Search

Vasohibin-1 and small vasohibin-binding protein (SVBP) form an intermolecular heterotetramer in the crystal. The heterotetramer was stabilized by exchange of the conserved N-terminal region., Vasohibins regulate angiogenesis, tumor growth, metastasis and neuronal differentiation. They form a complex with small vasohibin-binding protein (SVBP) and show tubulin tyrosine carboxypeptidase activity. Recent crystal structure determinations of vasohibin–SVBP complexes have provided a molecular basis for complex formation, substrate binding and catalytic activity. However, the regulatory mechanism and dynamics of the complex remain elusive. Here, the crystal structure of the VASH1–SVBP complex and a molecular-dynamics simulation study are reported. The overall structure of the complex was similar to previously reported structures. Importantly, however, the structure revealed a domain-swapped heterotetramer that was formed between twofold symmetry-related molecules. This heterotetramerization was stabilized by the mutual exchange of ten conserved N-terminal residues from the VASH1 structural core, which was intramolecular in other structures. Interestingly, a comparison of this region with previously reported structures revealed that the patterns of hydrogen bonding and hydrophobic interactions vary. In the molecular-dynamics simulations, differences were found between the heterotetramer and heterodimer, where the fluctuation of the N-terminal region in the heterotetramer was suppressed. Thus, heterotetramer formation and flexibility of the N-terminal region may be important for enzyme activity and regulation.

Familial Alzheimer’s disease (FAD) is a rare early-onset genetic form of common dementia of old age. Striking in middle age, FAD is caused by missense mutations in three genes: APP (encoding the amyloid precursor protein) and PSEN1 and PSEN2 (encoding presenilin-1 and presenilin-2). APP is proteolytically processed successively by β-secretase and γ-secretase to produce the amyloid β-peptide (Aβ). Presenilin is the catalytic component of γ-secretase, a membrane-embedded aspartyl protease complex that cleaves APP within its single transmembrane domain to produce Aβ of varying lengths. Thus, all FAD mutations are found in the substrate and the enzyme that produce Aβ. The 42-residue variant Aβ42 has been the primary focus of Alzheimer drug discovery for over two decades, as this particular peptide is highly prone to aggregation, is the major protein deposited in the characteristic cerebral plaques of Alzheimer’s disease and is proportionately elevated in FAD. Despite extensive efforts, all agents targeting Aβ and Aβ42 have failed in the clinic, including γ-secretase inhibitors, leading to questioning of the amyloid hypothesis of Alzheimer pathogenesis. However, processing of the APP transmembrane domain by γ-secretase is complex, involving initial endoproteolysis followed by successive carboxypeptidase trimming steps to secreted Aβ peptides such as Aβ42. Recent findings reveal that FAD mutations in PSEN1 and in APP result in the deficient trimming of initially formed long Aβ peptides. A logical drug discovery strategy for FAD could therefore involve the search for compounds that rescue this deficient carboxypeptidase activity. The rare early-onset FAD arguably presents a simpler path to developing effective therapeutics compared to the much more complex heterogeneous sporadic Alzheimer’s disease.

Background and Aims Serum diagnostic markers of early-stage pancreatic ductal adenocarcinoma (PDAC) are needed, especially for stage I disease. As tumors grow and cause pancreatic atrophy, markers derived from pancreatic parenchyma such as serum carboxypeptidase A (CPA) activity lose diagnostic performance. We evaluated, with CA19-9, serum CPA as a marker of early pancreatic cancer. Methods Serum CPA activity levels were measured in 345 controls undergoing pancreatic surveillance, divided into 2 sets, set 1 being used to establish a reference range. Variants within the CPA1 locus were sought for their association with pancreatic CPA1 expression to determine if such variants associated with serum CPA levels. A total of 190 patients with resectable PDAC were evaluated. Results Among controls, those having 1 or more minor alleles of CPA1 variants rs6955723 or rs2284682 had significantly higher serum CPA levels than did those without (P = .001). None of the PDAC cases with pancreatic atrophy had an elevated CPA. Among 122 PDAC cases without atrophy, defining serum CPA diagnostic cutoffs by a subject’s CPA1 variants yielded a diagnostic sensitivity of 18% at 99% specificity (95% confidence interval [CI], 11.7–26) (vs 11.1% sensitivity using a uniform diagnostic cutoff); combining CPA with variant-stratified CA19-9 yielded a sensitivity of 68.0% (95% CI, 59.0–76.2) vs 63.1% (95% CI, 53.9– 71.7) for CA19-9 alone; and among stage I PDAC cases, diagnostic sensitivity was 51.9% (95% CI, 31.9–71.3) vs 37.0% (95% CI, 19.4–57.6) for CA19-9 alone. In the validation control set, the variant-stratified diagnostic cutoff yielded a specificity of 98.2%. Conclusion Serum CPA activity has diagnostic utility before the emergence of pancreatic atrophy as a marker of localized PDAC, including stage I disease.

