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1.
Sci Rep ; 13(1): 338, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611064

RESUMO

Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin binding protein with deubiquitinase (DUB) catalytic activity. Rare MYSM1 mutations in human patients result in an inherited bone marrow failure syndrome, highlighting the biomedical significance of MYSM1 in the hematopoietic system. We and others characterized Mysm1-knockout mice as a model of this disorder and established that MYSM1 regulates hematopoietic function and leukocyte development in such models through different mechanisms. It is, however, unknown whether the DUB catalytic activity of MYSM1 is universally required for its many functions and for the maintenance of hematopoiesis in vivo. To test this, here we generated a new mouse strain carrying a Mysm1D660N point mutation (Mysm1DN) and demonstrated that the mutation renders MYSM1 protein catalytically inactive. We characterized Mysm1DN/DN and Mysm1fl/DN CreERT2 mice, against appropriate controls, for constitutive and inducible loss of MYSM1 catalytic function. We report a profound similarity in the developmental, hematopoietic, and immune phenotypes resulting from the loss of MYSM1 catalytic function and the full loss of MYSM1 protein. Overall, our work for the first time establishes the critical role of MYSM1 DUB catalytic activity in vivo in hematopoiesis, leukocyte development, and other aspects of mammalian physiology.


Assuntos
Endopeptidases , Proteases Específicas de Ubiquitina , Humanos , Camundongos , Animais , Endopeptidases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Diferenciação Celular , Hematopoese/genética , Mutação , Células-Tronco Hematopoéticas/metabolismo , Camundongos Knockout , Mamíferos/metabolismo , Transativadores/metabolismo
2.
Neoplasia ; 36: 100871, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36610378

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive and lethal malignancies worldwide with an urgent need for new diagnostic and therapeutic strategies. One major risk factor for PDAC is the pre-indication of chronic pancreatitis (CP), which represents highly inflammatory pancreatic tissue. Kallikreins (KLKs) are secreted serine proteases that play an important role in various cancers as components of the tumor microenvironment. Previous studies of KLKs in solid tumors largely relied on either transcriptomics or immunodetection. We present one of the first targeted mass spectrometry profiling of kallikrein proteases in PDAC, CP, and normal pancreas. We show that KLK6 and KLK10 are significantly upregulated in PDAC (n=14) but not in CP (n=7) when compared to normal pancreas (n=16), highlighting their specific intertwining with malignancy. Additional explorative proteome profiling identified 5936 proteins in our pancreatic cohort and observed disease-specific proteome rearrangements in PDAC and CP. As such, PDAC features an enriched proteome motif for extracellular matrix (ECM) and cell adhesion while there is depletion of mitochondrial energy metabolism proteins, reminiscent of the Warburg effect. Although often regarded as a PDAC hallmark, the ECM fingerprint was also observed in CP, alongside with a prototypical inflammatory proteome motif as well as with an increased wound healing process and proteolytic activity, thereby possibly illustrating tissue autolysis. Proteogenomic analysis based on publicly accessible data sources identified 112 PDAC-specific and 32 CP-specific single amino acid variants, which among others affect KRAS and ANKHD1. Our study emphasizes the diagnostic potential of kallikreins and provides novel insights into proteomic characteristics of PDAC and CP.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatite Crônica , Humanos , Proteoma , Proteômica/métodos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Pâncreas/patologia , Endopeptidases/metabolismo , Calicreínas/genética , Microambiente Tumoral , Proteínas de Ligação a RNA/metabolismo
3.
Biosens Bioelectron ; 224: 115058, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36630744

RESUMO

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.


