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1.
PLoS Negl Trop Dis ; 14(3): e0007755, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163418

RESUMO

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 µM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.


Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Leishmania mexicana/enzimologia , Nitrilos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Cisteína Endopeptidases , Cisteína Proteases/metabolismo , Humanos
2.
J Agric Food Chem ; 68(8): 2467-2476, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32031791

RESUMO

Enzymatic browning is a major issue affecting the quality of processed potato (Solanum tuberosum L.). To understand the molecular mechanism of browning, transcriptional analyses were performed by employing potatoes that differed in browning. Coexpression analysis indicated that 9 out of 15 upregulated genes in browning-less groups encoded for potato protease inhibitors (StPIs). In addition, gene otology analysis showed that the enriched terms were mainly involved in protease inhibitors. Overexpression of cysteine StPI 143 and StPI 146 individually reduced browning and lowered protease activities and tyrosine and total free amino acid (FAA) contents, but they could not decrease polyphenol oxidase activity. Moreover, supplementing exogenous tyrosine or total FAAs into transgenic potato mash to wild-type amounts promoted mash browning, browning with total FAAs, more than with tyrosine, resembling wild-type levels. These results implied that cysteine StPIs reduced browning via lowering the accumulation of FAAs in addition to tyrosine. Our findings have enriched the knowledge about the roles and mechanisms of protease inhibitors in regulating enzymatic browning of potato, which provide new ways for controlling potato browning.


Assuntos
Aminoácidos/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteases/metabolismo , Solanum tuberosum/metabolismo , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Cor , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Proteínas de Plantas/genética , Solanum tuberosum/enzimologia , Solanum tuberosum/genética
3.
PLoS One ; 15(1): e0227341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923258

RESUMO

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.


Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Malária/enzimologia , Animais , Antimaláricos/química , Permeabilidade da Membrana Celular , Humanos , Malária/diagnóstico por imagem , Malária/tratamento farmacológico , Sondas Moleculares/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Sulfonas , Triptofano
4.
Biochim Biophys Acta Proteins Proteom ; 1868(3): 140362, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31927030

RESUMO

BACKGROUND: The malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review. SCOPE OF REVIEW: This review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites. MAJOR CONCLUSIONS: Based on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites. GENERAL SIGNIFICANCE: Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.


Assuntos
Cisteína Proteases/fisiologia , Plasmodium falciparum/enzimologia , Antimaláricos/uso terapêutico , Cisteína Proteases/química , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Plasmodium/enzimologia , Plasmodium falciparum/genética
5.
Phytochemistry ; 169: 112163, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605904

RESUMO

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest Vmax, Km, Kcat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC50 of around 50 µg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi.


Assuntos
Antifúngicos/farmacologia , Calotropis/enzimologia , Cisteína Proteases/metabolismo , Fusarium/efeitos dos fármacos , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/metabolismo , Biocatálise , Cisteína Proteases/química , Cisteína Proteases/genética , Relação Dose-Resposta a Droga , Fusarium/metabolismo , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Modelos Moleculares , Alinhamento de Sequência , Temperatura
6.
Food Chem ; 307: 125574, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31648178

RESUMO

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.


Assuntos
Calotropis/enzimologia , Caseínas/metabolismo , Queijo , Cisteína Proteases/metabolismo , Animais , Bovinos , Quimosina/metabolismo , Hidrólise , Látex/metabolismo , Proteínas de Plantas/metabolismo
7.
BMC Genomics ; 20(1): 996, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856729

RESUMO

BACKGROUND: The glasshouse whitefly, Trialeurodes vaporariorum, is a damaging crop pest and an invasive generalist capable of feeding on a broad range of host plants. As such this species has evolved mechanisms to circumvent the wide spectrum of anti-herbivore allelochemicals produced by its host range. T. vaporariorum has also demonstrated a remarkable ability to evolve resistance to many of the synthetic insecticides used for control. RESULTS: To gain insight into the molecular mechanisms that underpin the polyphagy of T. vaporariorum and its resistance to natural and synthetic xenobiotics, we sequenced and assembled a reference genome for this species. Curation of genes putatively involved in the detoxification of natural and synthetic xenobiotics revealed a marked reduction in specific gene families between this species and another generalist whitefly, Bemisia tabaci. Transcriptome profiling of T. vaporariorum upon transfer to a range of different host plants revealed profound differences in the transcriptional response to more or less challenging hosts. Large scale changes in gene expression (> 20% of genes) were observed during adaptation to challenging hosts with a range of genes involved in gene regulation, signalling, and detoxification differentially expressed. Remarkably, these changes in gene expression were associated with significant shifts in the tolerance of host-adapted T. vaporariorum lines to natural and synthetic insecticides. CONCLUSIONS: Our findings provide further insights into the ability of polyphagous insects to extensively reprogram gene expression during host adaptation and illustrate the potential implications of this on their sensitivity to synthetic insecticides.