Gram-negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM protecting cells from turgor and environmental stress conditions. In several bacteria including E. coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun lipoprotein) that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery Lpp has been studied extensively and although L,D-transpeptidases, the enzymes that catalyse the formation of PG–Lpp linkages have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly-identified paralog of L,D-transpeptidases in E. coli is a murein hydrolytic enzyme that catalyses cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF is earlier presumed to be an L,D-transpeptidase; however, our results show that it is indeed an L,D-endopeptidase that hydrolyses the products generated by the L,D-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG–Lpp cross-links suggesting a role for LdtF in regulation of PG-OM linkages to maintain the structural integrity of the bacterial cell envelope.Significance statementBacterial cell walls contain a unique protective exoskeleton, peptidoglycan, which is a target of several clinically important antimicrobials. In Gram-negative bacteria, peptidoglycan is covered by an additional lipid layer, outer membrane that serves as permeability barrier against entry of toxic molecules. In some bacteria, an extremely abundant lipoprotein, Lpp staples outer membrane to peptidoglycan to maintain the structural integrity of the cell envelope. In this study, we identify a previously unknown peptidoglycan hydrolytic enzyme that cleaves Lpp from the peptidoglycan sacculus and show how the outer membrane-peptidoglycan linkages are modulated in Escherichia coli. Overall, this study helps in understanding the fundamental bacterial cell wall biology and in identification of alternate drug targets for development of new antimicrobials.

Cysteine cathepsins are peptidases with housekeeping functions that play different specific roles in different tissues. Endogenous peptidase inhibitors, such as cystatins and thyropins are the ultimate way of controlling their activity. It appears, however, that cathepsin X, a monocarboxypeptidase, whose overexpression is associated with several patholog- ical processes, is not under the control of endogenous inhibitors. Inhibitors belonging to various groups inhibit other cathepsins tested, but none decrease the carboxypeptidase activity of cathepsin X. This absence of inhibitor control is another feature that distinguishes cathepsin X from other members of the cysteine peptidases. Cisteinski katepsini so peptidaze z vzdrževalno vlogo in z različnimi specifičnimi vlogami v različnih tkivih. Endogeni inhibitorji peptidaz, kot so cistatini in tiropini, so bistveni za nadzor njihove aktivnosti, vendar pa rezultati kažejo, da katepsin X, monokarboksipeptidaza, katere prekomerno izražanje je povezano z več patološkimi procesi, ni pod nad- zorom endogenih inhibitorjev. Inhibitorji, ki pripadajo različnim skupinam, so zavirali druge preizkušene katepsine, nobeden pa ni zmanjšal karboksipeptidazne aktivnosti katepsina X. Odsotnost takega nadzora z inhibitorji, je še ena značilnost, ki razlikuje katepsin X od drugih članov cisteinskih peptidaz.

SARS-CoV-2 infects a broader range of mammalian species than previously anticipated, suggesting there may be additional unknown hosts wherein the virus can evolve and potentially circumvent effective vaccines. We find that SARS-CoV-2 gains a wide host range by binding ACE2 sites essential for ACE2 carboxypeptidase activity. Six mutations found only in rodent species immune to SARS-CoV-2 are sufficient to abolish viral binding to human and dog ACE2. This is achieved through context-dependent mutational effects (intramolecular epistasis) conserved despite ACE2 sequence divergence between species. Across mammals, this epistasis generates sequence-function diversity, but through structures all bound by SARS-CoV-2. Mutational trajectories to the mouse conformation not bound by SARS-CoV-2 are blocked, by single mutations functionally deleterious in isolation, but compensatory in combination, explaining why human polymorphisms at these sites are virtually non-existent. Closed to humans, this path was opened to rodents via permissive cardiovascular phenotypes and ancient increases to ACE2 activity, serendipitously granting SARS-CoV-2 immunity. This reveals how ancient evolutionary trajectories are linked with unprecedented phenotypes such as COVID-19 and suggests extreme caution should be taken to monitor and prevent emerging animal reservoirs of SARS-CoV-2.One sentence summaryA conserved mechanism essential for ACE2 catalytic activity is exploited by SARS-CoV-2 binding, allowing the virus to infect a wide range of species.

