). The molecular weight of the purified mature active recombinant protein was 32 kDa, the N-terminal amino acid sequence was AKTSTGGEVI. The optimum pH for azocasein hydrolysis by protealysin was seven and it was completely inhibited by o-phenanthroline. The enzyme hydrolyzed 3-(2-furyl)acryloyl-glycyl-L-leucine amide, the standard substrate for TLPs, with k(cat)/K(m) ratio of (2.52 +/- 0.02) x 10(2) M(-1) s(-1). Protealysin maturation removes 50 AA from the N-terminus of the precursor. The removed region had no similarity with the preprosequence of thermolysin (232 AA) but was homologous to some other TLPs. These proteins shared a conserved 7-AA motif near the initial methionine. Such motif was also found in some nonproteolytic putative proteins; two of them were eukaryotic.     

Expression and protein chemistry yielding crystallization of the catalytic domain of ADAM17 complexed with a hydroxamate inhibitor

The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.     

Identification of a chitinase-modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

Chitinase-modifying proteins (cmps) are proteases secreted by fungal pathogens that truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. Here, we report that cmps are secreted by multiple species from the genus Fusarium, that cmp from Fusarium verticillioides (Fv-cmp) is a fungalysin metalloprotease, and that it cleaves within a sequence that is conserved in class IV chitinases. Protein extracts from Fusarium cultures were found to truncate ChitA and ChitB in vitro. Based on this activity, Fv-cmp was purified from F. verticillioides. N-terminal sequencing of truncated ChitA and MALDI-TOF-MS analysis of reaction products showed that Fv-cmp is an endoprotease that cleaves a peptide bond on the C-terminal side of the lectin domain. The N-terminal sequence of purified Fv-cmp was determined and compared with a set of predicted proteins, resulting in its identification as a zinc metalloprotease of the fungalysin family. Recombinant Fv-cmp also truncated ChitA, confirming its identity, but had reduced activity, suggesting that the recombinant protease did not mature efficiently from its propeptide-containing precursor. This is the first report of a fungalysin that targets a nonstructural host protein and the first to implicate this class of virulence-related proteases in plant disease.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Protein-protein interactions among human 20S proteasome subunits and proteassemblin

Immunoproteasomes and standard proteasomes assemble by alternative pathways that bias against the formation of certain "mixed" proteasomes. Differences between beta subunit propeptides contribute to assembly specificity and an assembly chaperone, proteassemblin, may be involved via differential propeptide interactions. We investigated possible mechanisms of biased proteasome assembly and the role of proteassemblin by identifying protein-protein interactions among human 20S proteasome subunits and proteassemblin using a yeast two-hybrid interaction assay. Forty-one interactions were detected, including five involving proteassemblin and contiguous beta subunits, which suggests that proteassemblin binds to preproteasomes via a beta subunit surface. Interaction between proteassemblin and beta5, but not beta5i, suggests that proteassemblin may be involved in the propeptide-dependent differential incorporation of these subunits. Interactions between proteassemblin and beta1, beta1i, and beta7 suggest that proteassemblin may regulate preproteasome dimerization via interactions with the C-termini of these subunits, which in the mature 20S structure extend to contact opposing beta subunit rings.     

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.