RESUMO
BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.
ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.
Assuntos
Aminopeptidases , Epilepsia , Lipofuscinoses Ceroides Neuronais , Serina Proteases , Tripeptidil-Peptidase 1 , Humanos , Lipofuscinoses Ceroides Neuronais/genética , Feminino , Masculino , Epilepsia/genética , Aminopeptidases/genética , Serina Proteases/genética , Criança , Adolescente , Adulto , Adulto Jovem , Pré-Escolar , Proteínas de Ligação a Telômeros/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Mutação , Testes Genéticos/métodos , Pessoa de Meia-Idade , LactenteRESUMO
The use of force spectroscopy approaches performed with optical tweezers can be very useful in determining the binding modes and the physical chemistry of DNA interactions with ligands, from small drugs to proteins. Helminthophagous fungi, on the other hand, have important enzyme secretion mechanisms for various purposes, and the interactions between such enzymes and nucleic acids are very poorly studied. Therefore, the main goal of the present work was to investigate, at the molecular level, the mechanisms of interaction between fungal serine proteases and the double-stranded (ds) DNA molecule. Experimental assays performed with this single molecule technique consist in exposing different concentrations of the protease of this fungus to dsDNA until saturation while monitoring the changes on the mechanical properties of the macromolecular complexes formed, from where the physical chemistry of the interaction can be deduced. It was found that the protease binds strongly to the double-helix, forming aggregates and changing the persistence length of the DNA molecule. The present work thus allowed us to infer information at the molecular level on the pathogenicity of these proteins, an important class of biological macromolecules, when applied to a target specimen.
Assuntos
Ascomicetos , Serina Proteases , Serina Proteases/genética , Ascomicetos/genética , Serina Endopeptidases , DNARESUMO
Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.
Assuntos
Autofagia , Caspase 1 , Interleucina-1beta , Neutrófilos , Serina Proteases , Autofagia/genética , Autofagia/imunologia , Caspase 1/genética , Caspase 1/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Serina Proteases/genética , Serina Proteases/imunologiaRESUMO
Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.
Assuntos
Bacteriemia , Toxinas Bacterianas , Proteínas de Escherichia coli , Escherichia coli Uropatogênica , Animais , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Camundongos , Serina Endopeptidases/metabolismo , Serina Proteases/genética , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/metabolismoRESUMO
OBJECTIVES: lncRNA ABHD11 antisense RNA 1 (ABHD11-AS1) acts as an oncogene involved in papillary thyroid carcinoma (PTC) occurrence and progression. ABHD11-AS1 exerts biologic functions by some miRNAs and proteins to regulate multiple targets. Identification of novel mechanism of ABHD11-AS1 could be helpful in therapeutic targeting for PTC treatment. METHODS: Differentially expressed lncRNAs were selected from TCGA database. qRT-PCR analysis was applied to examine the expression of ABHD11-AS1 in PTC cell lines and tissues. The relationship of ABHD11-AS1 expression and clinicopathological features was analyzed by Kaplan-Meier analysis. Two PTC cell lines (TPC-1 and KTC-1) were transfected with pcDNA 3.1, pcDNA3.1-ABHD11-AS1, si-NC and si-ABHD11-AS1, respectively, to verify the ABHD11-AS1 oncogene-regulating capacity to promote tumor progression. The cell metastasis and proliferation had been evaluated both in vitro and in vivo. RESULTS: High expression of ABHD11-AS1 was found in PTC tissues (P < 0.01), which was significantly correlated with lymph node metastasis (P < 0.05). ABHD11-AS1 overexpression noticeably promoted cell proliferation, migration, and invasion capabilities, which were obviously decreased upon ABHD11-AS1 knockdown. ABHD11-AS1 positively regulated EGFR/EPS15L1 pathway, as EGFR, EPS15L1, STAT3, and p-STAT3 were activated. CONCLUSION: ABHD11-AS1 promotes tumor progression in PTC by regulating EPS15L1/EGFR pathway.
Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias da Glândula Tireoide , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismo , Transdução de Sinais/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders-R) and relapse or therapeutic failure (Non-responders-NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.
Assuntos
Antimônio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/enzimologia , Serina Proteases/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Parasitologia , Serina Proteases/genéticaRESUMO
The aim of the study was to determine the frequency of six genes encoding serine protease autotransporter proteins Enterobacteriaceae (SPATE) in diffusely adherent Escherichia coli (DAEC) isolates from children with (WD, n=63) and without diarrhea (WOD, n=41) from Lima, Peru. WOD were considered a control group. For the detection of the genes, 2 multiple PCRs were standardized: triple A (sigA, pet, espP) and triple B (sat, pic, espC). In both groups, the most frequent SPATE gene was Sat (39.7% of WD and 41.5% of WOD), followed by spP (20.6% and 9.7% in WD and WOD respectively). The other genes were detected in proportions lower than 10.0%, in the following order of frequency: pet, sigA, espC and pic, without significant differences between the groups. It was concluded that Sat is the most frequent SPATE in DAEC and that these strains may possess SPATE genes regardless of whether they are isolated in WD or WOD.