Human Angiotensin I Converting Enzyme 2 (ACE2) that acts as a receptor for SARS-CoV-2 entry is highly expressed in human type II pneumocytes and enterocytes and similarly in other mammals and zebrafish (Danio rerio). The zebrafish genome has a highly conserved, one-to-one ortholog ofACE2, i.e.,ace2, whose expression profile however has not yet been studied during development or in pathologies relevant to COVID-19. Herein, we identified significant development-, tissue- and gender-specific modulations inace2expression based on meta-analysis of zebrafish Affymetrix transcriptomics datasets (ndatasets=107, GPL1319 in GEO database). Co-expression network analysis oface2revealed distinct positively correlated (carboxypeptidase activity and fibrin clot formation), and negatively correlated (cilia biogenesis/transport and chromatin modifications) STRING network modules. Using additional transcriptomics datasets, we showed zebrafish embryos before 3 days post fertilization (dpf) exhibited low levels oface2that increased significantly until 4 dpf implicating a role forace2in organogenesis. Re-analysis of RNA-seq datasets from zebrafish adult tissues demonstratedace2was expressed highly in intestines, variably in liver, and at lower levels in other organs. In addition, zebrafish females and males showed significant dimorphism in their age-dependent expression oface2, and between ovary and testis where the latter had higher levels. Moreover, we demonstratedace2expression was significantly modulated under different physiological and pathological conditions associated with development, diet, infection, and inflammation. Our findings implicate a novel translational role for zebrafishace2in differentiation and pathologies predominantly found in intestines and liver, in which the effects of SARS-CoV-2 could be detrimental.

A novel coronavirus (SARS-CoV-2) has emerged to a global pandemic and caused significant damages to public health. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides Ang II to control vasodilatation and permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we wonder how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ~3-10 fold when fluorogenic caspase-1 substrate and Bradykinin-analog peptides were used to characterize ACE2 activity. In addition, the enhancement was mediated by ACE2 binding of RBD domain of SARS-CoV-2 spike. These results highlighted the altered activity of ACE2 during SARS-CoV-2 infection and would shed new lights on the pathogenesis of COVID-19 and its complications for better treatments.

ACE2 is a type I membrane protein with extracellular carboxypeptidase activity displaying a broad tissue distribution with highest expression levels at the brush border membrane (BBM) of small intestine enterocytes and a lower expression in stomach and colon. In small intestinal mucosa, ACE2 mRNA expression appears to increase with age and to display higher levels in patients taking ACE-inhibitors (ACE-I). There, ACE2 protein heterodimerizes with the neutral amino acid transporter Broad neutral Amino acid Transporter 1 (B0AT1) (SLC6A19) or the imino acid transporter Sodium-dependent Imino Transporter 1 (SIT1) (SLC6A20), associations that are required for the surface expression of these transport proteins. These heterodimers can form quaternary structures able to function as binding sites for SARS-CoV-2 spike glycoproteins. The heterodimerization of the carboxypeptidase ACE2 with B0AT1 is suggested to favor the direct supply of substrate amino acids to the transporter, but whether this association impacts the ability of ACE2 to mediate viral infection is not known. B0AT1 mutations cause Hartnup disorder, a condition characterized by neutral aminoaciduria and, in some cases, pellagra-like symptoms, such as photosensitive rash, diarrhea, and cerebellar ataxia. Correspondingly, the lack of ACE2 and the concurrent absence of B0AT1 expression in small intestine causes a decrease in l-tryptophan absorption, niacin deficiency, decreased intestinal antimicrobial peptide production, and increased susceptibility to inflammatory bowel disease (IBD) in mice. Thus, the abundant expression of ACE2 in small intestine and its association with amino acid transporters appears to play a crucial role for the digestion of peptides and the absorption of amino acids and, thereby, for the maintenance of structural and functional gut integrity.

