Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.871
Filtrar
1.
Microb Pathog ; 131: 128-134, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959097

RESUMO

Quorum sensing (QS) is the cell density dependent communication network which coordinates the production of pathogenic determinants in majority of pathogenic bacteria. Pseudomonas aeruginosa causes hospital-acquired infections by virtue of its well-defined QS network. As the QS regulatory network in P. aeruginosa regulates the virulence determinants and antibiotic resistance, attenuating the QS system seems to be influential in developing next-generation anti-infective agents. In the current study, the QS attenuation potential of a flavonoid, mosloflavone was investigated against P. aeruginosa virulence and biofilm formation. Mosloflavone inhibited the pyocyanin production, LasB elastase and chitinase by 59.52 ±â€¯2.74, 35.90 ±â€¯4.34 and 61.18 ±â€¯5.52% respectively. The QS regulated biofilm formation and development was also reduced when supplemented with sub-MIC of mosloflavone. The gene expression studies of mosloflavone using RT-PCR depicted its ability to down-regulate the expression levels of QS regulated virulence genes such as lasI (60.64%), lasR (91.70%), rhlI (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%) and pelA (50.45%). The down-regulation of QS virulence phenotypes by mosloflavone could be attributed to its binding affinity with the QS regulatory proteins, LasR and RhlR by competitively inhibiting the binding of natural autoinducers as evidenced from simulation studies. Mosloflavone also exhibited promising potential in controlling bacterial infection in Caenorhabditis elegans model system, in vivo. The anti-biofilm and anti-QS potential of mosloflavone in the current study illustrated the candidature of mosloflavone as a promising biocide.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Flavonoides/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Quitinases/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Metaloendopeptidases/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Piocianina/metabolismo , Transativadores/genética , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética
2.
Nat Commun ; 10(1): 1198, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867416

RESUMO

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.


Assuntos
Proteínas de Bactérias/metabolismo , Doença Celíaca/imunologia , Proteínas na Dieta/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Metaloendopeptidases/metabolismo , Receptor PAR-2/imunologia , Adulto , Idoso , Animais , Antígenos/imunologia , Antígenos/metabolismo , Proteínas de Bactérias/genética , Biópsia , Estudos de Casos e Controles , Doença Celíaca/diagnóstico por imagem , Doença Celíaca/microbiologia , Doença Celíaca/patologia , Estudos de Coortes , Colonoscopia , Proteínas na Dieta/metabolismo , Modelos Animais de Doenças , Duodeno/imunologia , Duodeno/metabolismo , Duodeno/microbiologia , Duodeno/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Vida Livre de Germes , Glutens/imunologia , Glutens/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteólise , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptor PAR-2/metabolismo , Regulação para Cima , Adulto Jovem
3.
BMC Genomics ; 20(1): 118, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732584

RESUMO

BACKGROUND: The leishmaniasis are parasitic diseases caused by protozoans of the genus Leishmania, highly divergent eukaryotes, characterized by unique biological features. To survive in both the mammalian hosts and insect vectors, these pathogens make use of a number of mechanisms, many of which are associated with parasite specific proteases. The metalloprotease GP63, the major Leishmania surface antigen, has been found to have multiple functions required for the parasite's survival. GP63 is encoded by multiple genes and their copy numbers vary considerably between different species and are increased in those from the subgenus Viannia, including L. braziliensis. RESULTS: By comparing multiple sequences from Leishmania and related organisms this study sought to characterize paralogs in silico, evaluating their differences and similarities and the implications for the GP63 function. The Leishmania GP63 genes are encoded on chromosomes 10, 28 and 31, with the genes from the latter two chromosomes more related to genes found in insect or plant parasites. Those from chromosome 10 have experienced independent expansions in numbers in Leishmania, especially in L. braziliensis. These could be clustered in three groups associated with different mRNA 3' untranslated regions as well as distinct C-terminal ends for the encoded proteins, with presumably distinct expression patterns and subcellular localizations. Sequence variations between the chromosome 10 genes were linked to intragenic recombination events, mapped to the external surface of the proteins and predicted to be immunogenic, implying a role against the host immune response. CONCLUSIONS: Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes, possibly related to the pathogenesis of L. braziliensis and closely related species within the mammalian host. They also indicate different functions associated to genes mapped to different chromosomes. For the chromosome 10 genes, variable subcellular localizations were found to be most likely associated with multiple functions and target substrates for this versatile protease.


