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1.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
2.
World J Microbiol Biotechnol ; 35(8): 122, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346836

RESUMO

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Bacteriano/isolamento & purificação , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Bacteriano/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Nat Commun ; 10(1): 2876, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253842

RESUMO

Osteoarthritis (OA) is a common, painful disease. Currently OA is incurable, and its etiology largely unknown, partly due to limited understanding of OA as a whole-joint disease. Here we report that two homologous microRNAs, miR-204 and miR-211, maintain joint homeostasis to suppress OA pathogenesis. Specific knockout of miR-204/-211 in mesenchymal progenitor cells (MPCs) results in Runx2 accumulation in multi-type joint cells, causing whole-joint degeneration. Specifically, miR-204/-211 loss-of-function induces matrix-degrading proteases in articular chondrocytes and synoviocytes, stimulating articular cartilage destruction. Moreover, miR-204/-211 ablation enhances NGF expression in a Runx2-dependent manner, and thus hyper-activates Akt signaling and MPC proliferation, underlying multiplex non-cartilaginous OA conditions including synovial hyperplasia, osteophyte outgrowth and subchondral sclerosis. Importantly, miR-204/-211-deficiency-induced OA is largely rescued by Runx2 insufficiency, confirming the miR-204/-211-Runx2 axis. Further, intraarticular administration of miR-204-expressing adeno-associated virus significantly decelerates OA progression. Collectively, miR-204/-211 are essential in maintaining healthy homeostasis of mesenchymal joint cells to counteract OA pathogenesis.


Assuntos
Células-Tronco Mesenquimais/fisiologia , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Animais , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Osteoartrite/etiologia , Osteoartrite/patologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sinoviócitos/metabolismo
4.
Gene ; 711: 143934, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31228540

RESUMO

Phytopathogenic fungi secrete a wide range of enzymes to penetrate and colonize host tissues. Of them protease activity is reported to increase disease aggressiveness in the plant. With the aim to explore the reason of the higher infection potential of proteases, we have compared several genomic and proteomic attributes among different hydrolytic enzymes coded by five pathogenic fungal species which are the potent infectious agents of plant. Categorizing the enzymes into four major groups, namely protease, lipase, amylase and cell-wall degraders, we observed that proteases are evolutionary more conserved, have higher expression levels, contain more hydrophobic buried residues, short linear motifs and post-translational modified (PTM) sites than the other three groups of enzymes. Again, comparing these features of protease between pathogenic and non-pathogenic Aspergillus sps, we have hypothesized that protein structural properties could play significant roles in imposing infection potency to the fungal proteases.


Assuntos
Aspergillus/patogenicidade , Biologia Computacional/métodos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Aspergillus/classificação , Aspergillus/genética , Simulação por Computador , Sequência Conservada , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteômica/métodos
5.
Indian Pediatr ; 56(4): 311-313, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31064901

RESUMO

OBJECTIVE: To determine the diagnostic accuracy of polymerase chain reaction-based detection of sof gene compared to throat swab culture for S. pyogenes infection in patients with acute rheumatic fever and those with recurrence of rheumatic activity. METHODS: 40 patients between 3 to 18 years of age, with clinical diagnosis of acute rheumatic fever or new activity in established rheumatic heart disease were included. The amplicon of 228bp of sof gene was detected using a polymerase chain reaction-based technique and the results were compared with throat swab culture for Streptococcus pyogenes. RESULTS: 10 patients had a positive throat swab culture and 11 had sof gene detected. The sensitivity and specificity of the test was 100% and 96.7%, respectively compared to throat swab culture (P=0.001). The positive predictive value and the negative predictive value was 90.9% and 100% respectively. CONCLUSION: Polymerase chain reaction-based detection of sof gene provides an alternative to throat swab culture in diagnosing activity in Acute Rheumatic Fever or established Rheumatic heart disease.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Peptídeo Hidrolases/genética , Febre Reumática/diagnóstico , Infecções Estreptocócicas/diagnóstico , Streptococcus pyogenes , Adolescente , Criança , Pré-Escolar , Estudos Transversais , DNA Bacteriano/análise , DNA Bacteriano/genética , Humanos , Índia , Faringe/microbiologia , Reação em Cadeia da Polimerase , Febre Reumática/microbiologia , Sensibilidade e Especificidade , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação
6.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035313

