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Fibrosis is a common manifestation of most progressive and degenerative diseases, with myofibroblast activation and matrix accumulation playing a key role. In this issue of the JCI, Hoeft et al. identify the important role of ADAMTS12 in fibroblast activation. ADAMTS12, a secreted protein, is involved in extracellular matrix (ECM) remodeling, cell signaling, and inflammation. ADAMTS12 facilitates proteolysis by cleaving various substrates such as ECM components, which are vital for cellular signaling and remodeling. Additionally, it modulates cell-matrix interactions, influencing cell adhesion and migration, and plays an important role in the inflammatory processes. Understanding the role of ADAMTS12 offers potential therapeutic insights for targeting fibrosis in progressive diseases., Fibrosis in heart and kidney disease Fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix (ECM) components, particularly collagen, in organs, including the heart and kidneys, often [...]

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury- responsive fibroblast subset defined by aberrant high JAK/STAT signaling., Introduction Fibrosis represents the uncontrolled replacement of parenchymal tissue by extracellular matrix (ECM) (1). Whereas deposition of ECM sustains tissue integrity immediately after injury, continued deposition of ECM by myofibroblasts [...]

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy.sup.1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes.sup.5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy.sup.7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors. Fertilized Arabidopsis egg cells secrete endopeptidases into the extracellular space that cleave the pollen tube attractor LURE1, preventing polytubey., Author(s): Xiaobo Yu [sup.1] , Xuecheng Zhang [sup.1] , Peng Zhao [sup.1] , Xiongbo Peng [sup.1] , Hong Chen [sup.1] , Andrea Bleckmann [sup.2] , Anastasiia Bazhenova [sup.2] , Ce [...]

Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly.sup.1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions.sup.3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting-feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes. mTORC1 stabilizes HMG-CoA reductase, a rate-limiting enzyme in the cholesterol biosynthesis pathway, via the deubiquitylase USP20 in response to feeding., Author(s): Xiao-Yi Lu [sup.1] , Xiong-Jie Shi [sup.1] , Ao Hu [sup.1] , Ju-Qiong Wang [sup.1] , Yi Ding [sup.1] , Wei Jiang [sup.1] , Ming Sun [sup.1] , Xiaolu [...]

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread.sup.1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses.sup.3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-[kappa]B pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity. Biochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by preferentially cleaving the ubiquitin-like interferon-stimulated gene 15 protein (ISG15) and identify this protease as a potential therapeutic target for coronavirus disease 2019 (COVID-19)., Author(s): Donghyuk Shin [sup.1] [sup.2] [sup.3] , Rukmini Mukherjee [sup.1] [sup.2] , Diana Grewe [sup.2] , Denisa Bojkova [sup.4] , Kheewoong Baek [sup.5] , Anshu Bhattacharya [sup.1] [sup.2] , Laura [...]

During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-[8.sup.ECKO]) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-[8.sup.ECKO] pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP., Introduction Functional blood vessels are of vital importance. Impaired vessel formation contributes to many pathological situations, including ischemic and inflammatory disorders. Ischemic retinopathies are the main causes of severe visual [...]

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpCClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpCClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpCClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitinproteasome system., Author(s): Dbora Broch Trentini [1]; Marcin Jzef Suskiewicz [1]; Alexander Heuck [1]; Robert Kurzbauer [1]; Luiza Deszcz [1]; Karl Mechtler [1, 2]; Tim Clausen (corresponding author) [1] Proteins destined for [...]