Assuntos
Simulação por Computador , Variação Genética , Evasão da Resposta Imune/genética , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Metaloendopeptidases/genética , Sequência de Aminoácidos , Cromossomos/genética , Epitopos de Linfócito B/imunologia , Evolução Molecular , Leishmania braziliensis/patogenicidade , Metaloendopeptidases/química , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Virulência/genética
4.
Mol Cell ; 73(5): 1028-1043.e5, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733118

RESUMO

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.


Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Antiparkinsonianos/farmacologia , Transporte Biológico , Desenho de Drogas , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
Mol Plant Microbe Interact ; 32(7): 841-852, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30694091

RESUMO

Bacterial panicle blight caused by Burkholderia glumae is a major bacterial disease of rice. Our preliminary RNA-seq study showed that a serine metalloprotease gene, prtA, is regulated in a similar manner to the genes for the biosynthesis and transport of toxoflavin, which is a known major virulence factor of B. glumae. prtA null mutants of the virulent strain B. glumae 336gr-1 did not show a detectable extracellular protease activity, indicating that prtA is the solely responsible gene for the extracellular protease activity detected from this bacterium. In addition, inoculation of rice panicles with the prtA mutants resulted in a significant reduction of disease severity compared with the wild-type parent strain, suggesting the requirement of prtA for the full virulence of B. glumae. A double mutant deficient in both serine metalloprotease and toxoflavin (ΔtoxA/prtA-) exhibited a further numeric but not statistically significant decrease of disease development compared with the ΔtoxA strain. Both the prtA-driven extracellular protease activity and the toxoflavin production were dependent on both the tofI/tofR quorum-sensing and the global regulatory gene qsmR, indicating the important roles of the two global regulatory factors for the bacterial pathogenesis by this pathogen.


Assuntos
Burkholderia , Regulação Bacteriana da Expressão Gênica , Metaloendopeptidases , Virulência , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Burkholderia/genética , Burkholderia/patogenicidade , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Virulência/genética
6.
Cell Mol Life Sci ; 76(5): 1005-1025, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30599067

RESUMO

BACKGROUND: The ADAM10-mediated cleavage of transmembrane proteins regulates cellular processes such as proliferation or migration. Substrate cleavage by ADAM10 has also been implicated in pathological situations such as cancer or Morbus Alzheimer. Therefore, identifying endogenous molecules, which modulate the amount and consequently the activity of ADAM10, might contribute to a deeper understanding of the enzyme's role in both, physiology and pathology. METHOD: To elucidate the underlying cellular mechanism of the TBX2-mediated repression of ADAM10 gene expression, we performed overexpression, RNAi-mediated knockdown and pharmacological inhibition studies in the human neuroblastoma cell line SH-SY5Y. Expression analysis was conducted by e.g. real-time RT-PCR or western blot techniques. To identify the binding region of TBX2 within the ADAM10 promoter, we used luciferase reporter assay on deletion constructs and EMSA/WEMSA experiments. In addition, we analyzed a TBX2 loss-of-function Drosophila model regarding the expression of ADAM10 orthologs by qPCR. Furthermore, we quantified the mRNA level of TBX2 in post-mortem brain tissue of AD patients. RESULTS: Here, we report TBX2 as a transcriptional repressor of ADAM10 gene expression: both, the DNA-binding domain and the repression domain of TBX2 were necessary to effect transcriptional repression of ADAM10 in neuronal SH-SY5Y cells. This regulatory mechanism required HDAC1 as a co-factor of TBX2. Transcriptional repression was mediated by two functional TBX2 binding sites within the core promoter sequence (- 315 to - 286 bp). Analysis of a TBX2 loss-of-function Drosophila model revealed that kuzbanian and kuzbanian-like, orthologs of ADAM10, were derepressed compared to wild type. Vice versa, analysis of cortical brain samples of AD-patients, which showed reduced ADAM10 mRNA levels, revealed a 2.5-fold elevation of TBX2, while TBX3 and TBX21 levels were not affected. CONCLUSION: Our results characterize TBX2 as a repressor of ADAM10 gene expression and suggest that this regulatory interaction is conserved across tissues and species.


