RESUMEN
Schistosomiasis, or bilharzia, is a neglected tropical disease caused by Schistosoma spp. blood flukes that infects over 200 million people worldwide. Just one partially effective drug is available, and new drugs and drug targets would be welcome. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania. We previously showed that anticancer proteasome inhibitors that act through the Schistosoma mansoni 20S proteasome (Sm20S) kill the parasite in vitro. To advance these initial findings, we employed Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to define the substrate cleavage specificities of the three catalytic ß subunits of purified Sm20S. The profiles in turn were used to design and synthesize subunit-specific optimized substrates that performed two to eight fold better than the equivalent substrates used to measure the activity of the constitutive human proteasome (c20S). These specific substrates also eliminated the need to purify Sm20S from parasite extracts - a single step enrichment was sufficient to accurately measure substrate hydrolysis and its inhibition with proteasome inhibitors. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar, suggesting that data arising from an inhibitor development campaign that focuses on Sm20S can be extrapolated to the other two targets with consequent time and cost savings.
RESUMEN
The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the ß5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of ß5 plus either ß1 or ß2 results in improved potency.
RESUMEN
Proteolysis is a central regulator of many biological pathways and the study of proteases has had a significant impact on our understanding of both native biology and disease. Proteases are key regulators of infectious disease and misregulated proteolysis in humans contributes to a variety of maladies, including cardiovascular disease, neurodegeneration, inflammatory diseases, and cancer. Central to understanding a protease's biological role, is characterizing its substrate specificity. This chapter will facilitate the characterization of individual proteases and complex, heterogeneous proteolytic mixtures and provide examples of the breadth of applications that leverage the characterization of misregulated proteolysis. Here we present the protocol of Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), a functional assay that quantitatively characterizes proteolysis using a synthetic library of physiochemically diverse, model peptide substrates, and mass spectrometry. We present a detailed protocol as well as examples of the use of MSP-MS for the study of disease states, for the development of diagnostic and prognostic tests, for the generation of tool compounds, and for the development of protease-targeted drugs.
Asunto(s)
Péptido Hidrolasas , Proteómica , Humanos , Péptido Hidrolasas/metabolismo , Proteómica/métodos , Endopeptidasas/metabolismo , Proteolisis , Espectrometría de Masas/métodos , Especificidad por SustratoRESUMEN
BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.
Asunto(s)
Fasciola hepatica , Trematodos , Animales , Ratones , Trematodos/genética , Fasciola hepatica/genética , Schistosoma mansoni , Péptido Hidrolasas , MamíferosRESUMEN
Synaptic dysfunction and loss occur in Alzheimer's disease (AD) brains, which results in cognitive deficits and brain neurodegeneration. Neuropeptides comprise the major group of synaptic neurotransmitters in the nervous system. This study evaluated neuropeptide signatures that are hypothesized to differ in human AD brain compared to age-matched controls, achieved by global neuropeptidomics analysis of human brain cortex synaptosomes. Neuropeptidomics demonstrated distinct profiles of neuropeptides in AD compared to controls consisting of neuropeptides derived from chromogranin A (CHGA) and granins, VGF (nerve growth factor inducible), cholecystokinin, and others. The differential neuropeptide signatures indicated differences in proteolytic processing of their proneuropeptides. Analysis of cleavage sites showed that dibasic residues at the N-termini and C-termini of neuropeptides were the main sites for proneuropeptide processing, and data also showed that the AD group displayed differences in preferred residues adjacent to the cleavage sites. Notably, tau peptide signatures differed in the AD compared to age-matched control human brain cortex synaptosomes. Unique tau peptides were derived from the tau protein through proteolysis using similar and differential cleavage sites in the AD brain cortex compared to the control. Protease profiles differed in the AD compared to control, indicated by proteomics data. Overall, these results demonstrate that dysregulation of neuropeptides and tau peptides occurs in AD brain cortex synaptosomes compared to age-matched controls, involving differential cleavage site properties for proteolytic processing of precursor proteins. These dynamic changes in neuropeptides and tau peptide signatures may be associated with the severe cognitive deficits of AD.
