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1.
Commun Biol ; 7(1): 1055, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39191864

RESUMO

Aging is a consequence of complex molecular changes, but whether a single microRNA (miRNA) can drive aging remains unclear. A miRNA known to be upregulated during both normal and premature aging is miR-29. We find miR-29 to also be among the top miRNAs predicted to drive aging-related gene expression changes. We show that partial loss of miR-29 extends the lifespan of Zmpste24-/- mice, an established model of progeria, indicating that miR-29 is functionally important in this accelerated aging model. To examine whether miR-29 alone is sufficient to promote aging-related phenotypes, we generated mice in which miR-29 can be conditionally overexpressed (miR-29TG). miR-29 overexpression is sufficient to drive many aging-related phenotypes and led to early lethality. Transcriptomic analysis of both young miR-29TG and old WT mice reveals shared downregulation of genes associated with extracellular matrix organization and fatty acid metabolism, and shared upregulation of genes in pathways linked to inflammation. These results highlight the functional importance of miR-29 in controlling a gene expression program that drives aging-related phenotypes.


Assuntos
Envelhecimento , MicroRNAs , Fenótipo , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Envelhecimento/genética , Camundongos , Progéria/genética , Progéria/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Regulação da Expressão Gênica , Masculino , Longevidade/genética , Metaloendopeptidases
2.
Artigo em Russo | MEDLINE | ID: mdl-39113454

RESUMO

OBJECTIVE: To compare the effectiveness and safety of the use of non-immunogenic staphylokinase (NS) and alteplase (AP) for intravenous thrombolysis (IT) for ischemic stroke (IS) in real clinical practice at a regional vascular center. MATERIAL AND METHODS: Data from 100 patients with IS who received IT with NS and 100 patients who received IT with AP for the period 2022-2023 were analyzed. The groups were comparable on sociodemographic parameters, cardiovascular risk factors and diseases, and stroke characteristics. RESULTS: Door-to-needle time was 17 (13-22) min in the NS group and 38 (33-42) min in the AP group (p<0.001). During control neuroimaging, a cerebral infarction was detected in 46% of patients in the NS group and 61% of patients in the AP group (OR 0.479 [0.263; 0.875], p=0.035). When performing IT with NS, an NIHSS score of 0 points (no neurological deficit) was observed twice as often (OR 2.202 [1.079; 4.504], p=0.023). The incidence of hemorrhagic transformation, including symptomatic, as well as hospital mortality did not differ. CONCLUSION: IT with NS is associated with a lower probability of cerebral infarction and greater positive dynamics of the neurological status in comparison with the use of AP already within the first stage of treatment and rehabilitation.


Assuntos
Fibrinolíticos , AVC Isquêmico , Sistema de Registros , Terapia Trombolítica , Ativador de Plasminogênio Tecidual , Humanos , Masculino , Feminino , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tecidual/uso terapêutico , AVC Isquêmico/tratamento farmacológico , Idoso , Pessoa de Meia-Idade , Terapia Trombolítica/métodos , Fibrinolíticos/administração & dosagem , Fibrinolíticos/uso terapêutico , Resultado do Tratamento , Metaloendopeptidases , Administração Intravenosa
3.
Sci Rep ; 14(1): 18420, 2024 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-39117724

RESUMO

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.


Assuntos
Dinorfinas , Neurotensina , Humanos , Especificidade por Substrato , Dinorfinas/química , Dinorfinas/metabolismo , Neurotensina/química , Neurotensina/metabolismo , Metaloendopeptidases/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/antagonistas & inibidores , Ligação Proteica , Cristalografia por Raios X , Modelos Moleculares , Domínio Catalítico , Bradicinina/química , Bradicinina/metabolismo , Angiotensinas/metabolismo , Angiotensinas/química , Sequência de Aminoácidos
4.
J Pharm Biomed Anal ; 250: 116400, 2024 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-39126811

RESUMO

Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions.