RESUMO

Seed storage proteins must be hydrolyzed by proteases to deliver the amino acids essential for embryo growth and development. Several groups of proteases involved in this process have been identified in both the monocot and the dicot species. This review focuses on the implication of proteases during germination in two cereal species, barley and wheat, where proteolytic control during the germination process has considerable economic importance. Formerly, the participation of proteases during grain germination was inferred from reports of proteolytic activities, the expression of individual genes, or the presence of individual proteins and showed a prominent role for papain-like and legumain-like cysteine proteases and for serine carboxypeptidases. Nowadays, the development of new technologies and the release of the genomic sequences of wheat and barley have permitted the application of genome-scale approaches, such as those used in functional genomics and proteomics. Using these approaches, the repertoire of proteases known to be involved in germination has increased and includes members of distinct protease families. The development of novel techniques based on shotgun proteomics, activity-based protein profiling, and comparative and structural genomics will help to achieve a general view of the proteolytic process during germination.


Assuntos
Germinação/fisiologia , Hordeum/enzimologia , Hordeum/fisiologia , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Triticum/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Germinação/genética , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética
7.
BMC Genomics ; 20(1): 430, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138126

RESUMO

BACKGROUND: The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. RESULTS: The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. CONCLUSIONS: The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.


Assuntos
Genoma Fúngico , Lignina/metabolismo , Polyporales/genética , Transportadores de Cassetes de Ligação de ATP/genética , Metabolismo dos Carboidratos , Celulose/metabolismo , Genômica , Pectinas/metabolismo , Peptídeo Hidrolases/genética , Polyporales/enzimologia , Polissacarídeos/metabolismo , Metabolismo Secundário/genética
8.
Mol Phylogenet Evol ; 135: 78-85, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30836149

RESUMO

Cereblon (CRBN) is a substrate receptor for an E3 ubiquitin ligase that directly binds to target proteins resulting in cellular activities, such as energy metabolism, membrane potential regulation, and transcription factor degradation. Genetic mutations in human CRBN lead to intellectual disabilities. In addition, it draws pathological attention because direct binding with immunomodulatory drugs can cure multiple myeloma (MM) and lymphocytic leukemia. To further explore the function of CRBN, we focused on its molecular evolution. Since CRBN interacts directly with its substrates and is widely conserved in vertebrates, evolutionary study to identify the selective pressure on CRBN that occur during CRBN-substrate interaction is an effective approach to search for a novel active site. Using mammalian CRBN sequences, dN/dS analysis was conducted to detect positive selection. By multiple sequence alignment we found that the residue at position 366 was under positive selection. This residue is present in the substrate-binding domain of CRBN. Most mammals harbor cysteine at position 366, whereas rodents and chiroptera have serine at this site. Subsequently, we constructed a C366S human CRBN to confirm the potential of positive selection. Auto-ubiquitination activity occurs in E3 ubiquitin ligases, including CRBN, and increased in C366S CRBN, which lead to the conclusion that E3 ubiquitin ligase activity may have changed over the course of mammalian evolution. Furthermore, binding with AMP-activated protein kinase was augmented when the substitution was present, which is supported by coevolution analysis. These results suggest that the molecular evolution of CRBN occurred through codon-based positive selection, providing a new approach to investigate CRBN function.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Seleção Genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Códon/genética , Evolução Molecular , Humanos , Mamíferos/metabolismo , Mutação/genética , Filogenia , Ligação Proteica , Ubiquitinação
9.
PLoS Negl Trop Dis ; 13(3): e0007202, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30830907

RESUMO

Dengue virus (DENV) is the most important vector-borne virus globally. The safe and effective vaccines are still under development and there are no antiviral drugs for DENV induced diseases. In this study, we obtained five DENV1 isolates (DENV1 A to E) from the outbreak of dengue fever in 2014 of Guangzhou, China, and analyzed their replication efficiency and virulence in vitro and in vivo. The results suggested that among the five DENV1 strains, DENV1 B has the highest replication efficiency in both human and mosquito cells in vitro, also causes the highest mortality to suckling mice. Further study suggested that nonstructural proteins from DENV1B have higher capacity to suppress host interferon signaling. In addition, the NS2B3 protease from DENV1B has higher enzymatic activity compared with that from DENV1 E. Finally, we identified that the 64th amino acid of NS2A and the 55th amino acid of NS2B were two virulence determining sites for DENV1. This study provided new evidences of the molecular mechanisms of DENV virulence.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Dengue/virologia , Animais , China , Culicidae , Dengue/sangue , Dengue/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Virulência , Replicação Viral/genética
10.
Microb Pathog ; 131: 1-8, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30902730