Assuntos
Técnicas Biossensoriais , Hidrólise , Técnicas Biossensoriais/métodos , NAD , Imunoensaio/métodos , Oxirredução , Endopeptidases , Técnicas Eletroquímicas
4.
Molecules ; 28(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36677630

RESUMO

The African Swine Fever virus (ASFV) causes an infectious viral disease in pigs of all ages. The development of antiviral drugs primarily aimed at inhibition of proteases required for the proteolysis of viral polyproteins. In this study, the conformation of the pS273R protease in physiological states were investigated, virtually screened the multi-protein conformation of pS273R target proteins, combined various molecular docking scoring functions, and identified five potential drugs from the Food and Drug Administration drug library that may inhibit pS273R. Subsequent validation of the dynamic interactions of pS273R with the five putative inhibitors was achieved using molecular dynamics simulations and binding free energy calculations using the molecular mechanics/Poison-Boltzmann (Generalized Born) (MM/PB(GB)SA) surface area. These findings demonstrate that the arm domain and Thr159-Lys167 loop region of pS273R are significantly more flexible compared to the core structural domain, and the Thr159-Lys167 loop region can serve as a "gatekeeper" in the substrate channel. Leucovorin, Carboprost, Protirelin, Flavin Mononucleotide, and Lovastatin Acid all have Gibbs binding free energies with pS273R that were less than -20 Kcal/mol according to the MM/PBSA analyses. In contrast to pS273R in the free energy landscape, the inhibitor and drug complexes of pS273R showed distinct structural group distributions. These five drugs may be used as potential inhibitors of pS273R and may serve as future drug candidates for treating ASFV.


Assuntos
Vírus da Febre Suína Africana , Antivirais , Inibidores de Proteases , Animais , Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/enzimologia , Endopeptidases , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Hidrolases , Conformação Proteica , Suínos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Antivirais/química , Antivirais/farmacologia
5.
Science ; 379(6630): eabn8934, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36701450

RESUMO

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.


Assuntos
Linfócitos B , Peptídeo Hidrolases , Proteólise , Centro Germinativo , Antígenos , Linfonodos , Imunização , Vacinação , Endopeptidases
6.
Medicine (Baltimore) ; 102(3): e32548, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36701711

RESUMO

Laryngeal cancer (LC) is a malignant tumor that occurs in the head and neck. Laryngeal cancer is one of the most common cancers of the neck and head, and its prognosis has always been poor. The incidence of LC increased gradually and showed an early rising trend. Laryngeal cancer is rarely studied in relation to immunity, Malignant tumors will change the state of the human body in various ways to adapt to their own survival and avoid the immune system. This study aims to explore the immune molecular mechanism of laryngeal cancer through bioinformatics analysis. The gene expression data was downloaded for 3 microarray datasets: GSE27020, GSE59102, and GSE51985. CIBERSORT algorithm was performed to evaluate immune cell infiltration in tissues between LC and healthy control (HC). Differentially expressed genes (DEGs) were screened. Functional correlation of DEGs were analyzed by Gene Ontology, Gene Set Enrichment Analysis and Kyoto encyclopedia of genes and genomes. Candidate biomarkers were identified by cytoHubba of Cytoscape. Spearman correlations between the above biomarkers and infiltrating immune cells were explored using R software analysis. The immune cell types of LC and HC were significantly different. Twenty-one DEGs were obtained by cross-screening. The function of DEGs is closely related to the number of immune cells. Five central genes (TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4) were screened. The HUB gene was demonstrated to have the ability to diagnose LC and HC with good specificity and sensitivity. The correlation between immune cells and biomarkers showed that hub gene was positively correlated with macrophages and dendritic cells, and negatively correlated with CD4 + T cell. TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4 can be used as diagnostic biomarker for LC. Macrophages, dendritic cells and CD4 + T cell may participate in the occurrence and development of LC.


Assuntos
Carcinoma , Neoplasias Laríngeas , Humanos , Neoplasias Laríngeas/diagnóstico , Neoplasias Laríngeas/genética , Antígeno CTLA-4 , Desmina , Biologia Computacional , Endopeptidases , Metaloproteases , Biomarcadores , Perfilação da Expressão Gênica , Biomarcadores Tumorais/genética
7.
Cell Mol Life Sci ; 80(2): 43, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36646950

RESUMO

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-ß signaling by directly interacting with TGF-ß type I receptor (TßRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TßRI in the ER. By decreasing cell surface TßRI levels, USP19-ER inhibits TGF-ß/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-ß-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.