Asunto(s)
Enfermedad de Alzheimer , Neuropéptidos , Proteínas tau/análisis , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Neuropéptidos/análisis , Neuropéptidos/metabolismo , Péptidos/metabolismo , Proteolisis , Proteínas tau/metabolismoRESUMEN
Neuropeptides, functioning as peptide neurotransmitters and hormones, are generated from proneuropeptide precursors by proteolytic processing at dibasic residue sites (i.e., KR, RK, KK, RR). The cysteine proteases cathepsin L and cathepsin V, combined with the serine proteases proprotein convertases 1 and 2 (PC1/3 and PC2), participate in proneuropeptide processing to generate active neuropeptides. To compare the dibasic cleavage properties of these proteases, this study conducted global, unbiased substrate profiling of these processing proteases using a diverse peptide library in multiplex substrate profiling by mass spectrometry (MSP-MS) assays. MSP-MS utilizes a library of 228 14-mer peptides designed to contain all possible protease cleavage sites, including the dibasic residue sites of KR, RK, KK, and RR. The comprehensive MSP-MS analyses demonstrated that cathepsin L and cathepsin V cleave at the N-terminal side and between the dibasic residues (e.g., ↓K↓R, ↓R↓K, and K↓K), with a preference for hydrophobic residues at the P2 position of the cleavage site. In contrast, the serine proteases PC1/3 and PC2 displayed cleavage at the C-terminal side of dibasic residues of a few peptide substrates. Further analyses with a series of dipeptide-AMC and tripeptide-AMC substrates containing variant dibasic sites with hydrophobic P2 residues indicated the preferences of cathepsin L and cathepsin V to cleave between dibasic residue sites with preferences for flanking hydrophobic residues at the P2 position consisting of Leu, Trp, Phe, and Tyr. Such hydrophobic amino acids reside in numerous proneuropeptides such as pro-NPY and proenkephalin that are known to be processed by cathepsin L. Notably, cathepsin L displayed the highest specific activity that was 10-, 64-, and 1268-fold greater than cathepsin V, PC1/3, and PC2, respectively. Peptide-AMC substrates with dibasic residues confirmed that PC1/3 and P2 cleaved almost exclusively at the C-terminal side of dibasic residues. These data demonstrate distinct dibasic cleavage site properties and a broad range of proteolytic activities of cathepsin L and cathepsin V, compared to PC1/3 and PC2, which participate in producing neuropeptides for cell-cell communication.
Asunto(s)
Proteasas de Cisteína , Serina Proteasas , Secuencia de Aminoácidos , Catepsina L/metabolismo , Catepsinas , Procesamiento Proteico-Postraduccional , Serina EndopeptidasasRESUMEN
Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.
Asunto(s)
Catepsina B/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/química , Citosol/metabolismo , Lisosomas/metabolismo , Péptidos/química , Secuencia de Aminoácidos , Sitios de Unión , Catepsinas/metabolismo , Permeabilidad de la Membrana Celular , Inhibidores de Cisteína Proteinasa/metabolismo , Endopeptidasas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectrometría de Masas , Péptidos/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
Neuropeptides mediate cell-cell signaling in the nervous and endocrine systems. The neuropeptidome is the spectrum of peptides generated from precursors by proteolysis within dense core secretory vesicles (DCSV). DCSV neuropeptides and contents are released to the extracellular environment where further processing for neuropeptide formation may occur. To assess the DCSV proteolytic capacity for production of neuropeptidomes at intravesicular pH 5.5 and extracellular pH 7.2, neuropeptidomics, proteomics, and protease assays were conducted using chromaffin granules (CG) purified from adrenal medulla. CG are an established model of DCSV. The CG neuropeptidome consisted of 1239 unique peptides derived from 15 proneuropeptides that were colocalized with 64 proteases. Distinct CG neuropeptidomes were generated at the internal DCSV pH of 5.5 compared to the extracellular pH of 7.2. Class-specific protease inhibitors differentially regulated neuropeptidome production involving aspartic, cysteine, serine, and metallo proteases. The substrate cleavage properties of CG proteases were assessed by multiplex substrate profiling by mass spectrometry (MSP-MS) that uses a synthetic peptide library containing diverse cleavage sites for endopeptidases and exopeptidases. Parallel inhibitor-sensitive cleavages for neuropeptidome production and peptide library proteolysis led to elucidation of six CG proteases involved in neuropeptidome production, represented by cathepsins A, B, C, D, and L and carboxypeptidase E (CPE). The MSP-MS profiles of these six enzymes represented the majority of CG proteolytic cleavages utilized for neuropeptidome production. These findings provide new insight into the DCSV proteolytic system for production of distinct neuropeptidomes at the internal CG pH of 5.5 and at the extracellular pH of 7.2.