Assuntos
Regulação da Expressão Gênica de Plantas , Hemípteros/genética , Resistência a Inseticidas/genética , Adaptação Fisiológica/genética , Animais , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes de Insetos , Genoma de Inseto , Hemípteros/enzimologia , Hemípteros/metabolismo , Interações Hospedeiro-Patógeno/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas , Plantas , RNA-Seq , Transdução de Sinais/genética , Transcriptoma , Xenobióticos/metabolismo
8.
PLoS One ; 14(12): e0222055, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856175

RESUMO

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.


Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Domínio Catalítico , Catepsina L/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Doença de Chagas , Cisteína Endopeptidases/metabolismo , Cisteína Proteases/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Análise de Componente Principal/métodos , Ligação Proteica , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Trypanosoma cruzi/metabolismo
9.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527129

RESUMO

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1ß, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.


Assuntos
Entamoeba histolytica/patogenicidade , Entamebíase/patologia , Escherichia coli Enteropatogênica/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Animais , Células CACO-2 , Linhagem Celular , Cricetinae , Cisteína Proteases/metabolismo , Citocinas/metabolismo , Entamoeba histolytica/microbiologia , Células HT29 , Humanos , Inflamação , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Mucina-2/metabolismo , Fatores de Virulência/biossíntese
10.
Parasit Vectors ; 12(1): 383, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362766

RESUMO

BACKGROUND: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). RESULTS: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. CONCLUSIONS: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.


Assuntos
Antígenos de Helmintos/imunologia , Calpaína/genética , Calpaína/metabolismo , Schistosoma/enzimologia , Animais , Antígenos de Helmintos/genética , Cumarínicos/metabolismo , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Feminino , Imunização , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Schistosoma/genética , Esquistossomose/imunologia , Esquistossomose/parasitologia , Análise de Sequência de DNA
11.
Infect Genet Evol ; 75: 103975, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31344488

RESUMO

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cisteína Proteases/genética , Infecções por Enterovirus/veterinária , Enterovirus Suínos/genética , Proteínas Estruturais Virais/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Cisteína Proteases/metabolismo , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Fezes/virologia , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Filogenia , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia
12.
Biotechnol Lett ; 41(8-9): 1043-1050, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286326

RESUMO

OBJECTIVE: To determine the enzymatic properties of asclepain f, a plant cysteine protease isolated and purified from the latex of Asclepias fruticosa, and to investigate its potential application to hydrolyze soybean proteins. RESULTS: Kinetic parameters were determined by hydrolysis of p-Glu-Phe-Leu-p-nitroanilide (PFLNA). The Km value for asclepain f was 6 to 8 times higher than those achieved for papain, bromelain and ficin, the main plant cysteine proteases. Asclepain f showed 12 cut-off points toward the oxidized B chain insulin, revealing that the enzyme possesses broad substrate specificity. The cut specificity was governed by the presence of hydrophobic residues (F, L, V) in the P2 position. Asclepain f was able to selectively hydrolyze soybean proteins at pH 10, employing an enzyme/substrate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity. CONCLUSIONS: Asclepain f was revealed as a successful enzyme for biocatalysis of protein hydrolysis processes at alkaline pH. This new plant protease has a broad substrate specificity and is capable of selectively degrading the fractions of soy proteins and improving its functional properties.


Assuntos
Apocynaceae/enzimologia , Cisteína Proteases/metabolismo , Proteínas de Soja/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Proteólise , Especificidade por Substrato
13.
Eur J Med Chem ; 179: 765-778, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284086

RESUMO

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 µM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 µM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs.


Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Descoberta de Drogas , Malária/tratamento farmacológico , Schistosoma mansoni/metabolismo , Tripanossomicidas/farmacologia , Animais , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Malária/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Schistosoma mansoni/efeitos dos fármacos , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo
14.
mBio ; 10(4)2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337717

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTU's immunosuppressive function. Instead, the prolonged occupancy of the OTU with UbV-CC4 directly targets viral replication by interfering with CCHFV RNA synthesis. Together, our data provide mechanistic details supporting the development of antivirals targeting viral OTUs.IMPORTANCE Crimean-Congo hemorrhagic fever virus is an important human pathogen with a wide global distribution for which no therapeutic interventions are available. CCHFV encodes a cysteine protease belonging to the ovarian tumor (OTU) family which is involved in host immune suppression. Here we demonstrate that artificially prolonged binding of the OTU to a substrate inhibits virus infection. This provides novel insights into CCHFV OTU function during the viral replicative cycle and highlights the OTU as a potential antiviral target.


