Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to early lethality in mice.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either as a homodimer, or as a more active BMP4/7 heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of BMP4 homodimers or heterodimers generated from BMP4, BMP4S91C or BMP4E93G precursor proteins in Xenopus embryos and found that these mutations reduce the activity of BMP4 homodimers but not heterodimers. We generated Bmp4 S91C and Bmp4 E93G knock-in mice and found that Bmp4 S91C/S91C mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart at E10.5. Most Bmp4 E93G/E93G mice die before weaning and Bmp4 -/E93G mutants die prenatally with reduced or absent eyes, heart and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from Bmp4 S91C and Bmp4 E93G embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because Bmp7 is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of cleaved ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.

The prodomain of bone morphogenetic protein 2 promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus. We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides.

The neurotrophin nerve growth factor (NGF) and its precursor proNGF are both bioactive and exert similar or opposite actions depending on the cell target and its milieu. The balance between NGF and proNGF is crucial for cell and tissue homeostasis and it is considered an indicator of pathological conditions. Proteolytical cleavage of proNGF to the mature form results in different fragments, whose function and/or bioactivity is still unclear. The present study was conducted to investigate the distribution of proNGF fragments derived from endogenous cleavage in brain and peripheral tissues of adult rats in the healthy condition and following inflammatory lipopolysaccharide (LPS) challenge. Different anti-proNGF antibodies were tested and the presence of short peptides corresponding to the prodomain sequence (pdNGFpep) was identified. Processing of proNGF was found to be tissue-specific and accumulation of pdNGFpeps was found in inflamed tissues, mainly in testis, intestine and heart, suggesting a possible correlation between organ functions and a response to insults and/or injury. The bioactivity of pdNGFpep was also demonstrated in vitro by using primary hippocampal neurons. Our study supports a biological function for the NGF precursor prodomain and indicates that short peptides from residues 1-60, differing from the 70-110 sequence, induce apoptosis, thereby opening the way for identification of new molecular targets to study pathological conditions.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Cardiomyopathy related desmocollin-2 prodomain variants affect the intracellular cadherin transport and processing.

Background: Arrhythmogenic cardiomyopathy can be caused by genetic variants in desmosomal cadherins. Since cardiac desmosomal cadherins are crucial for cell-cell-adhesion, their correct localization at the plasma membrane is essential. Methods: Nine desmocollin-2 variants at five positions from various public genetic databases (p.D30N, p.V52A/I, p.G77V/D/S, p.V79G, p.I96V/T) and three additional conserved positions (p.C32, p.C57, p.F71) within the prodomain were investigated in vitro using confocal microscopy. Model variants (p.C32A/S, p.V52G/L, p.C57A/S, p.F71Y/A/S, p.V79A/I/L, p.I96l/A) were generated to investigate the impact of specific amino acids. Results: We revealed that all analyzed positions in the prodomain are critical for the intracellular transport. However, the variants p.D30N, p.V52A/I and p.I96V listed in genetic databases do not disturb the intracellular transport revealing that the loss of these canonical sequences may be compensated. Conclusion: As disease-related homozygous truncating desmocollin-2 variants lacking the transmembrane domain are not localized at the plasma membrane, we predict that some of the investigated prodomain variants may be relevant in the context of arrhythmogenic cardiomyopathy due to disturbed intracellular transport.

Dynamics of the personalities of PCSK9 on missense variants (rs505151 and rs562556) from elderly cohort studies in Brazil.

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptors (LDLR). Gain-of-function (GOF) variants of PCSK9 significantly affects lipid metabolism leading to coronary artery disease (CAD), owing to the raising the plasma low-density lipoprotein (LDL). Considering the public health matter, large-scale genomic studies have been conducted worldwide to provide the genetic architecture of populations for the implementation of precision medicine actions. Nevertheless, despite the advances in genomic studies, non-European populations are still underrepresented in public genomic data banks. Despite this, we found two high-frequency variants (rs505151 and rs562556) in the ABraOM databank (Brazilian genomic variants) from a cohort SABE study conducted in the largest city of Brazil, São Paulo. Here, we assessed the structural and dynamical features of these variants against WT through a molecular dynamics study. We sought fundamental dynamical interdomain relations through Perturb Response Scanning (PRS) and we found an interesting change of dynamical relation between prodomain and Cysteine-Histidine-Rich-Domain (CHRD) in the variants. The results highlight the pivotal role of prodomain in the PCSK9 dynamic and the implications for the development of new drugs depending on patient group genotype.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-ß precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Molecular Mechanisms of AMH Signaling.