Assuntos
Proteína ADAM10/genética , Doença de Alzheimer/etiologia , Regulação da Expressão Gênica , Proteínas com Domínio T/fisiologia , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Encéfalo/metabolismo , Células Cultivadas , Desintegrinas/genética , Drosophila , Proteínas de Drosophila/genética , Histona Desacetilase 1/fisiologia , Humanos , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Regiões Promotoras Genéticas , Proteínas com Domínio T/química , Transcrição Genética
7.
Neuron ; 101(3): 429-443.e4, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30578106

RESUMO

Normal vascular development includes the formation and specification of arteries, veins, and intervening capillaries. Vein of Galen malformations (VOGMs) are among the most common and severe neonatal brain arterio-venous malformations, shunting arterial blood into the brain's deep venous system through aberrant direct connections. Exome sequencing of 55 VOGM probands, including 52 parent-offspring trios, revealed enrichment of rare damaging de novo mutations in chromatin modifier genes that play essential roles in brain and vascular development. Other VOGM probands harbored rare inherited damaging mutations in Ephrin signaling genes, including a genome-wide significant mutation burden in EPHB4. Inherited mutations showed incomplete penetrance and variable expressivity, with mutation carriers often exhibiting cutaneous vascular abnormalities, suggesting a two-hit mechanism. The identified mutations collectively account for ∼30% of studied VOGM cases. These findings provide insight into disease biology and may have clinical implications for risk assessment.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Mutação , Malformações da Veia de Galeno/genética , Efrinas/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Metaloendopeptidases/genética , Linhagem , Penetrância , Receptor EphB4/genética , Transdução de Sinais , Malformações da Veia de Galeno/patologia
8.
Dev Comp Immunol ; 90: 176-185, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30261235

RESUMO

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Bombyx/imunologia , Metaloendopeptidases/metabolismo , Pseudomonas aeruginosa/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/genética , Bombyx/microbiologia , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Hemolinfa/metabolismo , Sistema Imunitário , Imunidade Inata , Larva , Metaloproteinase 12 da Matriz , Metaloendopeptidases/genética , Microrganismos Geneticamente Modificados , Mutação/genética , Proteínas/metabolismo , Pseudomonas aeruginosa/patogenicidade , Virulência
9.
Eur J Neurol ; 26(1): 80-86, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30098094

RESUMO

BACKGROUND AND PURPOSE: Hereditary ataxias are heterogeneous groups of neurodegenerative disorders, characterized by cerebellar syndromes associated with dysarthria, oculomotor and corticospinal signs, neuropathy and cognitive impairment. Recent reports have suggested mutations in the SPG7 gene, causing the most common form of autosomal recessive spastic paraplegia (MIM#607259), as a main cause of ataxias. The majority of described patients were homozygotes or compound heterozygotes for the c.1529C>T (p.Ala510Val) change. We screened a cohort of 895 Italian patients with ataxia for p.Ala510Val in order to define the prevalence and genotype-phenotype correlation of this variant. METHODS: We set up a rapid assay for c.1529C>T using restriction enzyme analysis after polymerase chain reaction amplification. We confirmed the diagnosis with Sanger sequencing. RESULTS: We identified eight homozygotes and 13 compound heterozygotes, including two novel variants affecting splicing. Mutated patients showed a pure cerebellar ataxia at onset, evolving in mild spastic ataxia (alternatively) associated with dysarthria (~80% of patients), urinary urgency (~30%) and pyramidal signs (~70%). Comparing homozygotes and compound heterozygotes, we noted a difference in age at onset and Scale for the Assessment and Rating of Ataxia score between the two groups, supporting an earlier and more severe phenotype in compound heterozygotes versus homozygotes. CONCLUSIONS: The SPG7 c.1529C>T (p.Ala510Val) mutants accounted for 2.3% of cerebellar ataxia cases in Italy, suggesting that this variant should be considered as a priority test in the presence of late-onset pure ataxia. Moreover, the heterozygous/homozygous genotype appeared to predict the onset of clinical manifestation and disease progression.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Ataxia Cerebelar/epidemiologia , Ataxia Cerebelar/genética , Metaloendopeptidases/genética , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Estudos de Associação Genética , Heterozigoto , Homozigoto , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Prevalência
10.
Proteomics Clin Appl ; 13(2): e1800135, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30548811