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Dissulfetos , Mapeamento de Peptídeos , Tripsina , Anticorpos Biespecíficos/química , Dissulfetos/química , Mapeamento de Peptídeos/métodos , Anticorpos Monoclonais/química , Tripsina/química , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas/métodos , Desnaturação Proteica , Guanidina/química , Metaloendopeptidases
5.
Adv Cancer Res ; 162: 75-97, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39069370

RESUMO

Our understanding of the roles that mitochondria play in cellular physiology has evolved drastically-from a mere cellular energy supplier to a crucial regulator of metabolic and signaling processes, particularly in the context of development and progression of human diseases such as cancers. The present review examines the role of OMA1, a conserved, redox-sensitive metallopeptidase in cancer biology. OMA1's involvement in mitochondrial quality control, redox activity, and stress responses underscores its potential as a novel target in cancer diagnosis and treatment. However, our incomplete understanding of OMA1's regulation and structural detail presents ongoing challenges to target OMA1 for therapeutic purposes. Further exploration of OMA1 holds promise in uncovering novel insights into cancer mechanisms and therapeutic strategies. In this chapter, we briefly summarize our current knowledge about OMA1, its redox-regulation, and emerging role in certain cancers.


Assuntos
Metaloendopeptidases , Mitocôndrias , Neoplasias , Humanos , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/enzimologia , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Animais , Oxirredução
6.
Cell Death Dis ; 15(7): 523, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39039044

RESUMO

The mechanism regulating cellular senescence of postmitotic muscle cells is still unknown. cGAS-STING innate immune signaling was found to mediate cellular senescence in various types of cells, including postmitotic neuron cells, which however has not been explored in postmitotic muscle cells. Here by studying the myofibers from Zmpste24-/- progeria aged mice [an established mice model for Hutchinson-Gilford progeria syndrome (HGPS)], we observed senescence-associated phenotypes in Zmpste24-/- myofibers, which is coupled with increased oxidative damage to mitochondrial DNA (mtDNA) and secretion of senescence-associated secretory phenotype (SASP) factors. Also, Zmpste24-/- myofibers feature increased release of mtDNA from damaged mitochondria, mitophagy dysfunction, and activation of cGAS-STING. Meanwhile, increased mtDNA release in Zmpste24-/- myofibers appeared to be related with increased VDAC1 oligomerization. Further, the inhibition of VDAC1 oligomerization in Zmpste24-/- myofibers with VBIT4 reduced mtDNA release, cGAS-STING activation, and the expression of SASP factors. Our results reveal a novel mechanism of innate immune activation-associated cellular senescence in postmitotic muscle cells in aged muscle, which may help identify novel sets of diagnostic markers and therapeutic targets for progeria aging and aging-associated muscle diseases.


Assuntos
Senescência Celular , DNA Mitocondrial , Proteínas de Membrana , Nucleotidiltransferases , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Camundongos , Progéria/metabolismo , Progéria/patologia , Progéria/genética , Transdução de Sinais , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Camundongos Knockout , Células Musculares/metabolismo , Mitofagia , Mitocôndrias/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Metaloendopeptidases
7.
BMC Vet Res ; 20(1): 337, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39080654

RESUMO

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.


Assuntos
Meningites Bacterianas , Sorogrupo , Infecções Estreptocócicas , Streptococcus suis , Doenças dos Suínos , Streptococcus suis/patogenicidade , Streptococcus suis/enzimologia , Animais , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Camundongos , Meningites Bacterianas/veterinária , Meningites Bacterianas/microbiologia , Feminino , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos BALB C , Metaloendopeptidases/metabolismo , Metaloendopeptidases/genética
8.
J Pineal Res ; 76(5): e12991, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39039850

RESUMO

Although rapid progression and a poor prognosis in influenza A virus (IAV) infection-induced acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are frequently associated with metabolic energy disorders, the underlying mechanisms and rescue strategies remain unknown. We herein demonstrated that the level of resting energy expenditure increased significantly in IAV-induced AECOPD patients and that cellular energy exhaustion emerged earlier and more significantly in IAV-infected primary COPD bronchial epithelial (pDHBE) cells. The differentially expressed genes were enriched in the oxidative phosphorylation (OXPHOS) pathway; additionally, we consistently uncovered much earlier ATP exhaustion, more severe mitochondrial structural destruction and dysfunction, and OXPHOS impairment in IAV-inoculated pDHBE cells, and these changes were rescued by melatonin. The level of OMA1-dependent cleavage of OPA1 in the mitochondrial inner membrane and the shift in energy metabolism from OXPHOS to glycolysis were significantly increased in IAV-infected pDHBE cells; however, these changes were rescued by OMA1-siRNA or melatonin further treatment. Collectively, our data revealed that melatonin rescued IAV-induced cellular energy exhaustion via OMA1-OPA1-S to improve the clinical prognosis in COPD. This treatment may serve as a potential therapeutic agent for patients in which AECOPD is induced by IAV.