Anti-Müllerian Hormone (AMH) is a secreted glycoprotein hormone with critical roles in reproductive development and regulation. Its chemical and mechanistic similarities to members of the Transforming Growth Factor ß (TGF-ß) family have led to its placement within this signaling family. As a member of the TGF-ß family, AMH exists as a noncovalent complex of a large N-terminal prodomain and smaller C-terminal mature signaling domain. To produce a signal, the mature domain will bind to the extracellular domains of two type I and two type II receptors which results in an intracellular SMAD signal. Interestingly, as will be discussed in this review, AMH possesses several unique characteristics which set it apart from other ligands within the TGF-ß family. In particular, AMH has a dedicated type II receptor, Anti-Müllerian Hormone Receptor Type II (AMHR2), making this interaction intriguing mechanistically as well as therapeutically. Further, the prodomain of AMH has remained largely uncharacterized, despite being the largest prodomain within the family. Recent advancements in the field have provided valuable insight into the molecular mechanisms of AMH signaling, however there are still many areas of AMH signaling not understood. Herein, we will discuss what is known about the biochemistry of AMH and AMHR2, focusing on recent advances in understanding the unique characteristics of AMH signaling and the molecular mechanisms of receptor engagement.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both - 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

The anti-Müllerian hormone prodomain is displaced from the hormone/prodomain complex upon bivalent binding to the hormone receptor.

Noncovalent complexes of transforming growth factor-ß family growth/differentiation factors with their prodomains are classified as latent or active, depending on whether the complexes can bind their respective receptors. For the anti-Müllerian hormone (AMH), the hormone-prodomain complex is active, and the prodomain is displaced upon binding to its type II receptor, AMH receptor type-2 (AMHR2), on the cell surface. However, the mechanism by which this displacement occurs is unclear. Here, we used ELISA assays to measure the dependence of prodomain displacement on AMH concentration and analyzed results with respect to the behavior expected for reversible binding in combination with ligand-induced receptor dimerization. We found that, in solution, the prodomain has a high affinity for the growth factor (GF) (Kd = 0.4 pM). Binding of the AMH complex to a single AMHR2 molecule does not affect this Kd and does not induce prodomain displacement, indicating that the receptor binding site in the AMH complex is fully accessible to AMHR2. However, recruitment of a second AMHR2 molecule to bind the ligand bivalently leads to a 1000-fold increase in the Kd for the AMH complex, resulting in rapid release of the prodomain. Displacement occurs only if the AMHR2 is presented on a surface, indicating that prodomain displacement is caused by a conformational change in the GF induced by bivalent binding to AMHR2. In addition, we demonstrate that the bone morphogenetic protein 7 prodomain is displaced from the complex with its GF by a similar process, suggesting that this may represent a general mechanism for receptor-mediated prodomain displacement in this ligand family.

The role of pro-domains in human growth factors and cytokines.

Many growth factors and cytokines are produced as larger precursors, containing pro-domains, that require proteolytic processing to release the bioactive ligand. These pro-domains can be significantly larger than the mature domains and can play an active role in the regulation of the ligands. Mining the UniProt database, we identified almost one hundred human growth factors and cytokines with pro-domains. These are spread across several unrelated protein families and vary in both their size and composition. The precise role of each pro-domain varies significantly between the protein families. Typically they are critical for controlling bioactivity and protein localisation, and they facilitate diverse mechanisms of activation. Significant gaps in our understanding remain for pro-domain function - particularly their fate once the bioactive ligand has been released. Here we provide an overview of pro-domain roles in human growth factors and cytokines, their processing, regulation and activation, localisation as well as therapeutic potential.

Identification and Clinical Associations of 3 Forms of Circulating T-cadherin in Human Serum.