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development (1-3); however, the key mechanisms mediating the perception and relay of the C[O.sub.2] signal to the stomatal development machinery remain elusive. To adapt C[O.sub.2] intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric C[O.sub.2] (ref. 4). To date, one mutant that exhibits deregulation of this C[O.sub.2]-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana b-carbonic anhydrase double mutants (ca1 ca4) (6) exhibit an inversion in their response to elevated C[O.sub.2], showing increased stomatal development at elevated C[O.sub.2] levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of C[O.sub.2]-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2) (7, 8), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated C[O.sub.2] levels. Moreover, EPF2 is essential for C[O.sub.2] control of stomatal development. Using cell-wall proteomic analyses and C[O.sub.2]-dependent transcriptomic analyses, we identified a novel C[O.sub.2]-induced extracellular protease, CRSP (C[O.sub.2] RESPONSE SECRETED PROTEASE), as a mediator of C[O.sub.2]-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric C[O.sub.2] elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding of how plants perceive and relay the elevated C[O.sub.2] signal and provides a framework to guide future research into how environmental challenges can modulate gas exchange in plants., C[O.sub.2] exchange between plants and the atmosphere, and water loss from plants to the atmosphere, depends on the density and the aperture size of plant stomata, and plants have evolved [...]

Soil microbial activity can be constrained by availability of energy because soil carbon (C) occurs mostly as complex soil organic matter (SOM), with relatively small quantities of high-energy, labile C. Decomposition of SOM is mediated by energy-requiring processes that need extracellular enzymes produced by soil microbial communities. We examined how an increase in energy status via sucrose supplementation affects the production of SOM-degrading enzymes, comparing matched soils under forest and sugarcane agriculture with histories of contrasting inputs of complex and labile C. Activities of SOM-degrading enzymes increased in both soils after sucrose addition, but C[O.sub.2] production increased more rapidly in the sugarcane soil. The forest soil had greater increases in phosphatase and glucosidase activities, whereas the sugarcane soil had greater increases in protease and urease activity. The contrasting microbial community-level physiological profiles of the soils further diverged at 30 and 61 days after sucrose amendment, before returning to near pre-treatment profiles by 150 days. We interpreted the increasing soil enzyme production as indicative that enzyme production was limited by energy availability in both soils, despite contrasting histories of labile v. recalcitrant C supply. Quicker responses in sugarcane soil suggest pre-selection towards populations that exploit labile inputs. Additional keywords: land use change, nitrification, nitrogen, soil function., Introduction The dynamics of the interactions between soil microbes, labile carbon (C) and old soil organic matter (SOM) are key to understanding C and nutrient turnover in soils, and have [...]

The striped rice stem borer, Chilo suppressalis (Walker), is a major pest for rice production in China and the rest of Southeast Asia. Chemical control is the main means to alleviate losses due to this pest, which causes serious environmental pollution. An effective and environmentally friendly approach is needed for the management of the striped rice stem borer. Cysteine proteases in insects could be useful targets for pest management either through engineering plant protease inhibitors, targeting insect digestive cysteine proteases, or through RNA interference-based silencing of cysteine proteases, disrupting developmental regulation of insects. In this study, eight cysteine protease-like genes were identified and partially characterized. The genes CCO2 and CCL4 were exclusively expressed in the larval gut, and their expression was affected by the state of nutrition in the insect. The expression of CCL2, CCL3, and CCO1 was significantly affected by the type of host plant, suggesting a role in host plant--insect interactions. Our initial characterization of the striped rice stem borer cysteine protease-like genes provides a foundation for further research on this important group of genes in this major insect pest of rice. Key words: striped rice stem borer, Chilo suppressalis, digestive enzymes, cysteine proteases, cathepsin-like genes. La pyrale du riz, Chilo suppressalis (Walker), est un ravageur important en riziculture en Chine et dans le reste de l'Asie du Sud-Est. La lutte chimique est le principal moyen pour limiter les pertes dues a ce ravageur, une pratique qui cause une pollution environnementale serieuse. Une approche efficace et plus respectueuse de l'environnement est requise pour lutter contre la pyrale du riz. Les proteases a cysteine chez les insectes pourraient constituer des cibles utiles pour la lutte contre cet insecte soit en produisant des inhibiteurs de proteases visant specifiquement les proteases digestives de l'insecte, soit en utilisant l'interference par ARN pour eliminer les proteases a cysteine et ainsi perturber la regulation du developpement chez l'insecte. Dans ce travail, les auteurs ont identifie et partiellement caracterise huit genes du type protease a cysteine. Les genes CCO2 et CCL4 etaient exprimes specifiquement dans l'estomac de la larve et leur expression etait affectee par l'etat nutritionnel de l'insecte. L'expression des genes CCL2, CCL3 et CCO1 etait affectee de maniere significative par le type de plante hote, chose qui suggere un role dans les interactions plante hote--insecte. Cette caracterisation initiale de proteases a cysteine chez la pyrale du riz fournit une assise en vue de futurs travaux sur un groupe de genes importants chez un ravageur majeur en riziculture. [Traduit par la Redaction] Mots-cles: pyrale du riz, Chilo suppressalis, enzymes digestives, proteases a cysteine, genes de type cathepsine., Introduction Proteases play roles in various biological processes such as DNA replication, cell-cycle progression, cell proliferation, morphogenesis and tissue remodeling, neuronal outgrowth, haemostasis, wound healing, immunity, angiogenesis, apoptosis, and food [...]

