RESUMEN
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.
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Fibrinolíticos , Accidente Cerebrovascular Isquémico , Sistema de Registros , Terapia Trombolítica , Activador de Tejido Plasminógeno , Humanos , Masculino , Femenino , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/uso terapéutico , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Anciano , Persona de Mediana Edad , Terapia Trombolítica/métodos , Fibrinolíticos/administración & dosificación , Fibrinolíticos/uso terapéutico , Resultado del Tratamiento , Metaloendopeptidasas , Administración IntravenosaRESUMEN
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.
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Dinorfinas , Neurotensina , Humanos , Especificidad por Sustrato , Dinorfinas/química , Dinorfinas/metabolismo , Neurotensina/química , Neurotensina/metabolismo , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/antagonistas & inhibidores , Unión Proteica , Cristalografía por Rayos X , Modelos Moleculares , Dominio Catalítico , Bradiquinina/química , Bradiquinina/metabolismo , Angiotensinas/metabolismo , Angiotensinas/química , Secuencia de AminoácidosRESUMEN
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.
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Metaloendopeptidasas , Mitocondrias , Neoplasias , Humanos , Neoplasias/patología , Neoplasias/metabolismo , Neoplasias/enzimología , Metaloendopeptidasas/metabolismo , Mitocondrias/metabolismo , Animales , Oxidación-ReducciónRESUMEN
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.
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Senescencia Celular , ADN Mitocondrial , Proteínas de la Membrana , Nucleotidiltransferasas , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , ADN Mitocondrial/metabolismo , ADN Mitocondrial/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Ratones , Progeria/metabolismo , Progeria/patología , Progeria/genética , Transducción de Señal , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genética , Ratones Noqueados , Células Musculares/metabolismo , Mitofagia , Mitocondrias/metabolismo , Humanos , Ratones Endogámicos C57BL , MetaloendopeptidasasRESUMEN
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.
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Metabolismo Energético , GTP Fosfohidrolasas , Virus de la Influenza A , Melatonina , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Metabolismo Energético/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/metabolismo , Gripe Humana/tratamiento farmacológico , Melatonina/farmacología , Metaloendopeptidasas , Fosforilación Oxidativa/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológicoRESUMEN
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.
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Meningitis Bacterianas , Serogrupo , Infecciones Estreptocócicas , Streptococcus suis , Enfermedades de los Porcinos , Streptococcus suis/patogenicidad , Streptococcus suis/enzimología , Animales , Infecciones Estreptocócicas/veterinaria , Infecciones Estreptocócicas/microbiología , Porcinos , Enfermedades de los Porcinos/microbiología , Ratones , Meningitis Bacterianas/veterinaria , Meningitis Bacterianas/microbiología , Femenino , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Ratones Endogámicos BALB C , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/genéticaRESUMEN
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.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Colágeno Tipo IV , Mutación , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/genética , Longevidad/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Membrana Basal/metabolismo , Fenotipo , Movimiento Celular/genética , Gónadas/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , DesintegrinasRESUMEN
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.
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Anticuerpos Monoclonales , Endocitosis , Simulación del Acoplamiento Molecular , Neuronas , Toxina Tetánica , Animales , Ratas , Neuronas/metabolismo , Humanos , Anticuerpos Monoclonales/inmunología , Toxina Tetánica/inmunología , Toxina Tetánica/metabolismo , Tétanos/prevención & control , Tétanos/inmunología , Epítopos/inmunología , Gangliósidos/inmunología , Gangliósidos/metabolismo , Células Cultivadas , Simulación por Computador , MetaloendopeptidasasRESUMEN
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.
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Leishmania donovani , Leishmaniasis Visceral , Ratones Endogámicos BALB C , Vacunas de Partículas Similares a Virus , Animales , Leishmania donovani/inmunología , Ratones , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Leishmaniasis Visceral/prevención & control , Leishmaniasis Visceral/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Vacunas contra la Leishmaniasis/inmunología , Vacunas contra la Leishmaniasis/administración & dosificación , Eficacia de las Vacunas , Inmunidad Celular , Bazo/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos B/inmunología , Inmunidad Humoral , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/genética , Citocinas/inmunología , MetaloendopeptidasasRESUMEN
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.
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Enfermedad de Charcot-Marie-Tooth , Metaloendopeptidasas , Empalme del ARN , Adulto , Femenino , Humanos , Masculino , Enfermedad de Charcot-Marie-Tooth/genética , Intrones , Metaloendopeptidasas/genética , Mutación , LinajeRESUMEN
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.