CONTEXT: T-cadherin (T-cad) is a glycosylphosphatidylinositol (GPI)-anchored cadherin that mediates adiponectin to induce exosome biogenesis and secretion, protect cardiovascular tissues, promote muscle regeneration, and stimulate therapeutic heart protection by transplanted mesenchymal stem cells. CDH13, the gene locus of T-cad, affects plasma adiponectin levels most strongly, in addition to affecting cardiovascular disease risk and glucose homeostasis. Recently, it has been suggested that T-cad exists in human serum, although the details are still unclear. OBJECTIVE: To validate the existence of T-cad forms in human serum and investigate the association with clinical parameters of type 2 diabetes patients. METHODS: Using newly developed monoclonal antibodies against T-cad, pooled human serum was analyzed, and novel T-cad enzyme-linked immunosorbent assays (ELISAs) were developed. The serum T-cad concentrations of 183 Japanese type 2 diabetes patients were measured in a cross-sectional observational study. The main outcome measure was the existence of soluble T-cad in human serum. RESULTS: There were 3 forms of soluble T-cad: a 130-kDa form with a prodomain, a 100-kDa mature form, and a 30-kDa prodomain in human serum. Using newly developed ELISAs to measure them simultaneously, we found that the 130-kDa form of T-cad positively correlated with plasma adiponectin (r = 0.28, P < .001), although a physiological interaction with adiponectin was not observed in serum. The unique 30-kDa prodomain was associated with several clinical parameters in diabetes patients. CONCLUSION: We identified 3 novel forms of soluble T-cad. Their importance as disease markers and/or biomarkers of adiponectin function and the possible bioactivity of the respective molecules require further investigation.

Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase.

BACKGROUND: Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. RESULTS: A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. CONCLUSIONS: We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

Structure of the Plasmodium falciparum PfSERA5 pseudo-zymogen.

PfSERA5, a significantly abundant protein present within the parasitophorous vacuole (PV) and essential for normal growth during the blood-stage life cycle of the malaria parasite Plasmodium falciparum, displays structural similarity to many other cysteine proteases. However, PfSERA5 does not exhibit any detectable protease activity and therefore the role of the PfSERA5 papain-like domain (PfSERA5E), thought to remain bound to its cognate prodomain, remains unknown. In this study, we present a revised structure of the central PfSERA5E domain at a resolution of 1.2 Å, and the first structure of the "zymogen" of this papain-like domain including its cognate prodomain (PfSERA5PE) to 2.2 Å resolution. PfSERA5PE is somewhat structurally similar to that of other known proenzymes, retaining the conserved overall folding and orientation of the prodomain through, and occluding, the archetypal papain-like catalytic triad "active-site" cleft, in the same reverse direction as conventional prodomains. Our findings are congruent with previously identified structures of PfSERA5E and of similar "zymogens" and provide a foundation for further investigation into the function of PfSERA5.

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis.

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Comparing sequence and structure of falcipains and human homologs at prodomain and catalytic active site for malarial peptide based inhibitor design.

BACKGROUND: Falcipains are major cysteine proteases of Plasmodium falciparum involved in haemoglobin degradation and remain attractive anti-malarial drug targets. Several inhibitors against these proteases have been identified, yet none of them has been approved for malaria treatment. Other Plasmodium species also possess highly homologous proteins to falcipains. For selective therapeutic targeting, identification of sequence and structure differences with homologous human cathepsins is necessary. The substrate processing activity of these proteins is tightly controlled via a prodomain segment occluding the active site which is chopped under low pH conditions exposing the catalytic site. Current work characterizes these proteases to identify residues mediating the prodomain regulatory function for the design of peptide based anti-malarial inhibitors. METHODS: Sequence and structure variations between prodomain regions of plasmodial proteins and human cathepsins were determined using in silico approaches. Additionally, evolutionary clustering of these proteins was evaluated using phylogenetic analysis. High quality partial zymogen protein structures were modelled using homology modelling and residue interaction analysis performed between the prodomain segment and mature domain to identify key interacting residues between these two domains. The resulting information was used to determine short peptide sequences which could mimic the inherent regulatory function of the prodomain regions. Through flexible docking, the binding affinity of proposed peptides on the proteins studied was evaluated. RESULTS: Sequence, evolutionary and motif analyses showed important differences between plasmodial and human proteins. Residue interaction analysis identified important residues crucial for maintaining prodomain integrity across the different proteins as well as the pro-segment responsible for inhibitory mechanism. Binding affinity of suggested peptides was highly dependent on their residue composition and length. CONCLUSIONS: Despite the conserved structural and catalytic mechanism between human cathepsins and plasmodial proteases, current work revealed significant differences between the two protein groups which may provide valuable information for selective anti-malarial inhibitor development. Part of this study aimed to design peptide inhibitors based on endogenous inhibitory portions of protease prodomains as a novel aspect. Even though peptide inhibitors may not be practical solutions to malaria at this stage, the approach followed and results offer a promising means to find new malarial inhibitors.