ABSTRACT
The rhomboid proteases were first discovered as regulators of Drosophila EGF receptor signaling; soon after, it was recognized that they represented the founder members of a widespread family of intramembrane serine proteases conserved in all kingdoms. More recently still, the family was promoted to a superfamily, encompassing a wide variety of distantly related proteins. One of the surprises has been that many members of the rhomboid-like superfamily are not active proteases. Given the size of this clan, and its relatively recent discovery, there is still much to learn. Nevertheless, we already understand much about how rhomboid proteases perform their surprising function of cleaving transmembrane domains. We also already know that members of the rhomboid-like superfamily participate in biological functions as diverse as growth factor signaling, mitochondrial dynamics, inflammation, parasite invasion, and the machinery of protein quality control. Their potential medical significance is now becoming apparent in several areas.
Subject(s)
Membrane Proteins/physiology , Multigene Family , Serine Proteases/physiology , Animals , Carrier Proteins/physiology , Catalytic Domain , Drosophila Proteins/physiology , Humans , Inflammation/enzymology , Mammals/metabolism , Membrane Proteins/classification , Mitochondria/enzymology , Mitochondrial Proteins/physiology , Parasitic Diseases/enzymology , Plant Proteins/physiology , Proteolysis , Serine Proteases/classification , Terminology as TopicABSTRACT
Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.
Subject(s)
Bacterial Proteins , Leucyl Aminopeptidase , Serine Proteases , Vibrio cholerae , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Leucyl Aminopeptidase/chemistry , Leucyl Aminopeptidase/genetics , Leucyl Aminopeptidase/physiology , Peptides , Serine Proteases/chemistry , Serine Proteases/genetics , Serine Proteases/physiology , Substrate Specificity , Vibrio cholerae/enzymology , Vibrio cholerae/genetics , Vibrio cholerae/physiology , CatalysisABSTRACT
Treatments are emerging for the neuronal ceroid lipofuscinoses (NCLs), a group of similar but genetically distinct lysosomal storage diseases. Clinical ratings scales measure long-term disease progression and response to treatment but clinically useful biomarkers have yet to be identified in these diseases. We have conducted proteomic analyses of brain and cerebrospinal fluid (CSF) from mouse models of the most frequently diagnosed NCL diseases: CLN1 (infantile NCL), CLN2 (classical late infantile NCL) and CLN3 (juvenile NCL). Samples were obtained at different stages of disease progression and proteins quantified using isobaric labeling. In total, 8303 and 4905 proteins were identified from brain and CSF, respectively. We also conduced label-free analyses of brain proteins that contained the mannose 6-phosphate lysosomal targeting modification. In general, we detect few changes at presymptomatic timepoints but later in disease, we detect multiple proteins whose expression is significantly altered in both brain and CSF of CLN1 and CLN2 animals. Many of these proteins are lysosomal in origin or are markers of neuroinflammation, potentially providing clues to underlying pathogenesis and providing promising candidates for further validation.
Subject(s)
Aminopeptidases/physiology , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Brain/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/physiology , Lysosomes/metabolism , Membrane Glycoproteins/physiology , Molecular Chaperones/physiology , Neuronal Ceroid-Lipofuscinoses/diagnosis , Serine Proteases/physiology , Thiolester Hydrolases/physiology , Animals , Disease Models, Animal , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuronal Ceroid-Lipofuscinoses/blood , Neuronal Ceroid-Lipofuscinoses/cerebrospinal fluid , Proteome/analysis , Tripeptidyl-Peptidase 1ABSTRACT
OBJECTIVE: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS. DESIGN: 39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states. RESULTS: Patients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota. CONCLUSION: A subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.