El objetivo del estudio fue determinar la frecuencia de seis genes que codifican proteínas autotransportadoras serin-proteasa de Enterobacteriaceae (SPATE) en aislamientos de Escherichia coli difusamente adherente (DAEC) provenientes de niños con diarrea (NCD, n=63) y sin diarrea (NSD, n=41) de Lima, Perú. Los NSD se consideraron como grupo control. Para la detección de los genes se estandarizaron 2 PCRs múltiples: triple A (sigA, pet, espP) y triple B (sat, pic, espC). En ambos grupos el gen SPATE más frecuente fue sat (39,7% de NCD y 41,5% de NSD), seguido de espP (20,6% y 9,7% en NCD y NSD respectivamente). Los otros genes se detectaron en proporciones inferiores al 10,0%, en el siguiente orden de frecuencia: pet, sigA, espC y pic, sin diferencias significativas entre los grupos. Se concluye que Sat es la SPATE más frecuente en cepas DAEC, y que estas cepas pueden poseer genes SPATE independientemente de si se aíslan en NCD o NSD.
Assuntos
Toxinas Bacterianas , Infecções por Escherichia coli , Proteínas de Escherichia coli , Criança , Diarreia , Enterobacteriaceae , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Peru , Serina Proteases/genética , Sistemas de Secreção Tipo VRESUMO
OBJECTIVE: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. RESULTS: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. CONCLUSION: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.
Assuntos
Mycobacterium tuberculosis/enzimologia , Pichia/crescimento & desenvolvimento , Serina Proteases/genética , Serina Proteases/metabolismo , Aldeído Oxidase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia de Afinidade , Proteínas Fúngicas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga , Humanos , Interleucina-10/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Mycobacterium tuberculosis/genética , Pichia/genética , Pichia/metabolismo , Domínios Proteicos , Engenharia de Proteínas , Serina Proteases/química , Serina Proteases/farmacologia , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Aging is a gradual biological process characterized by a decrease in cellular and organism functions. Aging-related processes involve changes in the expression and activity of several proteins. Here, we identified the transmembrane protease serine 11a (TMPRSS11a) as a new age-specific protein that plays an important role in skin wound healing. TMPRSS11a levels increased with age in rodent and human skin and gingival samples. Strikingly, overexpression of TMPRSS11a decreased cell migration and spreading, and inducing cellular senescence. Mass spectrometry, bioinformatics, and functional analyses revealed that TMPRSS11a interacts with integrin ß1 through an RGD sequence contained within the C-terminal domain and that this motif was relevant for cell migration. Moreover, TMPRSS11a was associated with cellular senescence, as shown by overexpression and downregulation experiments. In agreement with tissue-specific expression of TMPRSS11a, shRNA-mediated downregulation of this protein improved wound healing in the skin, but not in the skeletal muscle of old mice, where TMPRSS11a is undetectable. Collectively, these findings indicate that TMPRSS11a is a tissue-specific factor relevant for wound healing, which becomes elevated with aging, promoting cellular senescence and inhibiting cell migration and skin repair.
Assuntos
Envelhecimento/patologia , Movimento Celular , Fibroblastos/patologia , Proteínas de Membrana/metabolismo , Serina Proteases/metabolismo , Pele/patologia , Cicatrização , Adolescente , Adulto , Idoso , Envelhecimento/metabolismo , Animais , Proliferação de Células , Fibroblastos/metabolismo , Gengiva/metabolismo , Gengiva/patologia , Humanos , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Serina Proteases/genética , Transdução de Sinais , Pele/metabolismo , Adulto JovemRESUMO
Babesia, Theileria and Cytauxzoon are tick-borne apicomplexan protozoans of the order Piroplasmida, notorious for the diseases they cause in livestock, pets and humans. Host cell invasion is their Achilles heel, allowing for the development of drug or vaccine-based therapies. In other apicomplexans, cleavage of the transmembrane domain of adhesins by the serine rhomboid proteinase ROM4 is required for successful completion of invasion. In this study, we record and classify the rhomboid repertoire encoded in the genomes of 10 piroplasmid species pertaining to the lineages Babesia sensu stricto (s.s., Clade VI), Theileria sensu stricto (Clade IV), Theileria equi (Clade IV), Cytauxzoon felis (Clade IIIb) and Babesia microti (Clade I), as defined by Schnittger et al. (2012). Fifty-six piroplasmid rhomboid-like proteins were assigned by phylogenetic analysis and bidirectional best hit to the ROM4, ROM6, ROM7 or ROM8 groups, and their crucial motifs for conformation and function were identified. Forty-four of these rhomboids had either been incorrectly classified or misannotated. Babesia s.s. encode five or three ROM4 proteinase paralogs, whereas the remaining piroplasmids encode two ROM4 paralogs. All piroplasmids encode a single ROM6, ROM7 and ROM8. Thus, an increased paralog number of ROM4 is the only feature distinguishing Babesia s.s. from other piroplasmid lineages. Piroplasmid ROM6 is related to the mammalian mitochondrial rhomboid and, accordingly, N-terminal mitochondrial targeting signal sequences was found in some cases. ROM6 is the only rhomboid encoded by piroplasmids that is ubiquitous in other organisms. ROM8 represents a pseudoproteinase that is highly conserved between studied piroplasmids, suggesting that it is important in regulatory functions. ROM4, ROM6, ROM7 and ROM8 are exclusively present in Aconoidasida, which comprises piroplasmids and Plasmodium, suggesting a relevant functional role in erythrocyte invasion. The correct classification and designation of piroplasmid rhomboids presented in this study facilitates an informed choice for future in-depth study of their functions.