The newly emergent novel coronavirus disease 2019 (COVID-19) outbreak, which is caused by SARS-CoV-2 virus, has posed a serious threat to global public health and caused worldwide social and economic breakdown. Angiotensin-converting enzyme 2 (ACE2) is expressed in human vascular endothelium, respiratory epithelium, and other cell types, and is thought to be a primary mechanism of SARS-CoV-2 entry and infection. In physiological condition, ACE2 via its carboxypeptidase activity generates angiotensin fragments (Ang 1–9 and Ang 1–7), and plays an essential role in the renin-angiotensin system (RAS), which is a critical regulator of cardiovascular homeostasis. SARS-CoV-2 via its surface spike glycoprotein interacts with ACE2 and invades the host cells. Once inside the host cells, SARS-CoV-2 induces acute respiratory distress syndrome (ARDS), stimulates immune response (i.e., cytokine storm) and vascular damage. SARS-CoV-2 induced endothelial cell injury could exacerbate endothelial dysfunction, which is a hallmark of aging, hypertension, and obesity, leading to further complications. The pathophysiology of endothelial dysfunction and injury offers insights into COVID-19 associated mortality. Here we reviewed the molecular basis of SARS-CoV-2 infection, the roles of ACE2, RAS signaling, and a possible link between the pre-existing endothelial dysfunction and SARS-CoV-2 induced endothelial injury in COVID-19 associated mortality. We also surveyed the roles of cell adhesion molecules (CAMs), including CD209L/L-SIGN and CD209/DC-SIGN in SARS-CoV-2 infection and other related viruses. Understanding the molecular mechanisms of infection, the vascular damage caused by SARS-CoV-2 and pathways involved in the regulation of endothelial dysfunction could lead to new therapeutic strategies against COVID-19.

Entomopathogenic bacteria Serratia marcescens is widely used as an environmentally friendly biocontrol agent against various pests, including Spodoptera exigua. Understanding the immune defense mechanism of S. exigua through comparative proteomic analysis can identify the key proteins expressed in response to the microbial infection. Here, we employed the as isobaric tags for relative and absolute quantification (iTRAQ) technique to investigate the effects of S. marcescens on the proteomic expression of S. exigua. Based on the molecular functional analysis, the differentially expressed proteins (DEPs) were mainly involved in the binding process and catalytic activities. Further bioinformatics analysis revealed important DEPs that played a crucial role in innate immunity of S. exigua with recognition (C-type lectin), melanization (propanol oxidase 3, serine protease, Serine-type carboxypeptidase activity, clip domain serine protease 4), antimicrobial activity (lysozyme, lysozyme-like, gloverin, cecropin B), detoxification (acetyl-CoA C-acetyltransferase, 3-dehydroecdysone 3-alpha-reductase, glucuronosyltransferase, glutathione S-transferase) and others. The Quantitative real-time PCR (qRT-PCR) results further indicated the significant upregulation of the immune-related genes in Spodoptera exigua following S. marcescens infection. To the best of our knowledge, this is the first iTRAQ based study to characterize S. marcescens mediated proteomic changes in S. exigua and identified important immune-related DEPs. The results of this study will provide an essential resource for understanding the host-pathogen interactions and the development of novel microbial biopesticides against various pests.

Storage-induced changes in hydrolysed-lactose (LH) ultra-high-temperature (UHT)-treated milk were studied using proteomics. Five commercial lactase preparations were used to hydrolyse lactose in semi-skimmed milk after UHT treatment. The proteolytic side-effects of lactase preparations on UHT milk during storage for up to 60 days were compared with their effects on conventional UHT milk. The level of free amino groups increased more during storage in all LH UHT milk samples than in conventional UHT milk. The increase was partly ascribed to the release of C-terminal amino acid residues from intact β- and αS1-casein, indicating carboxypeptidase activity. During storage, three of the enzyme preparations increased release of peptides derived from the hydrophobic regions of β- and αS1-casein, which could potentially contribute to development of bitterness in the milk samples. The authors propose that residual protease side-activity in the lactase preparations accelerate product deterioration and decrease shelf-life of LH UHT milk.