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals (1). However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread (2). Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection (3'4). Viral genome analysis revealed close relatedness to coronaviruses found in bats (5). Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies., Coronaviruses infect a wide range of mammals and birds. Their tropism is primarily determined by the ability of the spike (S) entry protein to bind to a cell surface receptor. [...]

ABSTRACT Vitis amurensis (Vitaceae) has been reported to have anti-oxidant and anti-inflammatory activities. The present study investigated a methanol extract from the leaf and stem of V. amurensis for neuroprotective [...]

Different strategies of petal senescence and some important events associated with it have been discussed. On the basis of sensitivity to ethylene and associated symptoms of senescence, petal senescence has been classified into five different classes; besides changes in membrane permeability, autophagy and involvement of VPEs (Vacuolar processing enzymes), degradation of nucleic acids, protein turn over and remobilization of essential nutrients during petal senescence have been discussed. Nucleus appears to play a central role in administrating the execution of the events associated with petal senescence. Protein turn over appears to be an important factor governing petal senescence in both ethylene-sensitive and ethylene-insensitive flower systems and that the loss of membrane integrity, vacuolar autophagy and remobilization of essential nutrients being its important consequences. Autophagy seems to be a main process responsible for cell dismantling and remobilization of macromolecules besides final disintegration of nucleus. A large number of senescence-associated genes have been found to be differentially expressed during petal senescence. On the basis of the available literature, a schematic model representing some important events associated with petal senescence has been constructed. The review recommends that more elaborate work is required at cellular and organelle level to understand the ethylene-independent pathway and its execution in both ethylene-sensitive and ethylene-insensitive flower systems. It also recommends that ethylene sensitivity should not be generally assigned to plants at the family level on the basis of response of a few species in a family. Resumes des strategies differentes de petale senescence et certains evenements importants associes ont ete discutes. Sur la base d'une sensibilite d'ethylene et les symptomes associes de senescence, petale senescence ont ete classe en cinq differentes classes ; outre les modifications dans la permeabilite de la membrane, Autophagie et l'implication des VPEs (Vacuolar traitement des enzymes), la degradation des acides nucleiques, proteines tour sur re de nutriments essentiels au cours de la senescence petale ont ete discutes. Noyau semble jouer un role central dans l'administration de l'execution des evenements associes petale senescence. Tour de proteines sur semble etre un facteur important regissant petale senescence dans les systemes de fleur d'ethylene sensibles et ethylene-insensible et que la perte d'integrite de la membrane, Autophagie vacuolar et re de nutriments essentiels a ses consequences importantes. Autophagie semble etre un processus principal responsable du demante1ement de la cellule et re de macromolecules outre finale desintegration du noyau. Un grand hombre de genes associes senescence a ete trouve pour etre exprime differemment au cours de la senescence petale. Sur la base de la documentation disponible, un mode1e schematic representant certains evenements importants associes petale senescence a ete construit. L'examen recommande qu'un travail plus elabore est necessaire au niveau cellulaire et Organite pour comprendre la voie de l'ethylene-independante et son execution dans les deux systemes de fleur d'ethylene sensibles et ethylene-insensible. Elle recommande egalement que ethylene sensibilite ne doit pas etre generalement affectee aux plantes au niveau familial sur la base de la reponse de quelques especes dans une famille. Keywords Autophagy * Membrane integrity * Proteases * Proteasomes * Remobilization * Senescence Motscles autophagie * integrite de membrane * proteases * proteasomes * resenescence, Introduction Senescence comprises those processes that follow physiological maturity which lead to the event of death of a whole plant, organ or tissue at macroscopic level. At microscopic level the [...]