RESUMO

As one of the dominant bacteria in the ocean, Vibrio play important roles in maintaining the aquatic ecosystem. In this study, we studied the phylogenetic relationships of 32 Vibrio based on the 16S rRNA genes sequences and utilized substrate immersing zymography method to detect the trend of protease production and components of multiprotease system of Vibrio extracellular proteases. The result showed that different extracellular proteolytic profiles among various Vibrio strains demonstrated a large interspecific variation, and for strains from the same environments, the closer the evolutionary relationship of them, the more similar their zymograms were. In addition, these proteases displayed very different hydrolysis abilities to casein and gelatin. Moreover, the results of the inhibitor-substrate immersing zymography indicated that the proteases secreted by marine Vibrio mostly belonged to serine proteases or metalloproteases. These results implied that combined taxonomic information of the Vibrios with their extracellular protease zymograms maybe contributed to the study of the classification, phylogeny and pathogenic mechanism of Vibrio, and can serve as a theoretical basis for controlling the pathogenic Vibrio disease as well as exploiting proteases. More importantly, we can also eliminate many similar strains by this way, thus can greatly reduce the workload of the experiments for us.


Assuntos
Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/genética , Filogenia , Vibrio/enzimologia , Vibrio/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Peixes/microbiologia , Genes Essenciais/genética , Metaloproteases/genética , Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Análise de Sequência , Serina Proteases/genética , Serina Proteases/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Vibrio/classificação , Vibrio/patogenicidade
11.
Nat Commun ; 10(1): 1360, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911012

RESUMO

TonB-dependent transporters (TBDTs) are ubiquitous outer membrane ß-barrel proteins that import nutrients and bacteriocins across the outer membrane in a proton motive force-dependent manner, by directly connecting to the ExbB/ExbD/TonB system in the inner membrane. Here, we show that the TBDT Oar in Myxococcus xanthus is required for secretion of a protein, protease PopC, to the extracellular milieu. PopC accumulates in the periplasm before secretion across the outer membrane, and the proton motive force has a role in secretion to the extracellular milieu. Reconstitution experiments in Escherichia coli demonstrate that secretion of PopC across the outer membrane not only depends on Oar but also on the ExbB/ExbD/TonB system. Our results indicate that TBDTs and the ExbB/ExbD/TonB system may have roles not only in import processes but also in secretion of proteins.


Assuntos
Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Myxococcus xanthus/genética , Peptídeo Hidrolases/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Myxococcus xanthus/classificação , Myxococcus xanthus/metabolismo , Peptídeo Hidrolases/metabolismo , Periplasma/metabolismo , Filogenia , Força Próton-Motriz
12.
G3 (Bethesda) ; 9(4): 1065-1074, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30723102

RESUMO

Hybrid male sterility (HMS) is a form of postmating postzygotic isolation among closely related species that can act as an effective barrier to gene flow. The Dobzhansky-Muller model provides a framework to explain how gene interactions can cause HMS between species. Genomics highlights the preponderance of non-coding DNA targets that could be involved in gene interactions resulting in gene expression changes and the establishment of isolating barriers. However, we have limited knowledge of changes in gene expression associated with HMS, gene interacting partners linked to HMS, and whether substitutions in DNA regulatory regions (cis) causes misexpression (i.e., expression of genes beyond levels found in parental species) of HMS genes in sterile hybrids. A previous transcriptome survey in a pair of D. pseudoobscura species found male reproductive tract (MRT) proteases as the largest class of genes misregulated in sterile hybrids. Here we assay gene expression in backcross (BC) and introgression (IG) progeny, along with site of expression within the MRT, to identify misexpression of proteases that might directly contribute to HMS. We find limited evidence of an accumulation of cis-regulatory changes upstream of such candidate HMS genes. The expression of four genes was differentially modulated by alleles of the previously characterized HMS gene Ovd.


Assuntos
Drosophila/fisiologia , Hibridização Genética , Infertilidade Masculina/genética , Proteínas de Insetos/genética , Peptídeo Hidrolases/genética , Animais , Drosophila/genética , Fluxo Gênico , Proteínas de Insetos/metabolismo , Proteínas de Insetos/fisiologia , Masculino , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/fisiologia , Sequências Reguladoras de Ácido Nucleico , Testículo/metabolismo
13.
Food Chem ; 281: 197-203, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30658748

RESUMO

Aspartic proteases are promising fining agents used in the production of fruit juices. In this study, a novel aspartic protease gene (Tlap) was identified in Talaromyces leycettanus JCM12802 and heterologously expressed in Pichia pastoris. Using casein as the substrate, purified recombinant TlAP showed optimal activities at pH 3.0 and 55 °C with a specific activity of 1795.4 ±â€¯62.8 U/mg, and remained stable over a pH range of 3.0-6.0 and at temperatures of 45 °C and below. Moreover, the enzyme was highly resistant to most metal ions and chemical reagents except for Fe3+ and ß-mercaptoethanol. When added to apple, orange, grape and kiwi fruit juice, it showed excellent proteolytic activity against haze-forming proteins, decreasing the turbidity by up to 49.9 nephelometry turbidity units (NTU). These favorable enzymatic properties make TlAP attractive for potential use in the juice industry.