Angiotensin I-converting enzyme (also known as peptidyl dicarboxypeptidase A, ACE, and EC 3.4.15.1), which is found in a wide range of organisms, cleaves C-terminal dipeptides from relatively short oligopeptides. Mammalian ACE plays an important role in the regulation of blood pressure. However, the precise physiological functions of insect ACE homologs have not been understood. As part of our effort to elucidate new physiological roles of insect ACE, we herein report a soluble ACE protein in male reproductive secretions from the silkmoth, Bombyx mori. Seminal vesicle sperm are quiescent in vitro, but vigorous motility is activated by treatment with either a glandula (g.) prostatica homogenate or trypsin in vitro. When seminal vesicle sperm were pre-incubated with captopril, a strong and specific inhibitor of mammalian ACE, and then stimulated to initiate motility by the addition of the g. prostatica homogenate or trypsin, the overall level of acquired motility was reduced in an inhibitor-concentration-dependent manner. In the course of this project, we detected ACE-related carboxypeptidase activity that was inhibited by captopril in both the vesicular (v.) seminalis of the noncopulative male reproductive tract and in the spermatophore that forms in the female bursa copulatrix at the time of mating, just as in an earlier report on the tomato moth, Lacanobia oleracea, which belongs to a different lepidopteran species (Ekbote et al., 2003a). Two distinct genes encoding ACE-like proteins were identified by analysis of B. mori cDNA, and were named BmAcer and BmAcer2, respectively [the former was previously reported by Quan et al. (2001) and the latter was first isolated in this paper]. RT-qPCR and Western blot analyses indicated that the BmAcer2 was predominantly produced in v. seminalis and transferred to the spermatophore during copulation, while the BmAcer was not detected in the adult male reproductive organs. A recombinant protein of BmAcer2 (devoid of a signal peptide) that was expressed in Escherichia coli cells exhibited captopril-sensitive carboxypeptidase activities. Our findings show that the BmAcre2 gene encodes a secreted ACE protein included in the Bombyx seminal plasma. In particular, the silkworm ACE protein in the seminal fluid might be involved in the signaling pathway that leads to the activation and regulation of sperm motility.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

SummaryType VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized aSalmonellaSPI-6 T6SS antibacterial effector called Tae5STM(type VIamidaseeffector 5). Tae5STMis toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STMand the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STMdisplays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides betweenmeso-diaminopimelic acid3and D-alanine4. Phylogenetic analysis showed that Tae5STMhomologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among α-, β- and γ-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

Prostate cancer (PCa) is a common malignant tumor among adult males, and convenient intraoperative detection of PCa would reduce the risk of leaving positive surgical margins, especially during nerve-sparing procedures. To achieve rapid, fluorescence-based visualization of PCa, we focused on the glutamate carboxypeptidase (CP) activity of prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein that is attracting attention as a PCa biomarker. Based on our finding that aryl glutamate conjugates with an azoformyl linker are recognized by PSMA and have a sufficiently low LUMO (lowest unoccupied molecular orbital) energy level to quench the fluorophore through photoinduced electron transfer, we designed and synthesized a first-in-class activatable fluorescence probe for CP activity of PSMA. The developed probe allowed us to visualize the CP activity of PSMA in living cells and in clinical specimens from PCa patients and is expected to be useful for rapid intraoperative detection and diagnosis of PCa.

Carboxypeptidase A (CPAs) are a well-studied group of zinc-containing exopeptidases that facilitate the breakdown of proteins and peptides during metabolism. Carboxypeptidase A is typically produced in mammalian pancreatic, brain and other tissues. A new gene encoding carboxypeptidase A in the prokaryote Bacillus pumilus was amplified by polymerase chain reaction (PCR), ligated into the shuttle vector pMA5, and cloned in a GRAS bacteria-Bacillus subtilis 168 host. This gene sequence contained a 1621 bp open reading frame that encodes a protein of 540 amino acids. The optimum pH and temperature for enzyme activity were 7.5 and 50°C, respectively. The enzyme was quite stable at neutral pH and maintained about 65% activity following a 24 h incubation at 40°C. The Km of this CPA was 0.1 mM, much higher than in mammalian species. Glycerol, ammonium sulfate, and sodium citrate improved enzyme activity under optimal culture condition. The carboxypeptidase activity in recombinant B. subtilis 168 reached a maximum of 179 U ml-1 in a 5 L fermentator when cultured on improved medium. The over expression of carboxypeptidase A in Bacillus subtilis has commercial applications. Key words : Bacillus pumilus, Bacillus subtilis 168, over-expression, orthogonal arrays, carboxypeptidase A, metallocarboxypeptidase.