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (CIpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, CIpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than CgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis. DOI 10.1099/mic.0.042275-0

Enterohaemorrhagic Escherichia coil (EHEC) comprise a group of animal and zoonotic pathogens of worldwide importance. Our previous research established that intestinal colonization of calves by EHEC serotypes 05 :H- and O111 :H- requires EHEC factor for adherence (Efa-1), also known as lymphostatin (LifA). Towards an understanding of the mode of action of Efa-1/LifA, chromosomal in-frame deletions of predicted glycosyltransferase (DXD) and cysteine protease (CHD) motifs were created in a Astxl derivative of EHEC 026 : H-. The magnitude and duration of faecal excretion of EHEC 026 : H- were significantly reduced by null mutation of efa-1/lifA, but were not impaired by [DELTA]DXD or [DELTA]CHD mutations, in contrast to observations made with truncated Efa-1/LifA mutants of Citrobacter rodenfium in mice. Although C. rodenfium Efa-l/LifA influences the induction of colonic hyperplasia in mice, EHEC 026 : H- Efa-1/LifA was not required for fluid accumulation or neutrophil recruitment in bovine ileal loops. In contrast to observations with EHEC 05 : H- or O111 : H- mutants, inactivation of efa-1/lifA in EHEC 026 : H- did not significantly affect adherence or secretion of type III secreted proteins that play pivotal roles in calf colonization. Lymphostatin activity could not be reliably demonstrated in lysates of EHEC O26 :H-; however, deletion of the glycosyltransferase and cysteine protease motifs in Efa-1/LifA from enteropathogenic E. coil O127 : H6 abolished lymphostatin activity. Our data uncouple the role of Efa-1/LifA in calf colonization from effects on type III secretion and reinforce the potential for pathotype- and serotype-specific phenotypes. DOI 10.1099/mic.0.039685-0

Bacteroides fragilis is a bacterium that resides in the normal human gastro-intestinal tract; however, it is also the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses, and the most common cause of anaerobic bacteraemia. Abscess formation is important in bacterial containment, limiting dissemination of infection and bacteraemia. In this study, we investigated B. fragilis binding and degradation of human fibrinogen, the major structural component involved in fibrin abscess formation. We have shown that B. fragilis NCTC9343 binds human fibrinogen. A putative Bacteroides fragilis fibrinogen-binding protein, designated BF-FBP, identified in the genome sequence of NCTC9343, was cloned and expressed in Escherichia coil. The purified recombinant BF-FBP bound primarily to the human fibrinogen B[beta]-chain. In addition, we have identified fibrinogenolytic activity in B. fragilis exponential phase culture supernatants, associated with fibrinogenolytic metalloproteases in NCTC9343 and 638R, and cysteine protease activity in YCH46. All nine clinical isolates of B. fragilis examined degraded human fibrinogen; with eight isolates, initial A[alpha]-chain degradation was observed, with varying B[beta]-chain and [gamma]-chain degradation. With one blood culture isolate, B[beta]-chain and [gamma]-chain degradation occurred first, followed by subsequent A[alpha]-chain degradation. Our data raise the possibility that the fibrinogen-binding protein of B. fragilis, along with a variety of fibrinogenolytic proteases, may be an important virulence factor that facilitates dissemination of infection via reduction or inhibition of abscess formation. DOI 10.1099/mic.0.038588-0