Subject(s)
Irritable Bowel Syndrome/physiopathology , Serine Proteases/physiology , Adult , Animals , Biopsy , Caco-2 Cells , Case-Control Studies , Colon/pathology , Dysbiosis/enzymology , Feces/enzymology , Female , Gastrointestinal Microbiome , Humans , Intestinal Absorption/physiology , Intestinal Mucosa/metabolism , Irritable Bowel Syndrome/enzymology , Irritable Bowel Syndrome/microbiology , Irritable Bowel Syndrome/pathology , Male , Mice , Middle Aged , Permeability , Prospective Studies , Proteolysis , Severity of Illness Index , Tight Junction Proteins/metabolismABSTRACT
Multiple novel members of the genus Hepacivirus have recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of hepatitis C virus (HCV) is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing interferon (IFN) production and contributing to HCV persistence in the human host. To investigate the conservation of fundamental aspects of the hepaciviral life cycle, we explored if MAVS cleavage and suppression of innate immune signaling represent a common mechanism employed across different clades of the genus Hepacivirus to enhance viral replication. To estimate the zoonotic potential of these nonhuman hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS. NS3/4A proteases of viruses infecting colobus monkeys, rodents, horses, and cows cleaved the MAVS proteins of their cognate hosts and interfered with the ability of MAVS to induce the IFN-ß promoter. All NS3/4A proteases from nonhuman viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genus Hepacivirus Human MAVS is susceptible to cleavage by these nonhuman viral proteases, indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans. IMPORTANCE: Virus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best-characterized member of the genus Hepacivirus within the family Flaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses have been discovered in various animal species, including wild, livestock, and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses, and human MAVS is likely unable to limit replication of these nonhuman viruses upon zoonotic exposure.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Hepacivirus/enzymology , Hepatitis C/transmission , Serine Proteases/physiology , Viral Nonstructural Proteins/physiology , Zoonoses/transmission , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Amino Acid Motifs , Amino Acid Sequence , Animals , Conserved Sequence , Evolution, Molecular , Genetic Variation , Hepacivirus/immunology , Hepacivirus/pathogenicity , Hepatitis C/immunology , Hepatitis C/virology , Host Specificity , Humans , Immunity, Innate , Serine Proteases/genetics , Signal Transduction , Viral Nonstructural Proteins/genetics , Virus Replication/immunology , Zoonoses/immunology , Zoonoses/virologyABSTRACT
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.
Subject(s)
Bacterial Proteins/physiology , Chaperonin 60/metabolism , Macrophages/immunology , Macrophages/metabolism , Mycobacterium tuberculosis/enzymology , Serine Endopeptidases/physiology , Serine Proteases/physiology , Animals , Bacterial Proteins/metabolism , Cells, Cultured , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Macrophage Activation , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/immunology , Protein Binding , Protein Multimerization , Proteolysis , Serine Endopeptidases/metabolism , Serine Proteases/metabolismABSTRACT
BACKGROUND: The related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown. METHODS: The effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid. RESULTS: Subtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors. CONCLUSIONS: Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention.
Subject(s)
Colorectal Neoplasms/metabolism , Diet , Membrane Proteins/metabolism , Receptors, Cell Surface/metabolism , Serine Proteases/physiology , Tumor Suppressor Proteins/metabolism , Animals , Cell Line, Tumor , Cell Movement , Colorectal Neoplasms/etiology , Colorectal Neoplasms/pathology , DCC Receptor , Environmental Microbiology , Humans , Male , Proteolysis , Rats, WistarABSTRACT
Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.
Subject(s)
Biomarkers, Tumor/immunology , Complement C3b/metabolism , Complement C5/metabolism , Prostate-Specific Antigen/physiology , Prostate/immunology , Proteolysis , Semen/immunology , Serine Proteases/physiology , Animals , Body Fluids/enzymology , Body Fluids/immunology , Cell Line , Humans , Male , Prostate/metabolism , Prostate/pathology , Prostate-Specific Antigen/immunology , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/immunology , Semen/enzymology , SheepABSTRACT
Proteinase-activated receptors (PARs) are a family of G protein-coupled receptor that are activated by extracellular cleavage of the receptor in the N-terminal domain. This slicing of the receptor exposes a tethered ligand which binds to a specific docking point on the receptor surface to initiate intracellular signalling. PARs are expressed by numerous tissues in the body, and they are involved in various physiological and pathological processes such as food digestion, tissue remodelling and blood coagulation. This chapter will summarise how serine proteinases activate PARs leading to the development of pain in several chronic pain conditions. The potential of PARs as a drug target for pain relief is also discussed.