Wild relatives represent a source of variation for many traits of interest for eggplant (Solanum melongena) breeding, as well as for broadening its genetic base. However, interspecific hybridization with wild relatives has been barely used in eggplant breeding programs, and reproductive barriers have resulted in reduced seed numbers in interspecific combinations. The mechanism underlying this phenomenon remains unclear. We hybridized females of cultivated eggplant 177 (Solanum melongena) with males of wild relatives 53 and Y11 (Solanum aethiopicum). Self-crossed 177 was the control. The seed number per control fruit was significantly higher than that of the hybrids. Paraffin sections showed no significant difference between control and 177×53 and 177×Y11. Double fertilization began 4 days post-pollination. Sperm cells were fused with egg cells 6 days post-pollination. To understand the differences in molecular mechanisms underlying this process, transcriptomes of ovaries at 0, 4, and 6 days after self-crossing and hybridization were analyzed. We screened 22,311 differentially expressed genes (DEGs) between the control and hybrids 4 and 6 days post-pollination. A total of 497 DEGs were shared among all pollination combinations. These DEGs were enriched in plant hormone transduction, cell senescence, metabolism, and biosynthesis pathways. DEG clustering analysis indicated distinct expression patterns between the control and hybrids but not between the hybrids. The DEGs in hybrids involved secondary metabolic process, phenylpropanoid metabolic process, and carboxypeptidase activity, while those in the control involved xyloglucan metabolic process, auxin-activated signaling pathway, cell wall polysaccharide metabolic process, and xyloglucosyl transferase activity. Additionally, 1683 transcription factors, including members of the AP2-ERF, MYB, bHLH, and B3 families may play important roles in self-crossing and hybridization. Our results provide insights into the regulatory mechanisms underlying variations between ovaries of self-crossed and hybrid eggplants and a basis for future studies on crossbreeding Solanum and genetic mechanisms underlying double fertilization.

Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-A resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Tubulin carboxypeptidase identity revealed Enzymes of the α-tubulin detyrosination/tyrosination cycle create landmarks on microtubules that are essential for their multiple cellular functions and are altered in disease. Tubulin carboxypeptidases (TCPs) responsible for detyrosination have remained elusive for 40 years (see the Perspective by Akhmanova and Maiato). Aillaud et al. identified vasohibins as enzymes that perform the TCP function and found that their small interacting partner SBVP was essential for their activity. Vasohibin/SVBP complexes were involved in neuron polarization and brain cortex development. The authors also developed an inhibitor targeting this family of enzymes. Using a completely different strategy, Nieuwenhuis et al. also showed that vasohibins can remove the C-terminal tyrosine of α-tubulin. Science , this issue p. 1448 , p. 1453 ; see also p. 1381

Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (Ki = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.

Aspergillus oryzae was isolated from coffee pulp, screened for the production of carboxypeptidase and confirmed by molecular studies. Coffee pulp posses high protease activity (1.4 × 103 nkat/mL) among the agro-substrates used for the production of proteases but wheat bran was used for further studies considering its easy availability in large volumes. Proximal composition, pre-treatment, auxiliary carbon and nitrogen with growth studies indicated carboxypeptidase activity (2.6 × 103 nkat/g). The RSM was employed to generate a predictive model of the combined effects of independent variables and indicated a significant impact on acid carboxypeptidase with 35% moisture, 30 °C temperature, 108 CFU mL−1 inoculum and 100 h duration with 4.8 × 103 nkat/g activity. The partially purified peptidase had a specific activity of 6.1 × 104 nkat/mg with a molecular weight of 50 kDa, optimal pH 3.6, temperature 35 °C and feature of a serine protease. Enzyme stability was maintained with 50% glycerol and 2 mg/mL BSA. The protease application to develop flavor profiles of coffee was noteworthy with an organoleptic score of 7.5 compared to 4.5 of the untreated sample. GC-MS highlighted enhanced flavor annotations of pyrazines contributing sweet fruit notes. Carboxypeptidase augmented the coffee bean with specialized flavor notes and has been reported for the first time.

Changes in chemical composition, grain quality parameters, oxidoreductase and exopeptidase activities of wheat grains infested by Sitophilus granarius were monitored during 1 year of storage. Significant increase in flour titratable acidity of infested grains occurred by day 90. Subsequently, infestation was followed by decrease of grain hectoliter weight on day 180, decrease of carboxypeptidase activity on day 240, polyphenol oxidase and catalase activities increased and total starch decreased on day 270, while significant changes of total protein content, extractable protein concentration, phenylalanyl-aminopeptidase and Zeleny sedimentation occurred between 300 and 360 days of storage. The data indicate that S. granarius infestation influenced chemical composition, quality parameters, as well as oxidoreductase and exopeptidase activity in wheat grains, but the extent of these changes was mostly detectable at higher levels of grain infestation. Among the examined parameters, flour titratable acidity and carboxypeptidase activity showed potential to be used as early indicators of grain infestation. Practical Applications Granary weevil, S. granarius, is one of the major grain-damaging insects. This primary infester feeds on grains as adult, or on grain endosperm as larvae causing significant economic losses, due to unsuitability of damaged grains for human food production. Although various methods of S. granarius detection in stored grains have been applied worldwide, there is still a need for cheap indicative methods for detecting insect infestation. This study investigating changes in chemical composition, quality parameters, oxidoreductase and exopeptidase activities during temporal development of infestation by S. granarius shows that flour titratable acidity and carboxypeptidase activity could be used as early indicators of wheat grain infestation.