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain--containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections. doi/ 10.1083/jcb.200912087

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5' of prcA-prtP. The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6xHis-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6 x His in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane. doi: 10.1128/JB.00274-10

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM. doi: 10.1128/JB.01315-09

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth (1,2). Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage (3,4) at a juxtamembrane site that otherwise lies buried within the quiescent NRR (5,6). Subsequent intramembrane proteolysis catalysed by the y-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer (7), making the Notch pathway a compelling target for new drugs. Although y-secretase inhibitors (GSIs) have progressed into the clinic (8), GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways (9) and cause intestinal toxicity (10), attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently., To enable independent antagonism of Notchl and Notch2, we used phage display to generate antibodies targeting the NRRs. Previous studies proposed that antibody targeting of the NRR might stabilize the [...]

Histone deacetylase (HDAC) inhibitors are emergent cancer drugs. HR23B is a candidate cancer biomarker identified in a genome-wide loss-of-function screen which influences sensitivity to HDAC inhibitors. Because HDAC inhibitors have found clinical utility in cutaneous T-cell lymphoma (CTCL), we evaluated the role of HR23B in CTCL cells. Our results show that HR23B governs the sensitivity of CTCL cells to HDAC inhibitors. Furthermore, proteasome activity is deregulated in HDAC inhibitor-treated CTCL cells through a mechanism dependent upon HR23B, and HDAC inhibitors sensitize CTCL cells to the effects of proteasome inhibitors. The predictive power of HR23B for clinical response to HDAC inhibitors was investigated through an analysis of a unique collection of CTCL biopsies taken from a phase II clinical trial, where there was a frequent coincidence between HR23B expression and clinical response to HDAC inhibitor. Our study supports the personalized medicine approach for treating cancer and the increasing drive to translate laboratory-based findings into clinical utility. cancer | histone deacetylase inhibitor | biomarker | proteasome | clinical trial doi/10.1073/pnas.0913912107

We investigated the expression of the [alpha] subunit of the Dictyostelium mitochondrial processing peptidase (Dd[alpha]-MPP) during development. Dd[alpha]-MPP mRNA is expressed at the highest levels in vegetatively growing cells and during early development, and is markedly downregulated after 10 h of development. The Dd[alpha]-MPP protein is expressed as two forms, designated [alpha]-MP[P.sub.H] and [alpha]-MP[P.sub.L], throughout the Dictyostelium life cycle. The larger form, [alpha]-MP[P.sub.H], is cleaved to produce the functional [alpha]-MP[P.sub.L] form. We were not able to isolate mutants in which the [alpha]-mpp gene had been disrupted. Instead, an antisense transformant, [alpha]A2, expressing [alpha]-MPP at a lower level than the wild-type AX-3 was isolated to examine the function of the [alpha]-MPP protein. Development of the [alpha]A2 strain was normal until the slug formation stage, but the slug stage was prolonged to ~24 h. In this prolonged slug stage, only [alpha]-MPPH was present, and [alpha]-MPPL protein and MPP activity were not detected. After 28 h, [alpha]-MP[P.sub.L] and MPP activity reappeared, and normal fruiting bodies were formed after a delay of approximately 8 h compared with normal development. These results indicate that MPP activity is controlled by the processing of [alpha]-MP[P.sub.H] to [alpha]-MP[P.sub.L] during development in Dictyostelium. DOI 10.1099/mic.0.094306-0

Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes. development | gene expression | non-apoptotic caspase activity | epigenetics doi/10.10731pnas.0913089107

Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined, mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation. doi: 10.1128/JB.01167-09

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyI-L-arginine p-nitroanilide (Bz-L-arginine pNA), as demonstrated by assaying culture supematants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-DL-arginine pNA rather than Bz-DL-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis. DOI 10.1099/mic.0.031609-0

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalioproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system. doi: 10.1128/JB.00264-09

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells ([Kit.sup.W-sh/W-sh] [Wsh]) or mast cell chymase ([Mcpt4.sup.-/-]) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not [Mcpt4.sup.-/-] mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function. homeostasis | intestinal permeability | mast cell protease-4 doi/10.1073/pnas.0906372106

Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)--dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPAl variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits. doi/10.1083/jcb.200906084

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network. doi/10.1083/jcb.200906083

The hexameric membrane-spanning ATP-dependent metal-Ioprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo-and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH. AAA+ protease | conformational rearrangement | pore residue | FtsH | metalloprotease doi/10.1073/pnas.0910708106

Phylogenomics reveals extreme gene loss in typhus group (TG) rickettsiae relative to the levels for other rickettsial lineages. We report here a curious protease-encoding gene (ppcE) that is conserved only in TG rickettsiae. As a possible determinant of host pathogenicity, ppcE warrants consideration in the development of therapeutics against epidemic and murine typhus. doi: 10.1128/JB.01077-09

The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways. doi: 10.1128/JB.00722-09

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a [beta]-barrel pore in the outer membrane implicated in translocation of the upstream 'passenger' domain across the outer membrane. However, recent structural data suggest that the diameter of the [beta]-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the [beta]-domain was not inserted and folded as an integral [beta]-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Barn (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane. DOI 10.1099/mic.0.034991-0

A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain. Phylogenetic analysis revealed that this enzyme belonged to the subtilisin family, but could not be grouped into any of its six known subfamilies. The mature protease CDF has an unusually high content of charged residues, which are mainly distributed on the enzyme surface. The recombinant proform of protease CDF formed inclusion bodies, but could be efficiently converted to the mature enzyme when the inclusion bodies were dissolved in alkaline buffers. The proform underwent a two-step maturation process, wherein the N-terminal part (85 residues) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residue peptide was further processed intermolecularly. Protease CDF exhibited optimal proteolytic activity at 50-55[degrees]C and pH 10.5-11.0. The enzyme was stable under high-pH conditions (pH 11.0-12.0), and NaCl could stabilize the enzyme at lower pH values. In addition, the enzyme was not dependent on calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with spores, and could be extracted from the spores with 2 M KCl and alkaline buffers without damaging the coat layer, demonstrating that the protease CDF is located on the surface of the spore coat. DOI 10.1099/mic.0.031336-0

The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc[gamma] receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the 'nick' of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc[gamma] family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised. antibody-dependent cellular cytotoxicity | complement-dependent cytotoxicity | Fc gamma Receptors | matrix metalloproteinases | monoclonal antibodies doi/ 10.1073/pnas.0904174106

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases' role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death. doi 10.1083/jcb.200905110

Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal seven-stranded jelly-roll fibronectin (Fn) Ill-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although [Ca.sup.2+]-binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be [Ca.sup.2+]-free and its thermo stability is [Ca.sup.2+]-independent. calcium | proprotein convertase | protease-associated domain | subtilisin | thermostability

The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment, cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of [Ca.sup.2+] and dipicolinic acid (DPA); the viability of these spores was ~[10.sup.4]-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants. DOI 10.1099/mic.0.030965-0

Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metalIoproteases. The Bacillus subtilis SpolVFB protein is a putative metalIoprotease that cleaves membrane-tethered Pro-[[sigma].sup.K], releasing [[sigma].sup.K] to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpolVFB, expression in E. coil was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-[[sigma].sup.K]. Uniquely for IPs examined so far, SpolVFB activity requires ATP, which binds to the Cterminal cystathionine-[beta]-synthase (CBS) domain of SpolVFB. Deleting just 10 residues from the C-terminal end of SpolVFB nearly eliminated cleavage of coexpressed Pro-[[sigma].sup.K] in E. coil The CBS domain of SpolVFB was shown to interact with Pro-[[sigma].sup.K] and ATP changed the interaction, suggesting that ATP regulates substrate access to the active site and renders cleavage sensitive to the cellular energy level, which may be a general feature of CBS-domain-containing IPs. Incorporation of SpolVFB into preformed liposomes stimulated its ability to cleave Pro-[[sigma].sup.K]. Cleavage depended on ATP and the correct peptide bond was shown to be cleaved using a rapid and sensitive mass spectrometry assay. A system for biochemical studies of RIP by a metalloprotease in a membrane environment has been established using methods that might be applicable to other IPs. Bacillus subtilis | intramembrane protease | regulated intramembrane proteolysis | SpolVFB | sporulation

Burkholderia cenocepacia secretes two zinc-dependent metalloproteases, designated ZmpA and ZmpB. Previously, ZmpA and ZmpB have been shown to cleave several proteins important in host defence. In this study, the ability of ZmpA and ZmpB to digest and inactivate antimicrobial peptides involved in innate immunity was examined. ZmpB but not ZmpA cleaved [beta]-defensin-1. ZmpA but not ZmpB cleaved the cathelicidin LL-37. Both enzymes cleaved elafin and secretory leukocyte inhibitor, which are antimicrobial peptides as well as neutrophil elastase inhibitors. Both ZmpA and ZmpB cleaved protamine, a fish antimicrobial peptide, and a zmpA zmpB mutant was more sensitive to protamine killing than the parental strain. ZmpA or ZmpB cleavage of elafin inactivated its anti-protease activity. The effect of ZmpA and ZmpB on the neutrophil proteases elastase and cathepsin G was also examined but neither enzyme was active against these host proteases. These studies suggest that ZmpA and ZmpB may influence the resistance of B. cenocepacia to host antimicrobial peptides as well as alter the host protease/anti-protease balance in chronic respiratory infections.

The anorexigenic neuromodulator [alpha]-melanocyte--stimulating hormone ([alpha]-MSH; referred to here as [alpha]-[MSH.sub.1-13]) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of [alpha]-[MSH.sub.1-13] maturation and inactivation is incompletely understood. Here we have provided insight into [alpha]-[MSH.sub.1-13] inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of [alpha]-[MSH.sub.1-13], producing [alpha]-[MSH.sub.1-12], which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where [alpha]-[MSH.sub.1-13] is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of [alpha]-[MSH.sub.1-13] in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet--induced obesity. Our results suggest that PRCP is an important component of mela-nocortin signaling and weight maintenance via control of active [alpha]-[MSH.sub.1-13] levels., Introduction Analysis of congenic mouse strains provides a general method to identify genes for complex traits using positional genetics tools. Congenic mouse strain analyses have successfully identified several positional candidate [...]

The level of neurotransmitters present in the synaptic cleft is a function of the delicate balance among neurotransmitter synthesis, recycling, and degradation. While much is known about the processes controlling neurotransmitter synthesis and release, the enzymes that degrade peptide neurotransmitters are poorly understood. A new study in this issue of the JCI reveals the important role of neuropeptide degradation in regulating obesity (see the related article beginning on page 2291). Wallingford et al. provide evidence that, in mice, the enzyme prolylcarboxypeptidase (PRCP) degrades [alpha]-melanocyte--stimulating hormone ([alpha]-MSH) to an inactive form that is unable to inhibit food intake. Their studies indicate that PRCP expression promotes obesity, while inhibitors of the enzyme counteract obesity., Neurons in the arcuate region of the hypothalamus that make the polypeptide hormone precursor proopiomelanocortin (POMC) are very important for body weight regulation, as revealed by the dramatic development of [...]