Assuntos
Neoplasias da Mama , Fator de Crescimento Transformador beta , Humanos , Feminino , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Transição Epitelial-Mesenquimal/genética , Neoplasias da Mama/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Membrana Celular/metabolismo , Movimento Celular/genética , Linhagem Celular Tumoral , Endopeptidases/metabolismo
8.
Sci Rep ; 13(1): 677, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635354

RESUMO

Orthodontic tooth movement (OTM) occurs through proteolytic remodelling within the periodontium following the application of external force to the tooth. This study describes the first characterization of the salivary peptidome and protease profile during the alignment stage of fixed appliance orthodontic treatment. Unstimulated whole mouth saliva from 16 orthodontic patients (10 males, 6 females, mean (SD) age 15.2 (1.6) years) was collected prior to fixed appliance placement (T1), 1-h (T2), 1-week (T3) following fixed appliance placement and on completion of mandibular arch alignment (T4). Salivary peptides were extracted using filtration followed by mass spectrometry to identify amino acid sequences. Protease prediction was carried out in silico using Proteasix and validated with gelatin zymography and enzyme-linked immunosorbent assay. A total of 2852 naturally-occurring peptides were detected, originating from 436 different proteins. Both collagen and statherin-derived peptide levels were increased at T2. Proteasix predicted 73 proteases potentially involved in generating these peptides, including metalloproteinases, calpains and cathepsins. Changes in predicted activity of proteases over time were also observed, with most metalloproteinases showing increased predicted activity at T2-T3. Increased gelatinolytic activity and MMP8/MMP9 levels were detected at T3. Collectively, multiple protein targets and changes in protease-predicted activity during OTM have been identified.


Assuntos
Endopeptidases , Peptídeo Hidrolases , Técnicas de Movimentação Dentária , Adolescente , Feminino , Humanos , Masculino , Endopeptidases/metabolismo , Boca/metabolismo , Aparelhos Ortodônticos Fixos , Peptídeo Hidrolases/metabolismo , Saliva/metabolismo
9.
Bioorg Chem ; 130: 106264, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36395603

RESUMO

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.


Assuntos
Pirimidinas , Compostos de Amônio Quaternário , SARS-CoV-2 , Inibidores de Protease Viral , Humanos , Antivirais/farmacologia , Endopeptidases , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/metabolismo , Compostos de Amônio Quaternário/farmacologia , Pirimidinas/farmacologia , Inibidores de Protease Viral/farmacologia
10.
J Am Chem Soc ; 145(1): 234-246, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36542079

RESUMO

We investigated the use of amphiphilic, protease-cleavable peptides as encapsulation moieties for hydrophobic metallodrugs, in order to enhance their bioavailability and consequent activity. Two hydrophobic, gold-containing anticancer agents varying in aromatic ligand distribution (Au(I)-N-heterocyclic carbene compounds 1 and 2) were investigated. These were encapsulated into amphiphilic decapeptides that form soluble filamentous structures with hydrophobic cores, varying supramolecular packing arrangements and surface charge. Peptide sequence strongly dictates the supramolecular packing within the aromatic core, which in turn dictates drug loading. Anionic peptide filaments can effectively load 1, and to a lesser extent 2, while their cationic counterparts could not, collectively demonstrating that loading efficiency is dictated by both aromatic and electrostatic (mis)matching between drug and peptide. Peptide nanofilaments were nontoxic to cancerous and noncancerous cells. By contrast, those loaded with 1 and 2 displayed enhanced cytotoxicity in comparison to 1 and 2 alone, when exposed to Caki-1 and MDA-MB-231 cancerous cell lines, while no cytotoxicity was observed in noncancerous lung fibroblasts, IMR-90. We propose that the enhanced in vitro activity results from the enhanced proteolytic activity in the vicinity of the cancer cells, thereby breaking the filaments into drug-bound peptide fragments that are taken up by these cells, resulting in enhanced cytotoxicity toward cancer cells.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Endopeptidases , Ouro/química , Peptídeo Hidrolases , Peptídeos/farmacologia , Peptídeos/química , Cápsulas
11.
Pathol Res Pract ; 241: 154288, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36566600

RESUMO

Microsatellite instability-high (MSI-H) colorectal cancer (CRC) is different from microsatellite stable (MSS) CRC concerning biological, and clinical features. In MSI-H CRCs, defects of mismatch repair genes produce increased mutation accumulation in repetitive DNA sequences. To see whether candidate tumor suppressor genes (TSGs) are altered in MSI-H CRC, we studied frameshift mutation and protein expression of candidate TSGs of RGS2, HNF1A, HNF1B, CAPN12, RCBTB2, ATE1, PKNOX1, and USP19. We found frameshift mutations of RGS2 in 5 (5%), HNF1A in 6 (6%), HNF1B in 2 (2%), CAPN12 in 3 (3%), RCBTB2 in 4 (4%), ATE1 in 2 (2%), PKNOX1 in 2 (2%), and USP19 in 2 (2%) MSI-H CRCs. However, we found no such mutations in MSS CRCs. RCBTB2, CAPN12, HNF1A, and HNF1B frameshift mutations revealed the regional difference in the same tumors. In addition, we identified loss of RGS2, HNF1A, and CAPN12 protein expression irrespective of MSI phenotype in 13-29% of CRCs. The results indicate that many TSGs harbor concurrent inactivating mutations and protein loss in MSI-H CRCs with intratumoral mutational heterogeneity, and that MSS CRCs are altered by protein losses. These alterations could contribute to CRC development and underlying mechanisms and consequences of the TSG alterations remain to be clarified.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Proteínas RGS , Humanos , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA/métodos , Endopeptidases/genética , Mutação da Fase de Leitura , Genes Supressores de Tumor , Fator 1-alfa Nuclear de Hepatócito/genética , Instabilidade de Microssatélites , Proteínas RGS/genética
12.
Int J Biol Macromol ; 228: 333-345, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36565834

RESUMO

Proteases are a major virulence factor in pathogenic fungi and can serve as a potential therapeutic target. The interaction of gallic acid (GA) with Aspartic fungal protease (PepA) was investigated using biophysical and in silico approaches. UV-Vis and fluorescence spectroscopy showed complex formation and static quenching of PepA by GA with Ka of 7.4 × 105 M-1 and stoichiometric binding site (n) of 1.67. CD-spectroscopy showed marked changes in helical content and synchronous fluorescence spectra measurements indicated significant changes in the microenvironment around tryptophan residues in the GA-PepA complex. Outcomes of Isothermal Titration Calorimetry (ITC) measurement and molecular modelling studies validated the spectroscopic results. The binding of GA to Human Serum albumin (HSA) was moderate (Ka = 1.9 × 103 M-1) and did not cause structural disruption of HSA. To conclude, gallic acid is strongly bound to fungal protease leading to structural disruption and inhibition whereas HSA structure was largely conserved. Gallic acid thus appears to be a potential therapeutic agent against fungal proteases.


Assuntos
Ácido Aspártico Proteases , Albumina Sérica Humana , Humanos , Simulação de Acoplamento Molecular , Termodinâmica , Albumina Sérica Humana/química , Espectrometria de Fluorescência , Sítios de Ligação , Ligação Proteica , Ácido Aspártico Proteases/metabolismo , Peptídeo Hidrolases/metabolismo , Endopeptidases/metabolismo , Dicroísmo Circular
13.
J Immunol ; 210(3): 283-296, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36548461

RESUMO

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, one of the most highly infectious animal viruses throughout the world. The JAK-STAT signaling pathway is a highly conserved pathway for IFN-ß-induced antiviral gene expression. Previous studies have shown that FMDV can strongly suppress the innate immune response. Moreover, although STAT1 and STAT2 (STAT1/2) have been well established in JAK-STAT signaling-induced antiviral gene expression, whether FMDV proteins inhibit IFN-ß-induced JAK-STAT signaling remains poorly understood. In this study, we described the Lb leader protease (Lbpro) of FMDV as a candidate for inhibiting IFN-ß-induced signaling transduction via directly interacting with STAT1/2. We further showed that Lbpro colocalized with STAT1/2 to inhibit their nuclear translocation. Importantly, Lbpro cleaved STAT1/2 to inhibit IFN-ß-induced signal transduction, whereas the catalytically inactive mutant of LC51A (Lbpro with cysteine substituted with alanine at amino acid residue 51) had no effect on the stability of STAT1/2 proteins. The cleavage of the STAT1/2 proteins was also determined during FMDV infection in vitro. Lbpro could cleave the residues between 252 and 502 aa for STAT1 and the site spanning residues 140 - 150 aa (QQHEIESRIL) for STAT2. The in vivo results showed that Lbpro can cleave STAT1/2 in pigs. Overall, our findings suggest that FMDV Lbpro-mediated targeting of STAT1/2 may reveal a novel mechanism for viral immune evasion.


Assuntos
Endopeptidases , Vírus da Febre Aftosa , Interferon beta , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Animais , Vírus da Febre Aftosa/enzimologia , Imunidade Inata , Peptídeo Hidrolases , Transdução de Sinais , Suínos , Interferon beta/imunologia
14.
Nat Methods ; 20(1): 112-122, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36481965

RESUMO

Natural or engineered peptides serve important biological functions. A general approach to achieve chemical-dependent activation of short peptides will be valuable for spatial and temporal control of cellular processes. Here we present a pair of chemically activated protein domains (CAPs) for controlling the accessibility of both the N- and C-terminal portion of a peptide. CAPs were developed through directed evolution of an FK506-binding protein. By fusing a peptide to one or both CAPs, the function of the peptide is blocked until a small molecule displaces them from the FK506-binding protein ligand-binding site. We demonstrate that CAPs are generally applicable to a range of short peptides, including a protease cleavage site, a dimerization-inducing heptapeptide, a nuclear localization signal peptide, and an opioid peptide, with a chemical dependence up to 156-fold. We show that the CAPs system can be utilized in cell cultures and multiple organs in living animals.


Assuntos
Peptídeo Hidrolases , Peptídeos , Animais , Peptídeos/química , Endopeptidases/metabolismo , Proteínas de Ligação a Tacrolimo/genética
15.
Bioresour Technol ; 370: 128487, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36528180

RESUMO

Enzymatic treatment of food and vegetable waste (FVW) is an eco-friendly approach for producing industrially relevant value-added products. This review describes the sources, activities and potential applications of crucial enzymes in FVW valorization. The specific roles of amylase, cellulase, xylanase, ligninase, protease, pectinase, tannase, lipase and zymase enzymes were explained. The exhaustive list of value-added products that could be produced from FVW is presented. FVW valorization through enzymatic and whole-cell enzymatic valorization was compared. The note on global firms specialized in enzyme production reiterates the economic importance of enzymatic treatment. This review provides information on choosing an efficient enzymatic FVW treatment strategy, such as nanoenzyme and cross-linked based enzyme immobilization, to make the process viable, sustainable and cheaper. Finally, the importance of life cycle assessment of enzymatic valorization of FVW was impressed to prove this approach is a better option to shift from a linear to a circular economy.


Assuntos
Celulase , Verduras , Amilases , Peptídeo Hidrolases , Endopeptidases
16.
J Med Chem ; 66(1): 251-265, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36540942

RESUMO

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.


Assuntos
Doença de Parkinson , Peptídeo Hidrolases , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo , Mitofagia , Doença de Parkinson/tratamento farmacológico , Proteínas Quinases/metabolismo , Proteínas Mitocondriais/metabolismo , Endopeptidases , Ubiquitina-Proteína Ligases/metabolismo
17.
Eur Rev Med Pharmacol Sci ; 26(22): 8567-8575, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36459037

RESUMO

OBJECTIVE: Proteostasis is an important process occurring in all living cells and is highly indispensable for cell survival. The HslVU protease/chaperone complex's critical role in regulating proteostasis to maintain a healthy cellular proteome and its presence in pathogenic microbes made it an important drug target. This study aimed to identify small molecular inhibitors of the HslV protease. MATERIALS AND METHODS: Herein, a library of small molecules belonging to the triazine and chromone families has been evaluated for their inhibitory potential against the E. coli HslV protease using both in silico and in vitro techniques. RESULTS: Four compounds, i.e., SHS-II-123a, SHS-II-147a, US-IV-89, and US-IV-92, were identified as potential inhibitors of the HslV protease having IC50 values in the range of 0.1 to 0.32 µM. Additionally, these compounds' drug-likeness and ADMET profiles indicated their compatibility to be considered safer drug candidates. CONCLUSIONS: To the best of our knowledge, this is the first report on small molecules having inhibitory effects on the HslVU complex. These identified compounds can be efficiently subjected to further investigations to develop novel and safer antimicrobial agents.


Assuntos
Cromonas , Peptídeo Hidrolases , Humanos , Cromonas/farmacologia , Triazinas/farmacologia , Tiazóis , Escherichia coli , Endopeptidases , Chaperonas Moleculares
18.
Mol Cell ; 82(23): 4405-4406, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459983

RESUMO

In this issue, Liu et al. present an in-depth study aiming to unravel the structural, biochemical, and physiological aspects of how type III-E CRISPR-Cas systems trigger abortive infection by activating a protease upon target RNA recognition.1.


Assuntos
Sistemas CRISPR-Cas , Endopeptidases , Peptídeo Hidrolases , RNA , Biologia
19.
World J Microbiol Biotechnol ; 39(1): 33, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36469174

RESUMO

Pseudomonas fluorescens is considered among the main spoilage microorganisms due to its ability to produce proteases. Food deterioration caused by spoilage microorganisms has a major impact on food quality and the environment. The inactivation of Pseudomonas fluorescens growth and protease production was intensively investigated with the use of Salmide®, A Sodium Chlorite-Based Oxy-halogen Disinfectant. A unique M9 media was also developed to assure sufficient protease productions with different mutants of Pseudomonas fluorescens as a microbioreactor. Mutations were induced by classical whole-cell mutagenesis using N-methyl-N'- nitro-N-nitrosoguanidine (NTG). A dramatic decrease occurred in protease activity when different Salmide concentrations (5, 10, and 15 ppm) were added to the growth culture followed by a complete inhibition concentration (20, 25, 50, and 100 ppm) of Salmide. However, no significant inhibition occurred once it is secreted out of cells. Some mutants were resistant and remains highly stable with high protease production under stressful conditions of Sodium Chlorite-Based Oxy-halogen. The production of the protease showed a linear correlation with the increase in incubation time using a continuous culture bioreactor system and recorded maximum protease activity after 40 h. Our findings would offer alternative antimicrobial procedures for food and industrial sectors.


Assuntos
Pseudomonas fluorescens , Endopeptidases , Peptídeo Hidrolases , Halogênios
20.
BMC Cancer ; 22(1): 1350, 2022 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-36564767

RESUMO

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal form of prostate cancer, and the molecular mechanism driving mCRPC progression has not yet been fully elucidated. Immunotherapies such as chimeric antigen receptor, T-cell therapy and immune checkpoint blockade have exerted promising antitumor effects in hematological and solid tumor malignancies; however, no encouraging responses have been observed against mCRPC. The deubiquitinase USP13 functions as a tumor suppressor in many human cancers, as it sustains the protein stability of PTEN and TP53; however, its role in prostate cancer (PCa) and involvement in DNA damage and AR signaling remain unclear. In the current study, we explored the prognostic value of USP13 in PCa based on the TCGA database, and we analyzed the expression of USP13 in PCa tissues and adjacent normal tissues based on TCGA and our cohort. The results suggested that USP13 is overexpressed in PCa tumors and has the potential to be an independent biomarker for the overall survival of PCa patients. Additionally, enrichment analysis indicated that USP13 may participate in the AR pathway and PI3k/Wnt signaling, which are closely related to PCa progression. We also observed a significant correlation between the expression of USP13 and AR-related genes, DDR genes and mismatch repair genes based on the TCGA_PRAD dataset, which further supported the critical role of USP13 in AR activation and the DNA damage response of PCa. USP13 was also found to be enriched in protein neddylation, and expression of USP13 was significantly associated with infiltration of immune cells and expression of immunomodulators. Taken together, our study revealed a key role of USP13 in contributing to PCa progression by participating in multiple oncogenic signaling pathways, the DNA damage response and the immunosuppressive tumor microenvironment. Targeting USP13 may inhibit tumor growth and provide additional benefits in cooperation with DDR inhibitors and immunotherapy.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Neoplasias de Próstata Resistentes à Castração/patologia , Peptídeo Hidrolases , Ubiquitina/genética , Neoplasias da Próstata/metabolismo , Endopeptidases/genética , Dano ao DNA/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral , Proteases Específicas de Ubiquitina
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