The stability of wheat bran proteases for producing angiotensin I-converting enzyme (ACE) inhibitory peptides during storage was investigated. The aspartic proteinase activity upon storage at 15, 20, and 30°C was > 90% of initial levels after 30 days, while storage at 40 and 50°C resulted in levels of 87.3 and 63.1%, respectively. The carboxypeptidase activity remained constant at all temperatures during the storage period. The yields of ACE inhibitory peptides from wheat bran at 15, 20, 30, 40, and 50°C after 30 days were 99.0, 98.0, 97.1, 97.1, and 92.2% of the initial level, respectively. The ACE inhibitory activity of all fractions was > 95% of the initial level. These results indicated that the storage of wheat bran at 15 to 40°C results in maintenance of the ACE inhibitory peptide-producing activity, for at least 30 days.

Cyanobacteria possess a unique capacity for the production of structurally novel secondary metabolites compared to the biosynthetic abilities of other environmental prokaryotes such as bacteria of the genus Streptomyces. Two different strategies to explore cyanobacteria-derived natural products have been explored previously: (1) cultivation of single cyanobacterial strains, in bioreactors for example; (2) bulk collections from the environment of so called ‘algal blooms’ that are dominated by cyanobacteria. In this study a new environmentally friendly collection technique for obtaining large quantities of algal bloom biomass was utilized. Algal biomass derived from eight million liters of lake water was concentrated using a novel continuous countercurrent filtration system. Analysis of this freshwater algal bloom from Grand Lake-Saint Marys, Ohio led to the discovery of anabaenopeptin 679 ( 1 ), as well as the known anabaenopeptins B, F, H and 908. Anabaenopeptin 679 is unusual in that it possesses the classical anabaenopeptin-like cyclic pentapeptide core, but lacks the typical sidechain attached to the constitutive ureido group. Screening of all anabaenopeptin derivatives in an enzymatic assay for inhibitory activity toward carboxypeptidase A identified anabaenopeptin 679 as a strong inhibitor of carboxypeptidase A with an IC 50 value of 4.6 μg/mL. This result defines a new minimal core structure for carboxypeptidase activity among the anabaenopeptin class, and provides further insight into the structure–activity relationship of anabaenopeptin-like carboxypeptidase A inhibitors.

In breast cancer, hormonal changes are rather constant in post-menopausal women since they tend to vary only over long time spans. However, in pre-menopausal women, the development of breast cancer is associated with hormonal physiological variations. The aim of the present work was to analyse the changes in circulating levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pre- and post-menopausal women that were healthy or with breast cancer, and their connection to serum pyrrolidone carboxypeptidase (Pcp) activity. We observed significant changes in the hormonal profile in post-menopausal women with breast cancer compared to the control group. In pre-menopausal women, we found significant changes in circulating GnRH levels with respect to the healthy group. Our present results support the existence of neuroendocrine misregulation that could be involved in tumour progression, with Pcp being a potentially new pharmacological target in breast cancer treatments.

Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1 , termed kexA . A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.

The use of aspirin is widely recommended for the prevention of heart attacks owing to its ability to inhibit platelet activation by irreversibly blocking cyclooxygenase 1. However, aspirin also affects the fibrinolytic and hemostatic pathways by mechanisms that are not well understood, causing severe hemorrhagic complications. Here, we investigated the ability of aspirin and aspirin metabolites to inhibit thrombin-activatable fibrinolysis inhibitor (TAFI), the major inhibitor of plasma fibrinolysis. TAFI is activated via proteolytic cleavage by the thrombin-thrombomodulin complex to TAFIa, a carboxypeptidase B-like enzyme. TAFIa modulates fibrinolysis by removing the C-terminal arginine and lysine residues from partially degraded fibrin, which in turn inhibits the binding of plasminogen to fibrin clots. Aspirin and its major metabolites, salicylic acid, gentisic acid, and salicyluric acid, inhibit TAFIa carboxypeptidase activity. Salicyluric acid effectively blocks activation of TAFI by thrombin-thrombomodulin; however, salicylates do not inhibit carboxypeptidase N or pancreatic carboxypeptidase B. Aspirin and other salicylates accelerated the dissolution of fibrin clots and reduced thrombus formation in an in vitro model of fibrinolysis. Inhibition of TAFI represents a novel hemostatic mechanism that contributes to aspirin’s therapy-associated antithrombotic activity and hemorrhagic complications.

γ-Secretase is a membrane-embedded protease complex with presenilin as the catalytic component. Cleavage within the transmembrane domain of the amyloid β-protein precursor (APP) by γ-secretase produces the C-terminus of the amyloid β-peptide (Aβ), a proteolytic product prone to aggregation and strongly linked to Alzheimer’s disease (AD). Presenilin mutations are associated with early-onset AD, but their pathogenic mechanisms are unclear. One hypothesis is that these mutations cause AD through a toxic gain of function, changing γ-secretase activity to increase the proportion of 42-residue Aβ over the more soluble 40-residue form. A competing hypothesis is that the mutations cause AD through a loss of function, by reducing γ-secretase activity. However, γ-secretase apparently has two types of activities, an endoproteolytic function that first cuts APP to generate a 48/49-residue form of Aβ, and a carboxypeptidase activity that processively trims these longer Aβ intermediates approximately every three residues to form shorter, secreted forms. Recent studies suggest a resolution of the gain-of-function vs. loss-of-function debate: presenilin mutations may increase the proportion of longer, more aggregation-prone Aβ by specifically decreasing the trimming activity of γ-secretase. That is, the reduction of this particular proteolytic function of presenilin, not its endoproteolytic activity, may lead to the neurotoxic gain of function.

Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug-resistant tuberculosis. These β-lactams target the transpeptidases that introduce interpeptide cross-links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of M. tuberculosis (Mtb) with the β-lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3-3 cross-linkages (involving two diaminopimelate (DAP) molecules) predominate over 4-3 cross-linkages (involving one DAP and one D-alanine) in stationary-phase cells. We purified and analyzed peptidoglycan from Mtb and found that 3-3 cross-linkages predominate throughout all growth phases and the ratio of 4-3/3-3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D-carboxypeptidase and an L,D-transpeptidase. We purified a candidate D,D-carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem-treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3-cross-links while simultaneously limiting the pool of available substrates available for cross-linking.

The pH optimum and thermostability of both exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.

The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicated mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addi-tion of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

We have found that Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, expresses a dipeptidyl-carboxypeptidase with activity homologous to angiotensin-converting enzyme (ACE). The carboxypeptidase activity was purified to homogeneity as a complex/aggregate from a bacterial surface extract and was also active as a 165 kDa monomer. The specific activity for the carboxypeptidase activity was eightfold higher than that for recombinant human ACE. Selected ACE inhibitors, captopril, lisinopril and enalapril, did not inhibit the ACE activity. The carboxypeptidase also hydrolysed the Aα and Bβ-chains of human fibrinogen, which resulted in impaired fibrin formation by thrombin. The gene encoding ACE carboxypeptidase activity was sequenced and the inferred polypeptide product showed 99 % amino acid homology to SGO_0566, sgc, ‘challisin’ of S. gordonii CL1 Challis, and had no significant amino acid sequence homology to human ACE. Homologues of challisin ACE activity were commonly detected among the viridans group streptococci most often associated with IE.

The generation of anaphylatoxins, particularly C5a, is important in extracorporeal circulation therapies such as granulocyte/monocyte apheresis, which activates the complement system and elevates C5a levels. However, no side effects of granulocyte/monocyte apheresis using cellulose acetate beads have been reported. To investigate the mechanism of complement activation, we prepared plasma from cellulose acetate bead-treated blood (P-CAB) and compared it with zymosan-activated plasma (ZAP). Anaphylaxis activity was measured by skin test, and the activity of carboxypeptidase, which inactivates C5a, was measured by colorimetric assay. Pro-carboxypeptidase R and neutrophil elastase concentrations were measured by enzyme-linked immunosorbent assay. Although C5a was generated in P-CAB, the anaphylaxis activity of P-CAB was lower than that of ZAP. Carboxypeptidase activity and pro-carboxypeptidase R levels were suppressed in P-CAB, but not in ZAP. Furthermore, neutrophil elastase levels increased in P-CAB. The decreases in carboxypeptidase activity and inactivation of anaphylatoxin were inhibited by a neutrophil elastase inhibitor. These results suggest that cellulose acetate beads initiate the activation of carboxypeptidase R via elastase release, thereby inducing the inactivation of anaphylatoxin.

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.