Assuntos
Cisteína Proteases/metabolismo , Enzimas Desubiquitinantes/metabolismo , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Ubiquitina/farmacologia , Replicação Viral , Animais , Linhagem Celular Tumoral , Cisteína Proteases/genética , Citocinas/genética , Citocinas/metabolismo , Enzimas Desubiquitinantes/genética , Feminino , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Humanos , Camundongos , Mutação , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1866(12): 118517, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31340164

RESUMO

Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca2+ influx via store-operated Ca2+ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca2+-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca2+ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.


Assuntos
Cálcio/metabolismo , Catepsinas/metabolismo , Cisteína Proteases/metabolismo , Lisossomos/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Cicatrização/efeitos dos fármacos
16.
Biochimie ; 166: 184-193, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31194996

RESUMO

Dozens of studies have assessed the practical value of plant cystatins as ectopic inhibitors of Cys proteases in biological systems. The potential of these proteins in crop protection to control herbivorous pests and pathogens has been documented extensively over the past 25 years. Their usefulness to regulate endogenous Cys proteases in planta has also been considered recently, notably to implement novel traits of agronomic relevance in crops or to generate protease activity-depleted environments in plants or plant cells used as bioreactors for recombinant proteins. After a brief update on the basic structural characteristics of plant cystatins, we summarize recent advances on the use of these proteins in plant biotechnology. Attention is also paid to the molecular improvement of their structural properties for the improvement of their protease inhibitory effects or the fine-tuning of their biological target range.


Assuntos
Cistatinas , Inibidores de Cisteína Proteinase , Proteínas de Plantas , Plantas/metabolismo , Biotecnologia , Cistatinas/química , Cistatinas/fisiologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Controle Biológico de Vetores , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia
17.
Int J Parasitol ; 49(9): 697-704, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31254529

RESUMO

Trichomonas vaginalis is a primary urogenital parasite that causes trichomoniasis, a common sexually transmitted disease. As the first line of host defense, vaginal epithelial cells play critical roles in orchestrating vaginal innate immunity and modulate intracellular Cl- homeostasis via the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that plays positive roles in regulating nuclear factor-κB (NF-κB) signalling. However, the association between T. vaginalis infection and intracellular Cl- disequilibrium remains elusive. This study showed that after T. vaginalis infection, CFTR was markedly down-regulated by cysteine proteases in vaginal epithelial cells. The intracellular Cl- concentration ([Cl-]i) was consequently elevated, leading to NF-κB signalling activation via serum- and glucocorticoid-inducible kinase-1. Moreover, heightened [Cl-]i and activated NF-κB signalling could be sustained in a positive feedback regulatory manner resulting from decreased intracellular cAMP through NF-κB-mediated up-regulation of phosphodiesterase 4. The results conclusively revealed that the intracellular Cl- of the human vaginal epithelium could be dynamically modulated by T. vaginalis, which contributed to mediation of epithelial inflammation in the human vagina.


Assuntos
Cloretos/metabolismo , Vaginite por Trichomonas/prevenção & controle , Trichomonas vaginalis/efeitos dos fármacos , Vagina/patologia , Western Blotting , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Cisteína Proteases/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Epitélio/parasitologia , Epitélio/patologia , Feminino , Humanos , Proteínas Imediatamente Precoces/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Vaginite por Trichomonas/parasitologia , Vagina/metabolismo , Vagina/parasitologia
18.
Virology ; 533: 21-33, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31078932

RESUMO

Cavally virus (CavV) is a mosquito-borne plus-strand RNA virus in the family Mesoniviridae (order Nidovirales). We present X-ray structures for the CavV 3C-like protease (3CLpro), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the P4-to-P1 residues of a natural substrate. The 3CLpro structure (refined to 1.94 Å) shows that the protein forms dimers. The monomers are comprised of N-terminal domains I and II, which adopt a chymotrypsin-like fold, and a C-terminal α-helical domain III. The catalytic Cys-His dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic His and a conserved Asp located in the domain II-III junction and is suitably positioned to stabilize the developing positive charge of the catalytic His in the transition state during catalysis. The study also reveals the structural basis for the distinct P2 Asn-specific substrate-binding pocket of mesonivirus 3CLpros.


Assuntos
Culicidae/virologia , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Nidovirales/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Cisteína Proteases/genética , Nidovirales/química , Nidovirales/genética , Alinhamento de Sequência , Especificidade por Substrato , Proteínas Virais/genética
19.
Protein J ; 38(5): 598-607, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31119598

RESUMO

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M-1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10-100 µM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.


Assuntos
Cistatina C/química , Cistatina C/metabolismo , Cisteína Proteases/metabolismo , Cistatina C/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Termodinâmica
20.
Proc Natl Acad Sci U S A ; 116(16): 7831-7836, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30944220

RESUMO

Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value.


Assuntos
Ciclotídeos , Cisteína Proteases , Papaína , Proteínas de Plantas , Ciclotídeos/química , Ciclotídeos/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Defensinas/química , Defensinas/metabolismo , Modelos Moleculares , Papaína/química , Papaína/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
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