Subject(s)
Pain/etiology , Receptors, Proteinase-Activated/physiology , Animals , Humans , Pain/physiopathology , Receptors, Proteinase-Activated/antagonists & inhibitors , Serine Proteases/physiology , Signal TransductionABSTRACT
Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease.
Subject(s)
Cystic Fibrosis/metabolism , Protease Inhibitors/metabolism , Serine Proteases/physiology , Cathepsin G/physiology , Elafin/physiology , Humans , Leukocyte Elastase/physiology , Myeloblastin/physiology , Neutrophils/enzymology , Secretory Leukocyte Peptidase Inhibitor/physiology , alpha 1-Antitrypsin/physiologyABSTRACT
Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.
Subject(s)
DNA-Binding Proteins/chemistry , Endopeptidases/chemistry , Escherichia coli Proteins/chemistry , Membrane Proteins/chemistry , Serine Proteases/chemistry , DNA-Binding Proteins/physiology , Endopeptidases/physiology , Escherichia coli Proteins/physiology , Membrane Proteins/physiology , Protein Multimerization , Protein Structure, Tertiary , Serine Proteases/physiologyABSTRACT
Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.
Subject(s)
Drosophila melanogaster/enzymology , Metalloendopeptidases/genetics , Reproductive Physiological Phenomena/genetics , Semen/enzymology , Serine Proteases/genetics , Spermatozoa/enzymology , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Female , Fertility/genetics , Gene Knockdown Techniques , Intercellular Signaling Peptides and Proteins , Male , Metalloendopeptidases/metabolism , Metalloendopeptidases/physiology , Metalloproteases/genetics , Metalloproteases/metabolism , Ovulation/genetics , Ovulation/physiology , Peptides/genetics , Peptides/metabolism , Phenotype , Proteolysis , RNA Interference , Semen/physiology , Serine Proteases/metabolism , Serine Proteases/physiology , Sexual Behavior, Animal/physiology , Spermatozoa/physiologyABSTRACT
A growing family of virulence factors called serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by Shigella, Salmonella, and Escherichia coli pathotypes. SPATEs are subdivided into class 1 and class 2 based on structural features and phylogenetics. Class 1 SPATEs induce cytopathic effects in numerous epithelial cell lines, and several have been shown to cleave the cytoskeletal protein spectrin in vitro. However, to date the in vivo role of class 1 SPATEs in enteric pathogenesis is unknown. Citrobacter rodentium, a natural mouse pathogen, has recently been shown to harbor class 1 and class 2 SPATEs. To better understand the contribution of class 1 SPATEs in enteric infection, we constructed a class 1 SPATE null mutant (Δcrc1) in C. rodentium. Upon infection of C57BL/6 mice, the Δcrc1 mutant exhibited a hypervirulent, hyperinflammatory phenotype compared with its parent, accompanied by greater weight loss and a trend toward increased mortality in young mice; the effect was reversed when the crc1 gene was restored. Using flow cytometry, we observed increased infiltration of T cells, B cells, and neutrophils into the lamina propria of the distal colon in mice fed the Δcrc1 mutant, starting as early as 5 days after infection. No significant difference in epithelial cytotoxicity was observed. Reverse transcription-PCR (RT-PCR) analysis of distal colonic tissue on day 10 postinfection showed significant increases in mRNA encoding cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-1ß, and inducible nitric oxide synthase (iNOS) but not in mRNA encoding IL-17, IL-4, or IL-10 in the Δcrc1 mutant-infected mice. Our data suggest a previously unsuspected role for class 1 SPATEs in enteric infection.