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Fibrina , Inteínas , Metaloendopeptidasas , Ciclización , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fibrina/química , Fibrina/metabolismo , Estabilidad de Enzimas , Proteolisis , Activadores Plasminogénicos/química , Activadores Plasminogénicos/metabolismo , Activadores Plasminogénicos/farmacología , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Humanos , Plasminógeno/metabolismo , Plasminógeno/química , Fibrinolíticos/farmacología , Fibrinolíticos/químicaRESUMEN
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.
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Calcio , Precursores Enzimáticos , Péptido Hidrolasas , Pliegue de Proteína , Serratia marcescens , Calcio/metabolismo , Serratia marcescens/enzimología , Serratia marcescens/genética , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/genética , Modelos Moleculares , Conformación Proteica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Unión ProteicaRESUMEN
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.
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Técnicas Biosensibles , Colorimetría , Oro , Leishmaniasis , Nanopartículas del Metal , Colorimetría/métodos , Técnicas Biosensibles/métodos , Oro/química , Nanopartículas del Metal/química , Leishmaniasis/diagnóstico , Animales , Conejos , Humanos , Leishmania major/inmunología , Anticuerpos Antiprotozoarios/sangre , Sensibilidad y Especificidad , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/análisis , MetaloendopeptidasasRESUMEN
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.
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Proteínas Bacterianas , Multimerización de Proteína , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Dominio Catalítico , Metaloendopeptidasas/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/química , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/fisiología , Resistencia a la Vancomicina/genética , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/metabolismoRESUMEN
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.
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Quinasa de la Caseína II , Senescencia Celular , Daño del ADN , Longevidad , Proteínas de la Membrana , Metaloendopeptidasas , Animales , Senescencia Celular/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Ratones , Longevidad/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Daño del ADN/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/deficiencia , Apoptosis/efectos de los fármacos , Homólogo de la Proteína Chromobox 5/metabolismo , Histonas/metabolismo , Ratones Noqueados , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Fosforilación/efectos de los fármacosRESUMEN
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.
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Adipogénesis , Carboxipeptidasas , Sistema de Señalización de MAP Quinasas , Células Madre Mesenquimatosas , Osteogénesis , Análisis de la Célula Individual , Animales , Femenino , Humanos , Ratones , Carboxipeptidasas/metabolismo , Carboxipeptidasas/genética , Diferenciación Celular , Proteínas Ligadas a GPI , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Metaloendopeptidasas , Ratones Endogámicos C57BL , Osteogénesis/fisiología , Osteogénesis/genética , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , TranscriptomaRESUMEN
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.
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Alelos , Sistema del Grupo Sanguíneo Duffy , Glicoforinas , Sistema del Grupo Sanguíneo Rh-Hr , Femenino , Humanos , Masculino , Antígenos de Grupos Sanguíneos/genética , Sistema del Grupo Sanguíneo Duffy/genética , Etnicidad/genética , Frecuencia de los Genes , Perfil Genético , Genotipo , Glicoforinas/genética , Isoanticuerpos/sangre , Sistema del Grupo Sanguíneo de Kell/genética , Sistema del Grupo Sanguíneo de Kell/inmunología , Sistema del Grupo Sanguíneo de Kidd/genética , Glicoproteínas de Membrana , Metaloendopeptidasas , Sistema del Grupo Sanguíneo MNSs/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Pueblos del Sudeste AsiáticoRESUMEN
BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.
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Proteínas de Arabidopsis , Arabidopsis , MicroARNs , Solanum lycopersicum , Solanum lycopersicum/genética , Clorofila A/metabolismo , Filogenia , Cloroplastos/genética , Arabidopsis/genética , Mutación , Fenotipo , Hojas de la Planta/metabolismo , Carotenoides/metabolismo , MicroARNs/metabolismo , Precursores de Proteínas/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Proteínas de Arabidopsis/genéticaRESUMEN
Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.
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Presión Sanguínea , Calcio , Metaloendopeptidasas , Ratones Noqueados , Músculo Liso Vascular , Miocitos del Músculo Liso , Animales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Calcio/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/genética , Masculino , Ratones Endogámicos C57BL , Hipotensión/metabolismo , Células Cultivadas , Aorta/metabolismo , Aorta/citología , Vasoconstricción/efectos de los fármacos , Señalización del CalcioRESUMEN
Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.