Assuntos
Microbiologia de Alimentos , Sucos de Frutas e Vegetais , Proteínas Fúngicas/genética , Peptídeo Hidrolases/metabolismo , Talaromyces/enzimologia , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Int J Biol Macromol ; 128: 254-267, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30664968

RESUMO

Proteases are a group of large complex enzyme molecules that perform highly focused proteolysis functions. A vast quantity of the protease enzymes is predominantly sourced from microbial fermentation process, although proteases tend to natively present in plant, animals and humans. Proteases possess a pervasive importance in medical and pharmaceutical sector, because of its enriched specificity towards biomolecules. They are also actively encompassed in regulating certain physiological pathways. A distinct territory of human disorders is treated by substrate specific proteases. Enormous numbers of catalytic activities in habitual metabolism process of a living organism are protease dependent. Pilot scale researches and product development in industrial biotechnology sectors are wholly based on any one of the protease enzymes. The applications of the protease enzymes and its economic benefits of being an eco-friendly material are far-reaching. This review presents a brief overview on the classification and sources of various types of proteases. We describe the essential evidences of role of protease in different sectors. The proteases could be a potential relieves to harmful synthetic chemicals in distinctive industrial processes and thus gains global perception.


Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Animais , Biotecnologia , Indústria Farmacêutica , Fermentação , Indústria Alimentícia , Engenharia Genética , Humanos , Peptídeo Hidrolases/classificação , Peptídeo Hidrolases/genética , Plantas/enzimologia , Pesquisa
15.
J Biol Chem ; 294(11): 4259-4271, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30647130

RESUMO

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden on global human health. A vital process for norovirus replication is the processing of the nonstructural polyprotein by a viral protease into the viral components required to form the viral replication complex. This cleavage occurs at different rates, resulting in the accumulation of stable precursor forms. Here, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors, we demonstrated that all of them are proteolytically active in vitro, but that when expressed in cells, their activities are determined by both substrate and protease localization. Although all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking the NS4 protein were capable of efficiently cleaving a cytoplasmic substrate. By mapping the full range of protein-protein interactions among murine and human norovirus proteins with the LUMIER assay, we uncovered conserved interactions between replication complex members that modify the localization of a protease precursor subset. Finally, we demonstrate that fusion to the membrane-bound replication complex components permits efficient cleavage of a fused substrate when active polyprotein-derived protease is provided in trans These findings offer a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. Because the norovirus protease represents a key target in antiviral therapies, an improved understanding of its function and regulation, as well as identification of interactions among the other nonstructural proteins, offers new avenues for antiviral drug design.


Assuntos
Norovirus/enzimologia , Norovirus/metabolismo , Peptídeo Hidrolases/metabolismo , Poliproteínas/metabolismo , Replicação Viral , Animais , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Linhagem Celular , Células HeLa , Humanos , Camundongos , Norovirus/genética , Peptídeo Hidrolases/genética , Ligação Proteica , Replicação Viral/genética
16.
Viruses ; 11(1)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650571

RESUMO

Many plant viruses express their proteins through a polyprotein strategy, requiring the acquisition of protease domains to regulate the release of functional mature proteins and/or intermediate polyproteins. Positive-strand RNA viruses constitute the vast majority of plant viruses and they are diverse in their genomic organization and protein expression strategies. Until recently, proteases encoded by positive-strand RNA viruses were described as belonging to two categories: (1) chymotrypsin-like cysteine and serine proteases and (2) papain-like cysteine protease. However, the functional characterization of plant virus cysteine and serine proteases has highlighted their diversity in terms of biological activities, cleavage site specificities, regulatory mechanisms, and three-dimensional structures. The recent discovery of a plant picorna-like virus glutamic protease with possible structural similarities with fungal and bacterial glutamic proteases also revealed new unexpected sources of protease domains. We discuss the variety of plant positive-strand RNA virus protease domains. We also highlight possible evolution scenarios of these viral proteases, including evidence for the exchange of protease domains amongst unrelated viruses.


Assuntos
Peptídeo Hidrolases/química , Vírus de Plantas/enzimologia , Vírus de RNA/enzimologia , Proteínas Virais/química , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Evolução Molecular , Peptídeo Hidrolases/genética , Vírus de Plantas/genética , Poliproteínas/genética , Vírus de RNA/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Proteases/química , Serina Proteases/genética , Proteínas Virais/genética
17.
Gastroenterology ; 156(7): 1951-1968.e1, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30660731

RESUMO

Since the discovery of the first trypsinogen mutation in families with hereditary pancreatitis, pancreatic genetics has made rapid progress. The identification of mutations in genes involved in the digestive protease-antiprotease pathway has lent additional support to the notion that pancreatitis is a disease of autodigestion. Clinical and experimental observations have provided compelling evidence that premature intrapancreatic activation of digestive proteases is critical in pancreatitis onset. However, disease course and severity are mostly governed by inflammatory cells that drive local and systemic immune responses. In this article, we review the genetics, cell biology, and immunology of pancreatitis with a focus on protease activation pathways and other early events.


Assuntos
Pâncreas , Pancreatite , Animais , Apoptose , Ativação Enzimática , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação/metabolismo , Mutação , Necrose , Pâncreas/enzimologia , Pâncreas/imunologia , Pâncreas/patologia , Pâncreas/fisiopatologia , Pancreatite/enzimologia , Pancreatite/genética , Pancreatite/patologia , Pancreatite/fisiopatologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Fenótipo , Prognóstico , Dobramento de Proteína , Fatores de Risco , Transdução de Sinais
18.
Genome Biol Evol ; 11(3): 644-659, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30698789

RESUMO

The PSD-95/Dlg-A/ZO-1 (PDZ) domain is highly expanded, diversified, and well distributed across metazoa where it assembles diverse signaling components by virtue of interactions with other proteins in a sequence-specific manner. In contrast, in the microbial world they are reported to be involved in protein quality control during stress response. The distribution, functions, and origins of PDZ domain-containing proteins in the prokaryotic organisms remain largely unexplored. We analyzed 7,852 PDZ domain-containing proteins in 1,474 microbial genomes in this context. PDZ domain-containing proteins from planctomycetes, myxobacteria, and other eubacteria occupying terrestrial and aquatic niches are found to be in multiple copies within their genomes. Over 93% of the 7,852 PDZ domain-containing proteins were classified into 12 families including six novel families based on additional structural and functional domains present in these proteins. The higher PDZ domain encoding capacity of the investigated organisms was observed to be associated with adaptation to the ecological niche where multicellular life might have originated and flourished. Predicted subcellular localization of PDZ domain-containing proteins and their genomic context argue in favor of crucial roles in translation and membrane remodeling during stress response. Based on rigorous sequence, structure, and phylogenetic analyses, we propose that the highly diverse PDZ domain of the uncharacterized Fe-S oxidoreductase superfamily, exclusively found in gladobacteria and several anaerobes and acetogens, might represent the most ancient form among all the existing PDZ domains.


Assuntos
Evolução Biológica , Genoma Bacteriano , Genoma Fúngico , Família Multigênica , Domínios PDZ , Genes Microbianos , Oxirredutases/genética , Peptídeo Hidrolases/genética , Biossíntese de Proteínas , Estresse Fisiológico
19.
Braz J Microbiol ; 50(1): 85-97, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30623303

RESUMO

Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5-3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0-1.5 M NaCl concentration, pH 7-8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions.


Assuntos
Atriplex/microbiologia , Bactérias/enzimologia , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cloreto de Sódio/metabolismo , Microbiologia do Solo , Bactérias/classificação , Bactérias/metabolismo , Proteínas de Bactérias/genética , Biodiversidade , Celulases/genética , Celulases/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Lagos/microbiologia , Lipase/genética , Lipase/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Filogenia , Rizosfera
20.
PLoS Pathog ; 15(1): e1007515, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629698

RESUMO

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.


Assuntos
Nairovirus/genética , Neoplasias Ovarianas/virologia , Peptídeo Hidrolases/genética , Cristalografia por Raios X/métodos , Enzimas Desubiquitinantes/metabolismo , Feminino , Variação Genética/genética , Genômica , Humanos , Nairovirus/patogenicidade , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Peptídeo Hidrolases/metabolismo , Filogenia , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional/genética , Proteólise , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
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