The nature of the ligand's side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex.

The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp3-hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of CPB, the relative orientation of the catalytic amino acid residues, as well as the distance between Glu270 and SArg/SPhe, is much less dependent on the nature of the corresponding side chain of the substrate. The influence of the nature of the substrate side chain on the structural organization of the transition state determines catalytic activity and broad substrate specificity of the carboxypeptidase T.

Familial Cancer Variant Prioritization Pipeline version 2 (FCVPPv2) applied to a papillary thyroid cancer family.

Whole-genome sequencing methods in familial cancer are useful to unravel rare clinically important cancer predisposing variants. Here, we present improvements in our pedigree-based familial cancer variant prioritization pipeline referred as FCVPPv2, including 12 tools for evaluating deleteriousness and 5 intolerance scores for missense variants. This pipeline is also capable of assessing non-coding regions by combining FANTOM5 data with sets of tools like Bedtools, ChromHMM, Miranda, SNPnexus and Targetscan. We tested this pipeline in a family with history of a papillary thyroid cancer. Only one variant causing an amino acid change G573R (dbSNP ID rs145736623, NM_019609.4:exon11:c.G1717A:p.G573R) in the carboxypeptidase gene CPXM1 survived our pipeline. This variant is located in a highly conserved region across vertebrates in the peptidase_M14 domain (Pfam ID PF00246). The CPXM1 gene may be involved in adipogenesis and extracellular matrix remodelling and it has been suggested to be a tumour suppressor in breast cancer. However, the presence of the variant in the ExAC database suggests it to be a rare polymorphism or a low-penetrance risk allele. Overall, our pipeline is a comprehensive approach for prediction of predisposing variants for high-risk cancer families, for which a functional characterization is a crucial step to confirm their role in cancer predisposition.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1'). The mitochondrial processing activity involving cleavage of peptides lacking P1' proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Identification of carboxypeptidase X (CPX)-1 as a positive regulator of adipogenesis.

Adipose tissue expansion occurs through a combination of hypertrophy of existing adipocytes and generation of new adipocytes via the process of hyperplasia, which involves the proliferation and subsequent differentiation of preadipocytes. Deficiencies in hyperplasia contribute to adipose tissue dysfunction and the association of obesity with chronic cardiometabolic diseases. Thus, increased understanding of hyperplastic pathways may be expected to afford novel therapeutic strategies. We have reported that fibroblast growth factor (FGF)-1 promotes proliferation and differentiation of human preadipocytes and recently demonstrated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a central, proximal effector. Herein, we describe the identification and characterization of carboxypeptidase X (CPX)-1, a secreted collagen-binding glycoprotein, as a novel downstream effector in human primary and Simpson-Golabi-Behmel syndrome preadipocytes. CPX-1 expression increased after treatment of preadipocytes with FGF-1, BAMBI knockdown, or induction of differentiation. CPX-1 knockdown compromised preadipocyte differentiation coincident with reduced collagen expression. Furthermore, preadipocytes differentiated on matrix derived from CPX-1 knockdown cells exhibited reduced Glut4 expression and insulin-stimulated glucose uptake. Finally, CPX-1 expression was increased in adipose tissue from obese mice and humans. Collectively, these findings establish CPX-1 as a positive regulator of adipogenesis situated downstream of FGF-1/BAMBI that may contribute to hyperplastic adipose tissue expansion via affecting extracellular matrix remodeling.-Kim, Y.-H., Barclay, J. L., He, J., Luo, X., O'Neill, H. M., Keshvari, S., Webster, J. A., Ng, C., Hutley, L. J., Prins, J. B., Whitehead, J. P. Identification of carboxypeptidase X (CPX)-1 as a positive regulator of adipogenesis.

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein.

Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1. Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2. Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen. Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets. This polymerization occurs under near-physiological conditions, is rapid, and can be controlled by regulating calcium in solution. Fusion of the RTX domain to a soluble protein results in the incorporation of engineered protein function into these macromolecular assemblies. Applications of this protein sequence in bacterial adherence and colonization and the generation of biomaterials are discussed.

The molybdenum cofactor.

The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.

Increase in larval gut proteolytic activities and Bti resistance in the Dengue fever mosquito.

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.

Dipeptidyl peptidase III: a multifaceted oligopeptide N-end cutter.

Dipeptidyl peptidase III (DPP III), the sole member and representative of the M49 family of metallopeptidases, is a zinc-dependent aminopeptidase. It sequentially hydrolyses dipeptides from the N-terminal of oligopeptides ranging from three to 10 amino acid residues. Although implicated in an array of pathophysiological phenomena, the precise function of this peptidase is still unclear. However, a number of studies advocate its contribution in terminal stages of protein turnover. Altered expression of DPP III which suggests its involvement in primary ovarian carcinoma, oxidative stress (Nrf2 nuclear localization), pain, inflammation and cataractogenesis has recently led to resurgence of interest in delineating the role of the peptidase in these pathophysiological processes. This review article intends to bring forth the latest updates in this arena which may serve as a base for future studies on the peptidase.

Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization.

The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.

Effects of hydroxamate metalloendoprotease inhibitors on botulinum neurotoxin A poisoned mouse neuromuscular junctions.

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K(i) for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 muM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 muM DCH or 5 muM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40-90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 degrees C compared to 37 degrees C. Unlike DAP, neither DCH nor ABS 130 increased Ca(2+) levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca(2+) levels within motor nerve endings.

QM/MM X-ray refinement of zinc metalloenzymes.

Zinc metalloenzymes play an important role in biology. However, due to the limitation of molecular force field energy restraints used in X-ray refinement at medium or low resolutions, the precise geometry of the zinc coordination environment can be difficult to distinguish from ambiguous electron density maps. Due to the difficulties involved in defining accurate force fields for metal ions, the QM/MM (quantum-mechanical/molecular-mechanical) method provides an attractive and more general alternative for the study and refinement of metalloprotein active sites. Herein we present three examples that indicate that QM/MM based refinement yields a superior description of the crystal structure based on R and R(free) values and on the inspection of the zinc coordination environment. It is concluded that QM/MM refinement is an useful general tool for the improvement of the metal coordination sphere in metalloenzyme active sites.

Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain.

Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic P. fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence.

Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N.

Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel digestion with a second enzyme. We have benchmarked a relatively obscure basidomycete-derived zinc metalloendopeptidase, Lys-N, that selectively cleaves the amide bond N-terminal of lysine residues. We have found that Lys-N digestion yields peptides with easily assigned CID spectra. Using a mixture of purified proteins as well as a complex yeast lysate, we have shown that Lys-N efficiently digests all proteins at the predicted sites of cleavage. Shotgun proteomics analyses of Lys-N digests of both the standard mixture and yeast lysate yielded peptide and protein identification numbers that were generally comparable to trypsin digestion, whereas the combination data from Lys-N and trypsin digestion substantially enhanced protein coverage. During CID fragmentation, the additional amino terminal basicity enhanced b-ion intensity which was reflected in long b-ion tags that were particularly pronounced during CID in a quadrupole. Finally, immonium ion peaks produced from Lys-N digested peptides originate from the carboxy terminus in contrast to tryptic peptides where immonium ions originate from the amino terminus.

Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2.

L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.

Zinc regulation of aminopeptidase B involved in neuropeptide production.

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B.

Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.

Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.

Modeling and functional analysis of AEBP1, a transcriptional repressor.

Adipocyte enhancer binding protein 1 (AEBP1) is a transcriptional repressor of the aP2 gene, which encodes the adipocyte lipid binding protein and is involved in the differentiation of preadipocytes into mature adipocytes. It is an isoform of aortic carboxypeptidase-like protein (ACLP), which is a part of the extracellular matrix. AEBP1 and ACLP contain a conserved carboxypeptidase domain which is critical for the function of AEBP1 as a transcriptional repressor. Homology modeling and multiple alignment of AEBP1 homologues were performed to identify putative domains and critical residues that were then deleted or mutated in mouse AEBP1. Expression of wild-type and mutant AEBP1 proteins in CHO cells was performed, and their function in transcriptional repression was assayed by luciferase assay. All deletion forms of AEBP1 were able to repress transcription driven by the aP2 promoter. The DNA binding domain of AEBP1 was mapped by electrophoretic mobility shift assays to a region of the C-terminus rich in basic residues. However, wild-type AEBP1 was not able to interact strongly with DNA, suggesting that AEBP1 might function predominantly as a corepressor, independent of DNA binding. AEBP1 was also found to interact with Ca2+/calmodulin through this basic region, suggesting another mechanism of functional regulation.

Elucidation of CPX-1 involvement in RANKL-induced osteoclastogenesis by a proteomics approach.

To identify proteins potentially involved in osteoclast differentiation, we conducted a proteomics-based analysis using the osteoclastogenesis model cell line RAW264.7. Total proteins from undifferentiated cells, committed pre-osteoclasts, and differentiated osteoclasts were resolved by two-dimensional gel electrophoresis. Protein spots showing differential expression levels were processed for peptide mass fingerprinting. Among them, we identified the metallocarboxypeptidase CPX-1, which was prominently increased in pre-osteoclasts and then decreased in mature osteoclasts. Results of reverse transcription polymerase chain reaction, Western blot, and confocal microscopy were in agreement with the proteomics data. Notably, the forced overexpression of CPX-1 led to the inhibition of osteoclast formation, but not pre-osteoclast generation. Therefore, the transient up-regulation pattern of CPX-1 expression may be important for the successful progression from pre-osteoclasts to mature osteoclasts.