Assuntos
Metabolismo Energético , GTP Fosfo-Hidrolases , Vírus da Influenza A , Melatonina , Doença Pulmonar Obstrutiva Crônica , Humanos , Metabolismo Energético/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/metabolismo , Influenza Humana/tratamento farmacológico , Melatonina/farmacologia , Metaloendopeptidases , Fosforilação Oxidativa/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico
9.
PLoS One ; 19(7): e0305396, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38980840

RESUMO

The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloprotease MIG-17 plays a crucial role in the migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans. MIG-17 is secreted from the body wall muscle cells and localizes to the basement membranes (BMs) of various tissues including the gonadal BM where it regulates DTC migration through its catalytic activity. Missense mutations in the BM protein genes, let-2/collagen IV a2 and fbl-1/fibulin-1, have been identified as suppressors of the gonadal defects observed in mig-17 mutants. Genetic analyses indicate that LET-2 and FBL-1 act downstream of MIG-17 to regulate DTC migration. In addition to the control of DTC migration, MIG-17 also plays a role in healthspan, but not in lifespan. Here, we examined whether let-2 and fbl-1 alleles can suppress the age-related phenotypes of mig-17 mutants. let-2(k196) fully and fbl-1(k201) partly, but not let-2(k193) and fbl-1(k206), suppressed the senescence defects of mig-17. Interestingly, fbl-1(k206), but not fbl-1(k201) or let-2 alleles, exhibited an extended lifespan compared to the wild type when combined with mig-17. These results reveal allele specific interactions between let-2 or fbl-1 and mig-17 in age-related phenotypes, indicating that basement membrane physiology plays an important role in organismal aging.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Colágeno Tipo IV , Mutação , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/genética , Longevidade/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Basal/metabolismo , Fenótipo , Movimento Celular/genética , Gônadas/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Desintegrinas
10.
Int J Biol Macromol ; 275(Pt 1): 133448, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38945328

RESUMO

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.


Assuntos
Fibrina , Inteínas , Metaloendopeptidases , Ciclização , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Fibrina/química , Fibrina/metabolismo , Estabilidade Enzimática , Proteólise , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/metabolismo , Ativadores de Plasminogênio/farmacologia , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Humanos , Plasminogênio/metabolismo , Plasminogênio/química , Fibrinolíticos/farmacologia , Fibrinolíticos/química
11.
Braz J Microbiol ; 55(3): 2715-2726, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38926315

RESUMO

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.


Assuntos
Antibacterianos , Proteínas de Bactérias , Dipeptídeos , Imipenem , Testes de Sensibilidade Microbiana , Elastase Pancreática , Infecções por Pseudomonas , Pseudomonas aeruginosa , Percepção de Quorum , Fatores de Virulência , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Antibacterianos/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Dipeptídeos/farmacologia , Imipenem/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Farmacorresistência Bacteriana , Metaloendopeptidases
12.
Int J Mol Sci ; 25(11)2024 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-38891974

RESUMO

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.


Assuntos
Anticorpos Monoclonais , Endocitose , Simulação de Acoplamento Molecular , Neurônios , Toxina Tetânica , Animais , Ratos , Neurônios/metabolismo , Humanos , Anticorpos Monoclonais/imunologia , Toxina Tetânica/imunologia , Toxina Tetânica/metabolismo , Tétano/prevenção & controle , Tétano/imunologia , Epitopos/imunologia , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Células Cultivadas , Simulação por Computador , Metaloendopeptidases
13.
PLoS Negl Trop Dis ; 18(6): e0012229, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38857253

RESUMO

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Camundongos Endogâmicos BALB C , Vacinas de Partículas Semelhantes a Vírus , Animais , Leishmania donovani/imunologia , Camundongos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Feminino , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Vacinas contra Leishmaniose/imunologia , Vacinas contra Leishmaniose/administração & dosagem , Eficácia de Vacinas , Imunidade Celular , Baço/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos B/imunologia , Imunidade Humoral , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/genética , Citocinas/imunologia , Metaloendopeptidases
14.
J Peripher Nerv Syst ; 29(2): 262-274, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38860315

RESUMO

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.


Assuntos
Doença de Charcot-Marie-Tooth , Metaloendopeptidases , Splicing de RNA , Adulto , Feminino , Humanos , Masculino , Doença de Charcot-Marie-Tooth/genética , Íntrons , Metaloendopeptidases/genética , Mutação , Linhagem
15.
Transfus Med ; 34(4): 287-297, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38804163

RESUMO

BACKGROUND: Antibodies against blood group antigens play a key role in the pathophysiology of haemolytic transfusion reactions (HTRs) and haemolytic disease of the fetus and newborn (HDFN). This study aimed to determine the frequencies of alleles, genotypes, and risk of alloimmunisation of clinically significant blood group systems in ethnic northeastern Thais. METHODS: In total, 345 unrelated, healthy, ethnic northeastern Thais were tested using the in-house PCR-sequence specific primers (PCR-SSP) method for simultaneously genotyping of RHCE, Kell, Duffy, Kidd, Diego and MNS glycophorin hybrids and results confirmed by Sanger sequencing. RESULTS: In this cohort, the alleles RHCE*C (81.0%) and RHCE*e (84.8%) were more prevalent than RHCE*c (19.0%) and RHCE*E (15.2%). The most common predicted haplotype combinations of the RHCE alleles were C+c-E-e+(R1R1) (59.4%) followed by the C+c+E+e+ (R1R2) (20.6%) and C+c+E-e+ (R1r) (11.3%). The KEL*01 allele was not found in this study. The frequencies of FY*01 and FY*02 were 88.3% and 11.7%, respectively. The genotype FY*02/02 was found in four samples (1.2%). The frequencies of JK*01 and JK*02 were 52.5% and 47.5%, respectively. Homozygous JK*02/02 was found in 81 samples (23.5%). The frequencies of DI*01 and DI*02 were 0.6% and 99.4%, respectively. In total, 64 samples (18.6%) were found to carry the MNS glycophorin hybrids. CONCLUSIONS: Our results indicated a possible high risk of c, E, Fyb, Jka, Jkb and Mia alloimmunisation in these populations. Moreover, methods established for genotyping clinically significant blood groups in this study can now be utilised in routine clinical application.


Assuntos
Alelos , Sistema do Grupo Sanguíneo Duffy , Glicoforinas , Sistema do Grupo Sanguíneo Rh-Hr , Feminino , Humanos , Masculino , Antígenos de Grupos Sanguíneos/genética , Sistema do Grupo Sanguíneo Duffy/genética , Etnicidade/genética , Frequência do Gene , Perfil Genético , Genótipo , Glicoforinas/genética , Isoanticorpos/sangue , Sistema do Grupo Sanguíneo de Kell/genética , Sistema do Grupo Sanguíneo de Kell/imunologia , Sistema do Grupo Sanguíneo Kidd/genética , Glicoproteínas de Membrana , Metaloendopeptidases , Sistema do Grupo Sanguíneo MNSs/genética , Sistema do Grupo Sanguíneo Rh-Hr/genética , População do Sudeste Asiático
16.
FEBS J ; 291(9): 1958-1973, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38700222

RESUMO

Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.


Assuntos
Cálcio , Precursores Enzimáticos , Peptídeo Hidrolases , Dobramento de Proteína , Serratia marcescens , Cálcio/metabolismo , Serratia marcescens/enzimologia , Serratia marcescens/genética , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Metaloendopeptidases/genética , Modelos Moleculares , Conformação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ligação Proteica
17.
Cell Death Dis ; 15(5): 380, 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38816370