RESUMO

BACKGROUND: Progeria-like syndromes offer a unique insight into aging. Here the case of a boy affected with mandibuloacral dysplasia and compound heterozygous mutations in ZMPSTE24 is presented. METHODS: Capillary electrophoresis-mass spectroscopy is used for proteome analysis to analyze peptides previously found to be differentially regulated in chronic kidney disease (273 peptides defining the CKD273 classifier), coronary artery disease (238 peptides defining the CAD238 classifier), and aging (116 peptides defining the AGE116 classifier). RESULTS: No evidence of renal disease is identified. Although the boy has no overt cardiovascular disease other than a raised carotid intima media thickness relative to his age, a proteomic classifier for the diagnosis of coronary artery disease is mildly raised. The biological age based on the proteomic AGE116 classifier is 24 years compared to the chronological ages of 5 and 10 years. In contrast, a control group of healthy children has a significantly lower (p < 0.0001) calculated mean age of 13. CONCLUSION: Urinary proteomic analysis is effective in confirming advanced biological age and to identify early evidence of renal or cardiovascular damage. This case highlights the value of proteomic approaches in aging research and may represent a method for non-invasive monitoring of the effects of early aging.


Assuntos
Envelhecimento/genética , Doenças do Desenvolvimento Ósseo/genética , Anormalidades Craniofaciais/genética , Heterozigoto , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mutação , Proteômica , Criança , Pré-Escolar , Humanos
11.
Anticancer Res ; 39(1): 215-223, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30591461

RESUMO

BACKGROUND/AIM: Etomidate, an intravenous anesthetic, has been shown to have anticancer effects, including induction of cell-cycle arrest and apoptosis. However, to our knowledge, there are no reports about the anti-metastasis effects of etomidate on A549 human lung adenocarcinoma cells. MATERIALS AND METHODS: The cell viability, cell adhesion, gelatin zymography assay, transwell migration and invasion assay, and western blotting analysis were used to investigate the effects of etomidate on A549 cells. RESULTS: In our study, etomidate showed low cytotoxicity, inhibited cell adhesion, and suppressed the migration and invasion in A549 cells. The activity of matrix metallopeptidase 2 (MMP2) was reduced by 48 h treatment of etomidate. Results of western blotting analysis indicated that etomidate down-regulated the expression of protein kinase C, MMP7, MMP1, MMP9, and p-p-38, but up-regulated that of RAS, phosphoinositide 3-kinase, and phosphor-extracellular-signal related kinase after 24 and 48 h treatment, in A549 cells. CONCLUSION: Etomidate suppressed the migration and invasion of lung adenocarcinoma A549 cells via inhibiting the expression of MMP1, MMP2, MMP7 and MMP9, and provides potential therapeutic targets for lung cancer treatment.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Etomidato/farmacologia , Metaloendopeptidases/genética , Invasividade Neoplásica/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/genética
12.
Orphanet J Rare Dis ; 13(1): 226, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30558655

