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1.
FEBS Lett ; 598(6): 684-701, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38426217

RESUMO

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.


Assuntos
Geobacillus stearothermophilus , Peptídeo Hidrolases , Geobacillus stearothermophilus/metabolismo , Peptídeo Hidrolases/metabolismo , Endopeptidases , Aminopeptidases , Proteólise
2.
Bull Exp Biol Med ; 176(4): 457-460, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38491258

RESUMO

The effect of Kamchatka crab hepatopancreas containing three collagenolytic isoenzymes Collagenase KK and proteinases of Streptomyces lavendulae on metabolic activity and cell death were carried out on in vitro models. It was shown that changes in the protein structure under the influence of Collagenase KK occurred earlier than under the effect of bacterial proteinases. At the same time, activity of Collagenase KK was significantly higher than that of bacterial proteinases (p<0.01). Both preparations had a pronounced time- and dose-dependent effects on metabolic activity of cells. Collagenase KK had low cytotoxic effect, and cells mainly died by apoptosis. Thus, hepatopancreas collagenase has a high activity and proapoptotic effect on cells and can be used in low concentrations for enzymatic disaggregation of tissues.


Assuntos
Braquiúros , Animais , Braquiúros/metabolismo , Hepatopâncreas/metabolismo , Colagenases/metabolismo , Endopeptidases , Peptídeo Hidrolases
3.
Methods Mol Biol ; 2774: 59-69, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38441758

RESUMO

Genetically encoded sensor-actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs). RBPs are proteins that recognize specific motifs on the mRNA target inducing mRNA decay or translation inhibition. The use of RBPs deriving from different species in mammalian cells has allowed to create sophisticated and multilayered regulatory networks, addressing the previous limitation of regulatory orthogonal parts that can be assembled in synthetic devices. In this chapter, we describe the engineering and tests of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7) to expand the toolbox of synthetic circuits in mammalian cells.


Assuntos
Peptídeo Hidrolases , RNA Catalítico , Animais , Endopeptidases , RNA/genética , Proteínas de Ligação a RNA/genética , Mamíferos
4.
Front Immunol ; 15: 1344878, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38444844

RESUMO

Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.


Assuntos
Dermatite , Psoríase , Humanos , Inibidores de Cisteína Proteinase , Mananas , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Inflamação/tratamento farmacológico , Inibidores de Proteases , Imunidade Inata , Endopeptidases , Peptídeo Hidrolases
5.
Commun Biol ; 7(1): 261, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38438476

RESUMO

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.


Assuntos
Bryopsida , Peptídeo Hidrolases , Humanos , Calpaína/genética , Endopeptidases , Diferenciação Celular/genética
6.
Eur J Med Chem ; 268: 116275, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38452725

RESUMO

USP2 and USP8 are crucial in the development and progression of breast cancer, primarily through the stabilization of protein substrates such as Her2 and ERα. The dual-target inhibitor ML364, targeting both USP2 and USP8, has garnered significant interest in recent research. In this study, we developed a series of ML364 derivatives using ligand-based drug design strategies. The standout compound, LLK203, demonstrated enhanced inhibitory activity, showing a 4-fold increase against USP2 and a 9-fold increase against USP8, compared to the parent molecule. In MCF-7 breast cancer cells, LLK203 effectively degraded key proteins involved in cancer progression and notably inhibited cell proliferation. Moreover, LLK203 exhibited potent in vivo efficacy in the 4T1 homograft model, while maintaining a low toxicity profile. These results underscore the potential of LLK203 as a promising dual-target inhibitor of USP2/USP8 for breast cancer treatment.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Células MCF-7 , Proliferação de Células , Ubiquitina Tiolesterase , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/farmacologia
7.
PLoS One ; 19(3): e0299238, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38483871

RESUMO

BACKGROUND: Currently, there is no antiviral medication for dengue, a potentially fatal tropical infectious illness spread by two mosquito species, Aedes aegypti and Aedes albopictus. The RdRp protease of dengue virus is a potential therapeutic target. This study focused on the in silico drug discovery of RdRp protease inhibitors. METHODS: To assess the potential inhibitory activity of 29 phenolic acids from Theobroma cacao L. against DENV3-NS5 RdRp, a range of computational methods were employed. These included docking, drug-likeness analysis, ADMET prediction, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. The aim of these studies was to confirm the stability of the ligand-protein complex and the binding pose identified during the docking experiment. RESULTS: Twenty-one compounds were found to have possible inhibitory activities against DENV according to the docking data, and they had a binding affinity of ≥-37.417 kcal/mol for DENV3- enzyme as compared to the reference compound panduratin A. Additionally, the drug-likeness investigation produced four hit compounds that were subjected to ADMET screening to obtain the lead compound, catechin. Based on ELUMO, EHOMO, and band energy gap, the DFT calculations showed strong electronegetivity, favouravle global softness and chemical reactivity with considerable intra-molecular charge transfer between electron-donor to electron-acceptor groups for catechin. The MD simulation result also demonstrated favourable RMSD, RMSF, SASA and H-bonds in at the binding pocket of DENV3-NS5 RdRp for catechin as compared to panduratin A. CONCLUSION: According to the present findings, catechin showed high binding affinity and sufficient drug-like properties with the appropriate ADMET profiles. Moreover, DFT and MD studies further supported the drug-like action of catechin as a potential therapeutic candidate. Therefore, further in vitro and in vivo research on cocoa and its phytochemical catechin should be taken into consideration to develop as a potential DENV inhibitor.


Assuntos
Aedes , Cacau , Catequina , Chalconas , Dengue , Animais , Peptídeo Hidrolases , Simulação de Dinâmica Molecular , Catequina/farmacologia , Endopeptidases , Fenóis , RNA Polimerase Dependente de RNA , Simulação de Acoplamento Molecular
8.
Front Endocrinol (Lausanne) ; 15: 1302667, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38487343

RESUMO

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.


Assuntos
Adenoma Hipofisário Secretor de ACT , Adenoma , Hipersecreção Hipofisária de ACTH , Neoplasias Hipofisárias , Humanos , Feminino , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Corticotrofos/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Endopeptidases/genética , Adenoma Hipofisário Secretor de ACT/metabolismo , Hipersecreção Hipofisária de ACTH/metabolismo , Mutação , Adenoma/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Cell Mol Life Sci ; 81(1): 148, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38509419

RESUMO

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322-330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection.


Assuntos
Vírus da Febre Aftosa , Animais , Suínos , Vírus da Febre Aftosa/metabolismo , Peptídeo Hidrolases/metabolismo , Transdução de Sinais , Imunidade Inata , Endopeptidases/genética , Endopeptidases/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Antivirais/metabolismo
10.
Eur Rev Med Pharmacol Sci ; 28(5): 1847-1856, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38497867

RESUMO

OBJECTIVE: The purpose of the present study is to see if the presence of angiotensin-converting enzyme 2 (ACE-2), transmembrane serine protease 2 (TMPRSS), and P-selectin in platelets increases the risk of thrombosis in COVID-19 patients. PATIENTS AND METHODS: This was a cross-sectional study conducted in the COVID-19 isolation center between January and September 2021 and comprised 61 COVID-19-infected patients, 21 of whom were in the intensive care unit (ICU) and 40 of whom were non-ICU patients (non-ICUP) in the isolation center. The coagulation profile, as well as the ACE-2, TMPRES, and P-selectin receptors, were all assessed in addition to the complete blood count (CBC). A questionnaire was also utilized to collect social and demographic data. RESULTS: All platelet indices and coagulation profiles were significantly altered in COVID-19 ICUP and non-ICUP in this research; additionally, there is a significant association between the presence of ACE-2, P-selectin, and TMPRRS2 in COVID-19 patients with coagulation profile and platelet indices leading to hypercoagulable state. CONCLUSIONS: In summary, the interaction of ACE-2, TMPRSS, and P-selectin in platelets appears to be a key element contributing to COVID-19 severity via their impact on thrombus development. Further investigation into these pathways may provide possible treatment targets for reducing the severe consequences of the COVID-19 infection.


Assuntos
COVID-19 , Trombose , Humanos , Enzima de Conversão de Angiotensina 2 , COVID-19/complicações , Estudos Transversais , Endopeptidases , Selectina-P , Peptídeo Hidrolases , Serina
11.
Sci Adv ; 10(11): eadk2542, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38489364

RESUMO

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.


Assuntos
Retículo Endoplasmático , alfa-Sinucleína , Animais , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Transporte Proteico/fisiologia , Retículo Endoplasmático/metabolismo , Mamíferos/metabolismo , Endopeptidases/metabolismo
12.
Proc Natl Acad Sci U S A ; 121(11): e2307802121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38437557

RESUMO

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.


Assuntos
Neoplasias Ovarianas , Peptídeos , Humanos , Feminino , Interferência de RNA , Peptídeos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Peptídeo Hidrolases , RNA Interferente Pequeno/genética , Endopeptidases , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA
13.
Biochem Biophys Res Commun ; 706: 149730, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38461648

RESUMO

Peptides linking well-folded and non-interacting domains in fusion proteins can undergo proteolytic degradation. This leads to physical separation of the domains that were originally sought to be joined. In order to identify characteristics that determine linker degradation propensity, we selected a pair of thermostable, proteolytically-resistant domains, and joined them using five different linkers. We then assessed linker degradation propensities through size-exclusion chromatography, and denaturing and non-denaturing electrophoresis. The domains used were Coh2, an all-beta cohesin from C. thermocellum CipA, and BSX, a beta/alpha barrel xylanase from Bacillus sp. NG-27, while the linkers used were Rigid (3 repeats of N-EAAAK-C), Flexible (two repeats of N-SGGGG-C), Nat-full (42 residues of a Coh2-adjacent linker from CipA), Nat-half (a 21 residues-long derivative of Nat-full) and Nat-quarter (a 9 residues-long derivative of Nat-full). Both with proteolysis effected by proteases present in the environment, and with an exogenously-added protease (Subtilisin A), we found that Flexible underwent little or no degradation, whereas linkers of comparable length like Nat-quarter or Rigid underwent extensive degradation, as did longer linkers like Nat-Half and Nat-Full. Our analyses disfavor the likelihood of the sequence of Flexible being naturally resistant to proteolysis, and instead favor the explanation that the flexibility of Flexible facilitates movements of Coh2 relative to BSX which then serve to sterically prevent the approach of proteases. Thus, the construct incorporating Flexible appears to behave like a 'nunchuck' in which rods/spheres flanking a chain collide with approaching swords that are capable of severing the chain, to prevent severance.


Assuntos
Peptídeo Hidrolases , Peptídeos , Propionatos , Proteólise , Peptídeos/química , Indóis , Endopeptidases
14.
Biochem Biophys Res Commun ; 706: 149746, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38461646

RESUMO

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.


Assuntos
Peptídeo Hidrolases , Peptídeos , Peptídeos/química , Peptídeo Hidrolases/metabolismo , Endopeptidases/metabolismo , Proteólise
15.
Sci Adv ; 10(11): eadj2570, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38478622

RESUMO

Ubiquitination plays a crucial role throughout plant growth and development. The E3 ligase DA2 has been reported to activate the peptidase DA1 by ubiquitination, hereby limiting cell proliferation. However, the molecular mechanisms that regulate DA2 remain elusive. Here, we demonstrate that DA2 has a very high turnover and auto-ubiquitinates with K48-linkage polyubiquitin chains, which is counteracted by two deubiquitinating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13. Unexpectedly, we found that auto-ubiquitination of DA2 does not influence its stability but determines its E3 ligase activity. We also demonstrate that impairing the protease activity of DA1 abolishes the growth-reducing effect of DA2. Last, we show that synthetic, constitutively activated DA1-ubiquitin fusion proteins overrule this complex balance of ubiquitination and deubiquitination and strongly restrict growth and promote endoreduplication. Our findings highlight a nonproteolytic function of K48-linked polyubiquitination and reveal a mechanism by which DA2 auto-ubiquitination levels, in concert with UBP12 and UBP13, precisely monitor the activity of DA1 and fine-tune plant organ size.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Tamanho do Órgão , Endorreduplicação , Ubiquitinação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proliferação de Células , Endopeptidases/genética
16.
J Med Virol ; 96(3): e29523, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38483060

RESUMO

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.


Assuntos
Interferon Tipo I , Ubiquitina , Humanos , Ubiquitina/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Proteína Beclina-1 , Ubiquitinação , Imunidade Inata , Interferon Tipo I/metabolismo , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Endopeptidases/genética , Endopeptidases/metabolismo
17.
Arch Microbiol ; 206(4): 151, 2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38467842

RESUMO

Salmonella Typhimurium, a zoonotic pathogen, causes systemic and localized infection. The emergence of drug-resistant S. Typhimurium has increased; treating bacterial infections remains challenging. Phage endolysins derived from phages have a broader spectrum of bacteriolysis and better bacteriolytic activity than phages, and are less likely to induce drug resistance than antibiotics. LysST-3, the endolysin of Salmonella phage ST-3, was chosen in our study for its high lytic activity, broad cleavage spectrum, excellent bioactivity, and moderate safety profile. LysST-3 is a promising antimicrobial agent for inhibiting the development of drug resistance in Salmonella. The aim of this study is to investigate the molecular characteristics of LysST-3 through the prediction of key amino acid sites of LysST-3 and detection of its mutants' activity. We investigated its lytic effect on Salmonella and identified its key amino acid sites of interaction with substrate. LysST-3 may be a Ca2+, Mg2+ - dependent metalloenzyme. Its concave structure of the bottom "gripper" was found to be an important part of its amino acid active site. We identified its key sites (29P, 30T, 86D, 88 L, and 89 V) for substrate binding and activity using amino acid-targeted mutagenesis. Alterations in these sites did not affect protein secondary structure, but led to a significant reduction in the cleavage activity of the mutant proteins. Our study provides a basis for phage endolysin modification to target drug-resistant bacteria. Identifying the key amino acid site of the endolysin LysST-3 provides theoretical support for the functional modification of the endolysin and the development of subsequent effective therapeutic solutions.


Assuntos
Bacteriófagos , Fagos de Salmonella , Fagos de Salmonella/genética , Aminoácidos , Endopeptidases/genética , Endopeptidases/farmacologia , Endopeptidases/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Antibacterianos/farmacologia
18.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474092

RESUMO

Proteases are critical enzymes in cellular processes which regulate intricate events like cellular proliferation, differentiation and apoptosis. This review highlights the multifaceted roles of the serine proteases FAM111A and FAM111B, exploring their impact on cellular functions and diseases. FAM111A is implicated in DNA replication and replication fork protection, thereby maintaining genome integrity. Additionally, FAM111A functions as an antiviral factor against DNA and RNA viruses. Apart from being involved in DNA repair, FAM111B, a paralog of FAM111A, participates in cell cycle regulation and apoptosis. It influences the apoptotic pathway by upregulating anti-apoptotic proteins and modulating cell cycle-related proteins. Furthermore, FAM111B's association with nucleoporins suggests its involvement in nucleo-cytoplasmic trafficking and plays a role in maintaining normal telomere length. FAM111A and FAM111B also exhibit some interconnectedness and functional similarity despite their distinct roles in cellular processes and associated diseases resulting from their dysfunction. FAM111A and FAM111B dysregulation are linked to genetic disorders: Kenny-Caffey Syndrome type 2 and Gracile Bone Dysplasia for FAM111A and POIKTMP, respectively, and cancers. Therefore, the dysregulation of these proteases in diseases emphasizes their potential as diagnostic markers and therapeutic targets. Future research is essential to unravel the intricate mechanisms governing FAM111A and FAM111B and explore their therapeutic implications comprehensively.


Assuntos
Doenças do Desenvolvimento Ósseo , Nanismo , Humanos , Peptídeo Hidrolases/genética , Mutação , Proteínas de Ciclo Celular/metabolismo , Nanismo/genética , Endopeptidases/genética , Receptores Virais/metabolismo
19.
Molecules ; 29(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474583

RESUMO

Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp's ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.


Assuntos
Endopeptidases , Simulação de Dinâmica Molecular , Endopeptidases/metabolismo , Biotecnologia , Mutação
20.
Bioorg Chem ; 145: 107239, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38428282

RESUMO

Antimicrobial resistance (AMR) is a serious global concern and a huge burden on the healthcare system. Antimicrobial peptides (AMPs) are considered as a solution of AMR due to their membrane-lytic and intracellular mode of action and therefore resistance development against AMPs is less frequent. One such AMPs, temporin-L (TL) is a 13-mer peptide reported as a potent and broad-spectrum antibacterial agent with significant immunomodulatory activity. However, TL is toxic to human erythrocytes at their antibacterial concentrations and therefore various analogues were synthesized with potent antimicrobial activity and lower hemolytic activity. In this work, we have selected a non-toxic engineered analogue of TL (eTL) and performed hydrocarbon stapling of amino acid residues at i to i + 4 positions at different part of sequence. The synthesized peptides were investigated against both the gram-positive and gram-negative bacteria as well as methicillin resistant S. aureus, its MIC was measured in the concentrations range of 0.9-15.2 µM. All analogues were found equal or better antibacterial as compared to parent peptide. Interestingly one analogue eTL [5-9] was found to be non-cytotoxic and stable in presence of the human serum. Mode of action studies revealed membrane depolarizing and disruptive mode of action with live MRSA. Further in vivo studies of antimicrobial against MRSA infection and anti-endotoxin activities in mice model revealed potential activity of the stapled peptide analogue. Overall, this reports on stapled analogue of the AMPs highlights an important strategy for the development of new antibacterial therapeutics against AMR.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Animais , Camundongos , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeo Hidrolases , Bactérias Gram-Positivas , Bactérias Gram-Negativas , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/farmacologia , Endopeptidases , Hidrocarbonetos , Testes de Sensibilidade Microbiana
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