Subject(s)
Bacterial Proteins/physiology , Citrobacter rodentium/physiology , Colitis/microbiology , Serine Proteases/physiology , Analysis of Variance , Animals , B-Lymphocytes/cytology , Bacterial Toxins/metabolism , Citrobacter rodentium/genetics , Citrobacter rodentium/immunology , Citrobacter rodentium/pathogenicity , Colitis/immunology , Colon/cytology , Colon/metabolism , Cytokines/metabolism , Disease Models, Animal , Gene Deletion , Gene Expression Regulation, Bacterial , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Neutrophils/cytology , Protein Transport/physiology , RNA, Messenger/metabolism , T-Lymphocytes/cytologyABSTRACT
Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.
Subject(s)
Echinodermata/physiology , Fertilization/physiology , Serine Proteases/physiology , Amino Acid Sequence , Animals , Conserved Sequence , Echinodermata/classification , Echinodermata/genetics , Female , Fertilization/drug effects , Fertilization/genetics , Male , Molecular Sequence Data , Ovum/drug effects , Ovum/physiology , Phylogeny , Sequence Homology, Amino Acid , Serine Proteases/genetics , Serine Proteinase Inhibitors/pharmacology , Sperm-Ovum Interactions/drug effects , Sperm-Ovum Interactions/genetics , Sperm-Ovum Interactions/physiology , Strongylocentrotus purpuratus/drug effects , Strongylocentrotus purpuratus/genetics , Strongylocentrotus purpuratus/physiology , TranscriptomeABSTRACT
Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies.
Subject(s)
Aminopeptidases/deficiency , Cell Proliferation , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/deficiency , Disease Progression , Neuronal Ceroid-Lipofuscinoses/enzymology , Neuronal Ceroid-Lipofuscinoses/pathology , Serine Proteases/deficiency , Aminopeptidases/genetics , Aminopeptidases/physiology , Animals , Animals, Genetically Modified , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/physiology , Disease Models, Animal , Growth Inhibitors/deficiency , Growth Inhibitors/genetics , Growth Inhibitors/physiology , Humans , Motor Activity/physiology , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology , Neuronal Ceroid-Lipofuscinoses/genetics , Serine Proteases/genetics , Serine Proteases/physiology , Tripeptidyl-Peptidase 1 , ZebrafishABSTRACT
The HtrA protease of Streptococcus pneumoniae functions both in a general stress response role and as an error sensor that specifically represses genetic competence when the overall level of biosynthetic errors in cellular proteins is low. However, the mechanism through which HtrA inhibits development of competence has been unknown. We found that HtrA digested the pneumococcal competence-stimulating peptide (CSP) and constituted the primary extracytoplasmic CSP-degrading activity in cultures of S. pneumoniae. Mass spectrometry demonstrated that cleavage predominantly followed residue Phe-8 of the CSP-1 isoform of the peptide within its central hydrophobic patch, and in competition assays, both CSP-1 and CSP-2 interacted with HtrA with similar efficiencies. More generally, analysis of ß-casein digestion and of digestion within HtrA itself revealed a preference for substrates with non-polar residues at the P1 site. Consistent with a specificity for exposed hydrophobic residues, competition from native BSA only weakly inhibited digestion of CSP, but denaturation converted BSA into a strong competitive inhibitor of such proteolysis. Together these findings support a model in which digestion of CSP by HtrA is reduced in the presence of other unfolded proteins that serve as alternative targets for degradation. Such competition may provide a mechanism by which HtrA functions in a quality control capacity to monitor the frequency of biosynthetic errors that result in protein misfolding.