Asunto(s)
Médula Suprarrenal , Vesículas Secretoras , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Proteolisis , Vesículas Secretoras/metabolismoRESUMEN
The accumulation and propagation of hyperphosphorylated tau (p-Tau) is a neuropathological hallmark occurring with neurodegeneration of Alzheimer's disease (AD). Extracellular vesicles, exosomes, have been shown to initiate tau propagation in the brain. Notably, exosomes from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) are capable of inducing tau deposits in the mouse brain after in vivo injection. To gain insights into the exosome proteome cargo that participates in propagating tau pathology, this study conducted proteomic analysis of exosomes produced by human iPSC neurons expressing A246E mPS1. Significantly, mPS1 altered the profile of exosome cargo proteins to result in (1) proteins present only in mPS1 exosomes and not in controls, (2) the absence of proteins in the mPS1 exosomes which were present only in controls, and (3) shared proteins which were upregulated or downregulated in the mPS1 exosomes compared to controls. These results show that mPS1 dysregulates the proteome cargo of exosomes to result in the acquisition of proteins involved in the extracellular matrix and protease functions, deletion of proteins involved in RNA and protein translation systems along with proteasome and related functions, combined with the upregulation and downregulation of shared proteins, including the upregulation of amyloid precursor protein. Notably, mPS1 neuron-derived exosomes displayed altered profiles of protein phosphatases and kinases involved in regulating the status of p-tau. The dysregulation of exosome cargo proteins by mPS1 may be associated with the ability of mPS1 neuron-derived exosomes to propagate tau pathology.
RESUMEN
The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated ß-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.
Asunto(s)
Metaloproteasas/metabolismo , Metaloproteasas/fisiología , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Dominio Catalítico , Endopeptidasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Metaloproteasas/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Péptido Hidrolasas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ProteolisisRESUMEN
BACKGROUND: Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1ß (IL-1ß), but the molecular processes involved are not fully understood. METHODS: We examined IL-1ß activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1ß bioactivity. FINDINGS: Caspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1ß proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1ß activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection. INTERPRETATION: Preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1ß regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections. FUNDING: Full details are provided in the Acknowledgements section.
Asunto(s)
Interacciones Huésped-Patógeno , Interleucina-1beta/metabolismo , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/metabolismo , Animales , Biomarcadores , Fibrosis Quística/complicaciones , Fibrosis Quística/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Inmunohistoquímica , Inflamasomas/metabolismo , Mediadores de Inflamación , Metaloproteasas/antagonistas & inhibidores , Ratones , Ratones Noqueados , Modelos Biológicos , Neumonía Bacteriana/etiología , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/patología , Unión Proteica , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patologíaRESUMEN
Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.
Asunto(s)
Péptido Hidrolasas/sangre , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Péptido Hidrolasas/química , Proteómica , Especificidad por Sustrato , PorcinosRESUMEN
Accumulation and propagation of hyperphosphorylated Tau (p-Tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating Tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-Tau pathology after injection into mouse brain. To gain an understanding of the mTau exosome cargo involved in Tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in 1) proteins uniquely present only in mTau, and not control exosomes, 2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and 3) shared proteins which were significantly upregulated or downregulated in mTau compared with control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-Tau. Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes. Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or downregulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-Tau neuropathology in mouse brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Exosomas/metabolismo , Neuronas/metabolismo , Proteómica , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Cromatografía Liquida , Biología Computacional , Exosomas/patología , Ontología de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Neuronas/patología , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Mapas de Interacción de Proteínas , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Espectrometría de Masas en Tándem , Proteínas tau/genéticaRESUMEN
BACKGROUND: Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS: Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α-helix 3 and α-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 µg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION: This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry.