RESUMO

Senescent cell clearance is emerging as a promising strategy for treating age-related diseases. Senolytics are small molecules that promote the clearance of senescent cells; however, senolytics are uncommon and their underlying mechanisms remain largely unknown. Here, we investigated whether genomic instability is a potential target for senolytic. We screened small-molecule kinase inhibitors involved in the DNA damage response (DDR) in Zmpste24-/- mouse embryonic fibroblasts, a progeroid model characterized with impaired DDR and DNA repair. 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), which specifically inhibits casein kinase 2 (CK2), was selected and discovered to preferentially trigger apoptosis in Zmpste24-/- cells. Mechanistically, inhibition of CK2 abolished the phosphorylation of heterochromatin protein 1α (HP1α), which retarded the dynamic HP1α dissociation from repressive histone mark H3K9me3 and its relocalization with γH2AX to DNA damage sites, suggesting that disrupting heterochromatin remodeling in the initiation of DDR accelerates apoptosis in senescent cells. Furthermore, feeding Zmpste24-deficient mice with TBB alleviated progeroid features and extended their lifespan. Our study identified TBB as a new class senolytic compound that can reduce age-related symptoms and prolong lifespan in progeroid mice.


Assuntos
Caseína Quinase II , Senescência Celular , Dano ao DNA , Longevidade , Proteínas de Membrana , Metaloendopeptidases , Animais , Senescência Celular/efeitos dos fármacos , Caseína Quinase II/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Camundongos , Longevidade/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Dano ao DNA/efeitos dos fármacos , Metaloendopeptidases/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/deficiência , Apoptose/efeitos dos fármacos , Homólogo 5 da Proteína Cromobox/metabolismo , Histonas/metabolismo , Camundongos Knockout , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Fosforilação/efeitos dos fármacos
18.
FASEB J ; 38(9): e23657, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38713087

RESUMO

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP.


Assuntos
Adipogenia , Carboxipeptidases , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais , Osteogênese , Análise de Célula Única , Animais , Feminino , Humanos , Camundongos , Carboxipeptidases/metabolismo , Carboxipeptidases/genética , Diferenciação Celular , Proteínas Ligadas por GPI , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Metaloendopeptidases , Camundongos Endogâmicos C57BL , Osteogênese/fisiologia , Osteogênese/genética , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Transcriptoma
19.
Diagn Microbiol Infect Dis ; 109(4): 116352, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38768547

RESUMO

In this article, a colorimetric biosensor for detection of Leishmania major surface protease (Gp63) antibody (anti-gp63) was developed by using gold nanoparticle (AuNP) as a color reagent. The dispersion or aggregation of AuNPs leads to a distinct and sensitive change in UV-vis spectra and solution color. For this purpose, kinetoplastid membrane protein-11 (KMP-11) was labeled with AuNPs surface directly. After that, Gp63 antibody was added in the KMP-11@AuNP solution and a color change from red/pink to purple/violet was observed. As a result, anti-gp63 solution diluted at a ratio of 1:640 can be detected with the developed colorimetric leishmania biosensor. The relative standard deviation value for 1:320 diluted anti-gp63 was calculated as 1.29 %. Furthermore, the linear range of the developed colorimetric biosensor was determined as 1:80 to 1:640. Moreover, developed Leishmania biosensor was applied for detection of leishmania parasite crude antigen and rabbit serum which were used as positive and negative samples respectively. As a result, the recovery values for the measurements of aforementioned samples were calculated as 95.3 % ± 0.02, 103.1 % ± 0.02, 96.2 % ± 0.01 and 95.5 % ± 0.03 for dilutions of 1:200, 1:160, 1:320 and 1:640 anti-gp63 solutions respectively.


Assuntos
Técnicas Biossensoriais , Colorimetria , Ouro , Leishmaniose , Nanopartículas Metálicas , Colorimetria/métodos , Técnicas Biossensoriais/métodos , Ouro/química , Nanopartículas Metálicas/química , Leishmaniose/diagnóstico , Animais , Coelhos , Humanos , Leishmania major/imunologia , Anticorpos Antiprotozoários/sangue , Sensibilidade e Especificidade , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/análise , Metaloendopeptidases
20.
Protein Sci ; 33(6): e5002, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38723146

RESUMO

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.


Assuntos
Proteínas de Bactérias , Multimerização Proteica , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico , Metaloendopeptidases/química , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/fisiologia , Resistência a Vancomicina/genética , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/metabolismo
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