Subject(s)
Peptide Hydrolases/chemistry , Serine Proteases/physiology , Streptococcus pneumoniae/enzymology , Bacterial Physiological Phenomena , Binding Sites , Catalytic Domain , Fluorescence Resonance Energy Transfer , Gene Deletion , Kinetics , Mass Spectrometry/methods , Mutation , Peptides/chemistry , Polymerase Chain Reaction/methods , Protein Denaturation , Proteolysis , Quorum Sensing , Serine Proteases/chemistry , Signal TransductionABSTRACT
Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.
Subject(s)
Membrane Proteins/physiology , Plasmodium yoelii/growth & development , Serine Proteases/physiology , Vacuoles/parasitology , Animals , Hepatocytes/parasitology , Liver/parasitology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Plasmodium yoelii/genetics , Protozoan Proteins/genetics , Serine Proteases/deficiency , Serine Proteases/genetics , Sporozoites/growth & developmentABSTRACT
The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.
Subject(s)
Breast Neoplasms/pathology , Carcinoma/pathology , Lymphokines/metabolism , Platelet-Derived Growth Factor/metabolism , Serine Proteases/physiology , Animals , Autocrine Communication/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma/genetics , Carcinoma/metabolism , Cell Line, Tumor , Disease Progression , Female , Humans , Lymphokines/antagonists & inhibitors , Lymphokines/genetics , Lymphokines/physiology , Mice , NIH 3T3 Cells , Paracrine Communication/genetics , Platelet-Derived Growth Factor/antagonists & inhibitors , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/physiology , Protein Multimerization/genetics , Protein Multimerization/physiology , Protein Processing, Post-Translational , Serine Proteases/genetics , Serine Proteases/metabolism , TransfectionABSTRACT
Proteases play important roles in human physiology and pathology. The complement system is a proteolytic cascade, where serine proteases activate each other by limited proteolysis in a strictly ordered manner. Serine proteases are essential in both the initiation and the amplification of the cascade. Since uncontrolled complement activation contributes to the development of serious disease conditions, inhibition of the complement serine proteases could be an attractive therapeutic approach. In this chapter, we give a brief overview of the major types of natural serine protease inhibitors and their role in controlling the complement cascade. A special emphasis is laid on C1-inhibitor, a natural complement protease inhibitor, which is approved for clinical use in hereditary angioedema (HAE). We also examine the potential of developing artificial complement protease inhibitors. Synthetic small-molecule drugs can be very efficient serine protease inhibitors, but they usually lack sufficient specificity. A promising approach to yield more specific compounds is the alteration of natural protease inhibitors through engineering or directed evolution resulting in new variants with fine-tuned specificity and enhanced affinity.
Subject(s)
Complement System Proteins/physiology , Serine Proteinase Inhibitors/pharmacology , Animals , Complement C1 Inhibitor Protein/pharmacology , Humans , Protein Engineering , Serine Proteases/physiology , Serine Proteinase Inhibitors/therapeutic use , Small Molecule LibrariesABSTRACT
We investigated the involvement of serine protease and proteinase-activated receptor 2 (PAR(2)) in dermatophyte-induced itch in mice. An intradermal injection of an extract of the dermatophyte Arthroderma vanbreuseghemii (ADV) induced hind-paw scratching, an itch-related behavior. ADV extract-induced scratching was inhibited by the opioid receptor antagonists naloxone and naltrexone, the serine protease inhibitor nafamostat mesylate, and the PAR(2) receptor antagonist FSLLRY-NH(2). ADV extract-induced scratching was not inhibited by the H(1) histamine receptor antagonist terfenadine or by mast cell deficiency. Heat pretreatment of the ADV extract markedly reduced the scratch-inducing and serine protease activities. Proteolytic cleavage within the extracellular N terminus of the PAR(2) receptor exposes a sequence that serves as a tethered ligand for the receptor. The ADV extract as well as tryptase and trypsin cleaved a synthetic N-terminal peptide of the PAR(2) receptor. The present results suggest that serine protease secreted by dermatophytes causes itching through activation of the PAR(2) receptors, which may be a causal mechanism of dernatophytosis itch.