Asunto(s)
Bacillus thuringiensis , Escarabajos , Animales , Proteínas Bacterianas/genética , Quimotripsina , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Larva , Péptido Hidrolasas , TripsinaRESUMEN
Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven ß subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the ß1 and ß5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.
Asunto(s)
Antitricomonas/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Vaginitis por Trichomonas/tratamiento farmacológico , Trichomonas vaginalis/efectos de los fármacos , Trichomonas vaginalis/genética , Secuencia de Aminoácidos , Animales , Antiinfecciosos/farmacología , Citoplasma/parasitología , Resistencia a Medicamentos/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Parasitaria/métodos , Proteómica/métodos , Enfermedades de Transmisión Sexual/tratamiento farmacológico , Enfermedades de Transmisión Sexual/parasitología , Tricomoniasis/tratamiento farmacológico , Tricomoniasis/parasitología , Vaginitis por Trichomonas/parasitologíaRESUMEN
Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 µM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (ß1), trypsin-type (ß2), and chymotrypsin-type (ß5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the ß5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S ß2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 µM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Schistosoma mansoni/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Bortezomib/farmacología , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Células Hep G2 , Humanos , Leupeptinas , Oligopéptidos/farmacologíaRESUMEN
Proteolysis is an integral component of life and has been implicated in many disease processes. To improve our understanding of peptidase function, it is imperative to develop tools to uncover substrate specificity and cleavage efficiency. Here, we combine the quantitative power of tandem mass tags (TMTs) with an established peptide cleavage assay to yield quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS). This assay was validated with papain, a well-characterized cysteine peptidase, to generate cleavage efficiency values for hydrolysis of 275 unique peptide bonds in parallel. To demonstrate the breath of this assay, we show that qMSP-MS can uncover the substrate specificity of minimally characterized intramembrane rhomboid peptidases, as well as define hundreds of proteolytic activities in complex biological samples, including secretions from lung cancer cell lines. Importantly, our qMSP-MS library uses synthetic peptides whose termini are unmodified, allowing us to characterize not only endo- but also exo-peptidase activity. Each cleaved peptide sequence can be ranked by turnover rate, and the amino acid sequence of the best substrates can be used for designing fluorescent reporter substrates. Discovery of peptide substrates that are selectively cleaved by peptidases which are active at the site of disease highlights the potential for qMSP-MS to guide the development of peptidase-activating drugs for cancer and infectious disease.
Asunto(s)
Espectrometría de Masas/métodos , Péptido Hidrolasas/metabolismo , Aspergillus/metabolismo , Línea Celular Tumoral , Fluorescencia , Humanos , Neoplasias Pulmonares/metabolismo , Papaína/metabolismo , Proteolisis , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.
Asunto(s)
Ascomicetos/enzimología , Carboxipeptidasas/metabolismo , Quirópteros/microbiología , Micosis/metabolismo , Animales , Antipaína/farmacología , Carboxipeptidasas/antagonistas & inhibidores , Carboxipeptidasas/aislamiento & purificación , Leupeptinas/farmacología , Micosis/microbiología , SíndromeRESUMEN
Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.
Asunto(s)
Aminopeptidasas/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacteroides/enzimología , Microbioma Gastrointestinal , Glicina/metabolismo , Aminopeptidasas/química , Péptidos Catiónicos Antimicrobianos/química , Bacteroides/química , Bacteroides/metabolismo , Glicina/química , Humanos , Modelos Moleculares , Multimerización de Proteína , Proteolisis , Especificidad por SustratoRESUMEN
Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.