RESUMO

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is a rare autosomal recessive disease characterized by the combination of glomerulopathy with early-onset nephrotic syndrome and microcephaly with central nervous system anomalies. Given its clinical heterogeneity, GAMOS is believed to be a genetically heterogenous group of disorders. Recently, it has been reported that mutations in KEOPS-encoding genes, including the OSGEP gene, were responsible for GAMOS. RESULTS: Overall, 6 patients from 5 different Taiwanese families were included in our study; the patients had an identical OSGEP gene mutation (c.740G > A transition) and all exhibited a uniform clinical phenotype with early-onset nephrotic syndrome, craniofacial and skeletal dysmorphism, primary microcephaly with pachygyria, and death before 2 years of age. We reviewed their clinical manifestations, the prenatal and postnatal presentations and ultrasound findings, results of imaging studies, associated anomalies, and outcome on follow-up. All individuals were found to have an "aged face" comprising peculiar facial dysmorphisms. Arachnodactyly or camptodactyly were noted in all patients. Neurological findings consisted of microcephaly, hypotonia, developmental delay, and seizures. Brain imaging studies all showed pachygyria and hypomyelination. All patients developed early-onset nephrotic syndrome. The proteinuria was steroid-resistant and eventually resulted in renal function impairment. Prenatal ultrasound findings included microcephaly, intrauterine growth restriction, and oligohydramnios. Fetal MRI in 2 patients confirmed the gyral and myelin abnormalities. CONCLUSIONS: Our study suggests that a careful review of the facial features can provide useful clues for an early and accurate diagnosis. Prenatal ultrasound findings, fetal MRI, genetic counseling, and mutation analysis may be useful for an early prenatal diagnosis.


Assuntos
Hérnia Hiatal/genética , Metaloendopeptidases/genética , Microcefalia/genética , Nefrose/genética , Aracnodactilia/genética , Feminino , Humanos , Lisencefalia/genética , Masculino , Mutação/genética , Síndrome Nefrótica/genética , Diagnóstico Pré-Natal , Estudos Retrospectivos , Taiwan
13.
Int J Mol Sci ; 19(12)2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30544562

RESUMO

Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.


Assuntos
Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Western Blotting , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HEK293 , Humanos , Metaloendopeptidases/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética
14.
Vet Res ; 49(1): 109, 2018 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-30373658

RESUMO

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.


Assuntos
Metaloendopeptidases/metabolismo , Streptococcus suis/enzimologia , Animais , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/genética , Camundongos , Domínios Proteicos , Serina Endopeptidases/metabolismo , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Virulência
15.
PLoS One ; 13(4): e0196474, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29698456

RESUMO

Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.


Assuntos
Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Microscopia de Fluorescência , Filogenia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato
16.
Pathog Dis ; 76(4)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29684130

RESUMO

Clostridial neurotoxins, which include botulinum neurotoxins (BoNTs) and tetanus neurotoxins, have evolved a remarkably sophisticated structure and molecular mechanism fine-tuned for the targeting and cleavage of vertebrate neuron substrates leading to muscular paralysis. How and why did this toxin evolve? From which ancestral proteins are BoNTs derived? And what is, or was, the primary ecological role of BoNTs in the environment? In this article, we examine these questions in light of recent studies identifying homologs of BoNTs in the genomes of non-clostridial bacteria, including Weissella, Enterococcus and Chryseobacterium. Genomic and phylogenetic analysis of these more distantly related toxins suggests that they are derived from ancient toxin lineages that predate the evolution of BoNTs and are not limited to the Clostridium genus. We propose that BoNTs have therefore evolved from a precursor family of BoNT-like toxins, and ultimately from non-neurospecific toxins that cleaved different substrates (possibly non-neuronal SNAREs). Comparison of BoNTs with these related toxins reveals several unique molecular features that underlie the evolution of BoNT's unique function, including functional shifts involving all four domains, and gain of the BoNT gene cluster associated proteins. BoNTs then diversified to produce the existing serotypes, including TeNT, and underwent repeated substrate shifts from ancestral VAMP2 specificity to SNAP25 specificity at least three times in their history. Finally, similar to previous proposals, we suggest that one ecological role of BoNTs could be to create a paralytic phase in vertebrate decomposition, which provides a competitive advantage for necrophagous scavengers that in turn facilitate the spread of Clostridium botulinum and its toxin.


Assuntos
Clostridium botulinum/genética , Clostridium tetani/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Metaloendopeptidases/genética , Toxina Tetânica/genética , Chryseobacterium/classificação , Chryseobacterium/genética , Chryseobacterium/patogenicidade , Clostridium botulinum/classificação , Clostridium botulinum/patogenicidade , Clostridium tetani/classificação , Clostridium tetani/patogenicidade , Enterococcus/classificação , Enterococcus/genética , Enterococcus/patogenicidade , Evolução Molecular , Loci Gênicos , Interações Hospedeiro-Patógeno , Humanos , Metaloendopeptidases/biossíntese , Família Multigênica , Filogenia , Toxina Tetânica/biossíntese , Weissella/classificação , Weissella/genética , Weissella/patogenicidade
17.
Artigo em Inglês | MEDLINE | ID: mdl-29567411

RESUMO

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.


Assuntos
Núcleo Celular/patologia , Modelos Animais de Doenças , Proteínas de Peixes/deficiência , Proteínas de Membrana/deficiência , Metaloendopeptidases/deficiência , Oryzias/genética , Progéria/patologia , Nadadeiras de Animais/enzimologia , Nadadeiras de Animais/patologia , Nadadeiras de Animais/efeitos da radiação , Animais , Animais Geneticamente Modificados , Núcleo Celular/enzimologia , Núcleo Celular/efeitos da radiação , Forma do Núcleo Celular/efeitos da radiação , Células Cultivadas , Códon sem Sentido , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Oryzias/metabolismo , Progéria/enzimologia , Progéria/genética , Tolerância a Radiação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sobrevida , Encurtamento do Telômero/efeitos da radiação
18.
PLoS One ; 13(3): e0194612, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29579063

RESUMO

Protein myristoylation is a key protein modification carried out by N-Myristoyltransferase (NMT) after Methionine aminopeptidase 2 (MetAP2) removes methionine from the amino-terminus of the target protein. Protein myristoylation by NMT augments several signaling pathways involved in a myriad of cellular processes, including developmental pathways and pathways that when dysregulated lead to cancer or immune dysfunction. The emerging evidence pointing to NMT-mediated myristoylation as a major cellular regulator underscores the importance of understanding the framework of this type of signaling event. Various studies have investigated the role that myristoylation plays in signaling dysfunction by examining differential gene or protein expression between normal and diseased states, such as cancers or following HIV-1 infection, however no study exists that addresses the role of microRNAs (miRNAs) in the regulation of myristoylation. By performing a large scale bioinformatics and functional analysis of the miRNAs that target key genes involved in myristoylation (NMT1, NMT2, MetAP2), we have narrowed down a list of promising candidates for further analysis. Our condensed panel of miRNAs identifies 35 miRNAs linked to cancer, 21 miRNAs linked to developmental and immune signaling pathways, and 14 miRNAs linked to infectious disease (primarily HIV). The miRNAs panel that was analyzed revealed several NMT-targeting mRNAs (messenger RNA) that are implicated in diseases associated with NMT signaling alteration, providing a link between the realms of miRNA and myristoylation signaling. These findings verify miRNA as an additional facet of myristoylation signaling that must be considered to gain a full perspective. This study provides the groundwork for future studies concerning NMT-transcript-binding miRNAs, and will potentially lead to the development of new diagnostic/prognostic biomarkers and therapeutic targets for several important diseases.


Assuntos
Aciltransferases/metabolismo , Aminopeptidases/metabolismo , Doenças Transmissíveis/diagnóstico , Metaloendopeptidases/metabolismo , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Aciltransferases/genética , Aminopeptidases/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Análise por Conglomerados , Doenças Transmissíveis/enzimologia , Doenças Transmissíveis/genética , Receptores ErbB/metabolismo , Humanos , Metaloendopeptidases/genética , MicroRNAs/genética , Neoplasias/enzimologia , Neoplasias/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/metabolismo
20.
Mol Biotechnol ; 60(4): 302-309, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29502205

RESUMO

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.


Assuntos
Leptospira interrogans/enzimologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cromatografia de Afinidade , Clonagem Molecular , Inibidores de Cisteína Proteinase/metabolismo , Metaloendopeptidases/química , Engenharia de Proteínas , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inibidores de Serino Proteinase/metabolismo , Zinco/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA