RESUMO
Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin ß4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of α-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. α-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin ß4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Parkinson , alfa-Defensinas , Humanos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Cistatina B/análise , Cistatina B/metabolismo , Proteômica/métodos , Doença de Parkinson/diagnóstico , Doença de Parkinson/metabolismo , Doenças Neurodegenerativas/metabolismo , alfa-Defensinas/análise , alfa-Defensinas/metabolismo , Saliva/química , Proteínas e Peptídeos Salivares/metabolismo , Fatores de Transcrição/metabolismo , Biomarcadores/análiseRESUMO
EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease.
Assuntos
Cistatina B , Terapia Genética , Camundongos Knockout , Doenças Neuroinflamatórias , Animais , Camundongos , Terapia Genética/métodos , Cistatina B/genética , Doenças Neuroinflamatórias/terapia , Doenças Neuroinflamatórias/genética , Humanos , Ataxia/genética , Ataxia/terapia , Epilepsias Mioclônicas Progressivas/genética , Epilepsias Mioclônicas Progressivas/terapia , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagemRESUMO
Human stefin B, a member of the cystatin family of cysteine protease inhibitors, tends to form amyloid fibrils under relatively mild conditions, which is why it is used as a model protein to study amyloid fibrillation. Here, we show for the first time that bundles of amyloid fibrils, i.e., helically twisted ribbons, formed by human stefin B exhibit birefringence. This physical property is commonly observed in amyloid fibrils when stained with Congo red. However, we show that the fibrils arrange in regular anisotropic arrays and no staining is required. They share this property with anisotropic protein crystals, structured protein arrays such as tubulin and myosin, and other anisotropic elongated materials, such as textile fibres and liquid crystals. In certain macroscopic arrangements of amyloid fibrils, not only birefringence is observed, but also enhanced emission of intrinsic fluorescence, implying a possibility to detect amyloid fibrils with no labels by using optical microscopy. In our case, no enhancement of intrinsic tyrosine fluorescence was observed at 303 nm; instead, an additional fluorescence emission peak appeared at 425 to 430 nm. We believe that both phenomena, birefringence and fluorescence emission in the deep blue, should be further explored with this and other amyloidogenic proteins. This may allow the development of label-free detection methods for amyloid fibrils of different origins.
Assuntos
Amiloide , Cistatinas , Humanos , Cistatina B , Amiloide/metabolismo , Cistatinas/metabolismo , Vermelho Congo , Inibidores de Cisteína ProteinaseRESUMO
BACKGROUND: Gastric cancer (GC) is the fifth most common cancer and the third cause of cancer deaths globally, with late diagnosis, low survival rate, and poor prognosis. This case-control study aimed to evaluate the expression of cystatin B (CSTB) and deleted in malignant brain tumor 1 (DMBT1) in the saliva of GC patients with healthy individuals to construct diagnostic algorithms using statistical analysis and machine learning methods. METHODS: Demographic data, clinical characteristics, and food intake habits of the case and control group were gathered through a standard checklist. Unstimulated whole saliva samples were taken from 31 healthy individuals and 31 GC patients. Through ELISA test and statistical analysis, the expression of salivary CSTB and DMBT1 proteins was evaluated. To construct diagnostic algorithms, we used the machine learning method. RESULTS: The mean salivary expression of CSTB in GC patients was significantly lower (115.55 ± 7.06, p = 0.001), and the mean salivary expression of DMBT1 in GC patients was significantly higher (171.88 ± 39.67, p = 0.002) than the control. Multiple linear regression analysis demonstrated that GC was significantly correlated with high levels of DMBT1 after controlling the effects of age of participants (R2 = 0.20, p < 0.001). Considering salivary CSTB greater than 119.06 ng/mL as an optimal cut-off value, the sensitivity and specificity of CSTB in the diagnosis of GC were 83.87 and 70.97%, respectively. The area under the ROC curve was calculated as 0.728. The optimal cut-off value of DMBT1 for differentiating GC patients from controls was greater than 146.33 ng/mL (sensitivity = 80.65% and specificity = 64.52%). The area under the ROC curve was up to 0.741. As a result of the machine learning method, the area under the receiver-operating characteristic curve for the diagnostic ability of CSTB, DMBT1, demographic data, clinical characteristics, and food intake habits was 0.95. The machine learning model's sensitivity, specificity, and accuracy were 100, 70.8, and 80.5%, respectively. CONCLUSION: Salivary levels of DMBT1 and CSTB may be accurate in diagnosing GCs. Machine learning analyses using salivary biomarkers, demographic, clinical, and nutrition habits data simultaneously could provide affordability models with acceptable accuracy for differentiation of GC by a cost-effective and non-invasive method.
Assuntos
Neoplasias Gástricas , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Cistatina B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Saliva/metabolismo , Neoplasias Gástricas/patologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses.
Assuntos
Cyprinidae , Cistatinas , Doenças dos Peixes , Smegmamorpha , Animais , Cyprinidae/genética , Cistatina B/genética , Cistatinas/genética , Proteínas de Peixes/química , Masculino , Filogenia , Poli I-C/farmacologia , Alinhamento de SequênciaRESUMO
No robust biomarkers have yet been identified for autism spectrum disorder (ASD) or autistic traits. Familial factors likely influence biomarkers such as protein concentrations. Comparing twins with ASD or high autistic traits to the less affected co-twin allows estimating the impact of familial confounding. We measured 203 proteins in cerebrospinal fluid (n = 86) and serum (n = 127) in twins (mean age 14.2 years, 44.9% females) enriched for ASD and other neurodevelopmental conditions. Autistic traits were assessed by using the parent-report version of the Social Responsiveness Scale-2. In cerebrospinal fluid, autistic traits correlated negatively with three proteins and positively with one. In serum, autistic traits correlated positively with 15 and negatively with one. Also in serum, six were positively-and one negatively-associated with ASD. A pathway analysis of these proteins revealed immune system enrichment. In within twin pair analyses, autistic traits were associated with serum B-cell activating factor (BAFF) only, whereas Cystatin B (CSTB) remained significantly associated with ASD. These associations did not remain significant when only considering monozygotic twins. For the remainder, the within-pair analysis indicated familial confounding, including shared environment and genes, influencing both autism and protein levels. Our findings indicate proteins involved in immunity as putative biomarkers of autistic traits and ASD with partial genetic confounding. Although some results are in line with previous studies in general, further studies are needed for replication.
Assuntos
Transtorno do Espectro Autista/sangue , Transtorno do Espectro Autista/líquido cefalorraquidiano , Gêmeos Monozigóticos , Adolescente , Adulto , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Transtorno Autístico/sangue , Transtorno Autístico/líquido cefalorraquidiano , Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Fator Ativador de Células B/sangue , Fator Ativador de Células B/líquido cefalorraquidiano , Fator Ativador de Células B/genética , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Criança , Estudos de Coortes , Estudos Transversais , Cistatina B/sangue , Cistatina B/líquido cefalorraquidiano , Cistatina B/genética , Feminino , Humanos , Masculino , Mapas de Interação de Proteínas/fisiologia , Gêmeos Monozigóticos/genética , Adulto JovemRESUMO
Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency.
Assuntos
Cistatina B/deficiência , Histonas/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Animais , Células Cultivadas , Cistatina B/genética , Epigênese Genética/fisiologia , Histonas/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos KnockoutRESUMO
Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.
Assuntos
Colo do Útero/metabolismo , Infecções por HIV/transmissão , Proteômica/métodos , Infecções Sexualmente Transmissíveis/metabolismo , Vagina/metabolismo , Adulto , Colo do Útero/virologia , Análise por Conglomerados , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Cistatina B/metabolismo , Diagnóstico Precoce , Elafina/metabolismo , Feminino , Infecções por HIV/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Calicreínas/metabolismo , Estudos Longitudinais , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Parceiros Sexuais , Espectrometria de Massas em Tandem , Vagina/virologia , Adulto JovemRESUMO
BACKGROUND & AIMS: In a phase 3 trial (RESORCE), regorafenib increased overall survival compared with placebo in patients with hepatocellular carcinoma (HCC) previously treated with sorafenib. In an exploratory study, we analyzed plasma and tumor samples from study participants to identify genetic, microRNA (miRNA), and protein biomarkers associated with response to regorafenib. METHODS: We obtained archived tumor tissues and baseline plasma samples from patients with HCC given regorafenib in the RESORCE trial. Baseline plasma samples from 499 patients were analyzed for expression of 294 proteins (DiscoveryMAP) and plasma samples from 349 patients were analyzed for levels of 750 miRNAs (miRCURY miRNA PCR). Tumor tissues from 7 responders and 10 patients who did not respond (progressors) were analyzed by next-generation sequencing (FoundationOne). Forty-six tumor tissues were analyzed for expression patterns of 770 genes involved in oncogenic and inflammatory pathways (PanCancer Immune Profiling). Associations between plasma levels of proteins and miRNAs and response to treatment (overall survival and time to progression) were evaluated using a Cox proportional hazards model. RESULTS: Decreased baseline plasma concentrations of 5 of 266 evaluable proteins (angiopoietin 1, cystatin B, the latency-associated peptide of transforming growth factor beta 1, oxidized low-density lipoprotein receptor 1, and C-C motif chemokine ligand 3; adjusted P ≤ .05) were significantly associated with increased overall survival time after regorafenib treatment. Levels of these 5 proteins, which have roles in inflammation and/or HCC pathogenesis, were not associated with survival independently of treatment. Only 20 of 499 patients had high levels and a reduced survival time. Plasma levels of α-fetoprotein and c-MET were associated with poor outcome (overall survival) independently of regorafenib treatment only. We identified 9 plasma miRNAs (MIR30A, MIR122, MIR125B, MIR200A, MIR374B, MIR15B, MIR107, MIR320, and MIR645) whose levels significantly associated with overall survival time with regorafenib (adjusted P ≤ .05). Functional analyses of these miRNAs indicated that their expression level associated with increased overall survival of patients with tumors of the Hoshida S3 subtype. Next-generation sequencing analyses of tumor tissues revealed 49 variants in 27 oncogenes or tumor suppressor genes. Mutations in CTNNB1 were detected in 3 of 10 progressors and VEGFA amplification in 1 of 7 responders. CONCLUSION: We identified expression patterns of plasma proteins and miRNAs that associated with increased overall survival times of patients with HCC following treatment with regorafenib in the RESORCE trial. Levels of these circulating biomarkers and genetic features of tumors might be used to identify patients with HCC most likely to respond to regorafenib. ClinicalTrials.gov number NCT01774344. NCBI GEO accession numbers: mRNA data (NanoString): GSE119220; miRNA data (Exiqon): GSE119221.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/sangue , Compostos de Fenilureia/uso terapêutico , Piridinas/uso terapêutico , Idoso , Angiopoietina-1/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Quimiocina CCL3/sangue , Cistatina B/sangue , Feminino , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Mutação , Oncogenes/genética , Intervalo Livre de Progressão , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Receptores Depuradores Classe E/sangue , Taxa de Sobrevida , Transcriptoma , Fator de Crescimento Transformador beta1/sangue , Proteínas Supressoras de Tumor/genética , Fator A de Crescimento do Endotélio Vascular/genética , alfa-Fetoproteínas/metabolismo , beta Catenina/genéticaRESUMO
Viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. With the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. Viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. Studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (MVP) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and MVP to stimulate the interest of related researchers.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/fisiologia , Viroses/virologia , Antivirais/farmacologia , Cistatina B/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Hepatite E/tratamento farmacológico , Hepatite E/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/virologia , Interferon Tipo I/metabolismo , Triterpenos/farmacologia , Replicação ViralRESUMO
OBJECTIVE: Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very efficient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB). METHODS: We used confocal imaging and stereology-based quantification of apoptosis and phagocytosis of the hippocampus of Cstb knockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apoptosis, phagocytosis, and active hippocampal neurons. RESULTS: Microglial phagocytosis was impaired in the hippocampus of Cstb KO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present in Cstb KO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) in Cstb-deficient mice were at close proximity to active cFos+ neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. SIGNIFICANCE: These results suggest a complex crosstalk between apoptosis, phagocytosis, and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, these data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.
Assuntos
Giro Denteado/metabolismo , Modelos Animais de Doenças , Microglia/metabolismo , Neurônios/metabolismo , Fagocitose/fisiologia , Síndrome de Unverricht-Lundborg/metabolismo , Animais , Cistatina B/deficiência , Cistatina B/genética , Giro Denteado/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Neurônios/patologia , Síndrome de Unverricht-Lundborg/genética , Síndrome de Unverricht-Lundborg/patologiaRESUMO
Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention.
Assuntos
Cistatina B/genética , Cistatina B/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Cistatina B/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de SequênciaRESUMO
Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine (8GG9) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5'-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3'-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts.
Assuntos
Cistatina B/genética , Cistatina B/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Cistatina B/química , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peixes , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterináriaRESUMO
AIM OF THE STUDY: The aim of this study was to explore genetic findings and the phenotype in Polish patients with Unverricht-Lundborg disease (ULD). MATERIALS AND METHODS: We retrospectively evaluated mutations in the cystatin B (CSTB) gene and clinical presentation in a cohort of patients with ULD. The study population consisted of 19 (14 males) patients with genetically confirmed disease. RESULTS: Sixteen patients were homozygous for the expanded dodecamer repeat mutation alleles, one subject was compound heterozygous for the dodecamer repeat expansion and other mutation, in two, the type of mutation has not yet been established. The numbers of repeats in the CSTB gene varied from 60 to 81. Clinical information was available for 16 subjects. The disease course was progressive in all patients, leading to severe disability, mainly due to myoclonus, in nine. CONCLUSIONS AND CLINICAL IMPLICATIONS: Genetic findings and the clinical picture of our patients with ULD were in accordance with available studies. The most common genetic defect underlying ULD was homozygosity for an unstable expansion of a dodecamer repeat in the CSTB gene. Patients with action or/and stimulus sensitive myoclonus or intractable myoclonus epilepsy, especially with onset in late childhood/adolescence should be screened for ULD.
Assuntos
Síndrome de Unverricht-Lundborg , Adolescente , Criança , Estudos de Coortes , Cistatina B/genética , Testes Genéticos , Humanos , Masculino , Fenótipo , Polônia , Estudos Retrospectivos , Síndrome de Unverricht-Lundborg/genéticaRESUMO
Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Cistatina B/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/efeitos dos fármacos , Vitamina K 3/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/genética , Apoptossomas/efeitos dos fármacos , Apoptossomas/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina B/metabolismo , Catepsina C/antagonistas & inibidores , Catepsina C/genética , Catepsina C/metabolismo , Catepsina D/antagonistas & inibidores , Catepsina D/genética , Catepsina D/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Catepsinas/metabolismo , Cistatina B/metabolismo , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pepstatinas/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Transdução de Sinais , Células U937RESUMO
Endometrial cancer is one of the most common cancers in women worldwide, affecting more than 300,000 women annually. Dysregulated gene expression, especially those mediated by microRNAs, play important role in the development and progression of cancer. This study aimed to investigate differentially expressed genes in endometrial adenocarcinoma using next generation sequencing (NGS) and bioinformatics. The gene expression profiles and microRNA profiles of endometrial adenocarcinoma (cancer part) and normal endometrial tissue (non-cancer part) were assessed with NGS. We identified 56 significantly dysregulated genes, including 47 upregulated and 9 downregulated genes, in endometrial adenocarcinoma. Most of these genes were associated with defense response, response to stimulus, and immune system process, and further pathway analysis showed that human papillomavirus infection was the most significant pathway in endometrial adenocarcinoma. In addition, these genes were also associated with decreased cell death and survival as well as increased cellular movement. The analyses using Human Protein Atlas, identified 6 genes (PEG10, CLDN1, ASS1, WNT7A, GLDC, and RSAD2) significantly associated with poorer prognosis and 3 genes (SFN, PIGR, and CDKN1A) significantly associated with better prognosis. Combining with the data of microRNA profiles using microRNA target predicting tools, two significantly dysregulated microRNA-mediated gene expression changes in endometrial adenocarcinoma were identified: downregulated hsa-miR-127-5p with upregulated CSTB and upregulated hsa-miR-218-5p with downregulated HPGD. These findings may contribute important new insights into possible novel diagnostic or therapeutic strategies for endometrial adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Endométrio/genética , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/genética , Biologia Computacional , Cistatina B/genética , Regulação para Baixo , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/patologia , Endométrio/patologia , Feminino , Seguimentos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Estimativa de Kaplan-Meier , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para CimaRESUMO
Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease.
Assuntos
Catepsina B/metabolismo , Cistatina B/metabolismo , Degeneração Neural , Doença de Niemann-Pick Tipo C/metabolismo , Células de Purkinje/metabolismo , Animais , Apoptose , Proteínas de Transporte/genética , Catepsina B/antagonistas & inibidores , Cistatina B/genética , Cistatina B/farmacologia , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Lisossomos/patologia , Glicoproteínas de Membrana/genética , Camundongos , Mutação , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Estresse Oxidativo , Proteínas/genética , Células de Purkinje/patologiaRESUMO
OBJECTIVE/BACKGROUND: The development of an abdominal aortic aneurysm (AAA) involves extensive extracellular matrix remodelling, leading to aortic wall weakening. This process is mediated by proteases, including cysteinyl cathepsins. Cystatins are their endogenous inhibitors. This study tested whether plasma cystatin B levels in patients with AAA differed from those of healthy controls. METHODS: Plasma samples from patients with AAA and age matched controls were selected from the Viborg Vascular (VIVA) screening trial for AAA. Enzyme linked immunosorbent assay determined plasma cystatin B. T-test, logistic regression, Pearson's correlation and Cox regression tested whether plasma cystatin B correlates with AAA size and growth rate, or serves as a marker for AAA. RESULTS: Plasma cystatin B levels were significantly higher in patients with AAA than in controls (p < 0.001). Logistic regression analysis showed that cystatin B tertile at baseline was associated with the presence of AAA before (odds ratio [OR] 1.656; p < 0.001) and after adjustment for peripheral arterial disease (PAD), chronic obstructive pulmonary disease (COPD), and previous ischaemic events (OR 1.526; p < 0.001). A t-test showed a significant association between cystatin B and PAD at screening, hospital diagnosis of COPD, previous atherosclerotic events, and use of low dose aspirin. Pearson's correlation test showed positive and significant associations between cystatin B and AAA size (r = 0.15; p < 0.001). Cox regression test showed that plasma cystatin B tertile at baseline was associated with later AAA surgical repair before (hazard ratio [HR] 1.387; p < 0.001) and after adjustment for PAD, COPD, previous ischaemic event, and maximum infrarenal aortic diameter (HR 1.523; p < 0.001). CONCLUSION: In contrast to prior studies that showed that cystatin C is negatively associated with AAA development, this study demonstrated a positive association between cystatin B and AAA size and associations between cystatin B tertile at baseline and AAA presence and need for later surgical repair. It is possible that these two cystatins inhibit cathepsin activity and participate in AAA with different mechanisms.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/cirurgia , Cistatina B/sangue , Idoso , Aneurisma da Aorta Abdominal/patologia , Biomarcadores/sangue , Estudos de Casos e Controles , Humanos , Modelos Logísticos , Masculino , Estudos Prospectivos , Fatores de RiscoRESUMO
Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder (Paralichthys olivaceus) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1ß and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts.
Assuntos
Cistatina B/imunologia , Linguado/imunologia , Animais , Linhagem Celular , Cistatina B/genética , Cistatina B/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Citocinas/genética , DNA Complementar/genética , Feminino , Linguado/genética , Fatores Imunológicos/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Peptidoglicano/farmacologia , Poli I-C/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc.