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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207510

RESUMO

Interstitial lung diseases (ILDs) comprise different fibrotic lung disorders characterized by cellular proliferation, interstitial inflammation, and fibrosis. The JAK/STAT molecular pathway is activated under the interaction of a broad number of profibrotic/pro-inflammatory cytokines, such as IL-6, IL-11, and IL-13, among others, which are increased in different ILDs. Similarly, several growth factors over-expressed in ILDs, such as platelet-derived growth factor (PDGF), transforming growth factor ß1 (TGF-ß1), and fibroblast growth factor (FGF) activate JAK/STAT by canonical or non-canonical pathways, which indicates a predominant role of JAK/STAT in ILDs. Between the different JAK/STAT isoforms, it appears that JAK2/STAT3 are predominant, initiating cellular changes observed in ILDs. This review analyzes the expression and distribution of different JAK/STAT isoforms in ILDs lung tissue and different cell types related to ILDs, such as lung fibroblasts and alveolar epithelial type II cells and analyzes JAK/STAT activation. The effect of JAK/STAT phosphorylation on cellular fibrotic processes, such as proliferation, senescence, autophagy, endoplasmic reticulum stress, or epithelial/fibroblast to mesenchymal transition will be described. The small molecules directed to inhibit JAK/STAT activation were assayed in vitro and in in vivo models of pulmonary fibrosis, and different JAK inhibitors are currently approved for myeloproliferative disorders. Recent evidence indicates that JAK inhibitors or monoclonal antibodies directed to block IL-6 are used as compassionate use to attenuate the excessive inflammation and lung fibrosis related to SARS-CoV-2 virus. These altogether indicate that JAK/STAT pathway is an attractive target to be proven in future clinical trials of lung fibrotic disorders.


Assuntos
Janus Quinases/metabolismo , Doenças Pulmonares Intersticiais/patologia , Fatores de Transcrição STAT/metabolismo , Senescência Celular , Estresse do Retículo Endoplasmático , Humanos , Interleucinas/metabolismo , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Transdução de Sinais
2.
Nat Commun ; 12(1): 4230, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244494

RESUMO

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXß2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXß2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXß2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXß2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.


Assuntos
Processamento Alternativo , Carcinoma Epitelial do Ovário/genética , Proteínas da Matriz Extracelular/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Ovarianas/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ovário/patologia , Ovário/cirurgia , Fosforilação/genética , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nat Commun ; 12(1): 4212, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244496

RESUMO

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Feminino , Masculino , Metilação , Oogênese/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Processos de Determinação Sexual/genética
4.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207662

RESUMO

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.


Assuntos
Queratinas/metabolismo , Agregados Proteicos , Proteína Sequestossoma-1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Sequestossoma-1/genética
5.
Methods Enzymol ; 655: 85-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34183135

RESUMO

The development of high-throughput technologies has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization involving thousands of overlapping coding and non-coding RNA isoforms that challenge our traditional definitions of genes and functional regions of the genome. In this chapter, we discuss the application of an improved Transcript Isoform Sequencing approach (TIF-Seq2) able to concurrently determine the start and end sites of individual RNA molecules. We exemplify its use for the investigation of the human transcriptome and show how it is especially well suited to discriminate between overlapping molecules and accurately define their boundaries.


Assuntos
Genoma , Transcriptoma , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Análise de Sequência de RNA
6.
Nat Commun ; 12(1): 3285, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078899

RESUMO

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.


Assuntos
Nervo Femoral/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Tibial/metabolismo , Animais , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Axônios/metabolismo , Axônios/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Nervo Femoral/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Células de Schwann/patologia , Nervo Isquiático/patologia , Estresse Fisiológico , Nervo Tibial/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
7.
PLoS One ; 16(6): e0253458, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34185793

RESUMO

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Dopa Descarboxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Enzima de Conversão de Angiotensina 2/genética , Área Sob a Curva , COVID-19/virologia , Dopa Descarboxilase/genética , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Regulação para Cima , Carga Viral
8.
BMC Res Notes ; 14(1): 239, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34167576

RESUMO

OBJECTIVE: In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. RESULTS: In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the "regulation of signaling receptor activity" Gene Ontology annotation as a result of viral infection.


Assuntos
Perfilação da Expressão Gênica , Infecções por Poxviridae , Animais , Chlorocebus aethiops , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Isoformas de Proteínas/genética , Transcriptoma
9.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063460

RESUMO

The ubiquitously expressed adaptor protein Shc exists in three isoforms p46Shc, p52Shc, and p66Shc, which execute distinctly different actions in cells. The role of p46Shc is insufficiently studied, and the purpose of this study was to further investigate its functional significance. We developed unique rat mutants lacking p52Shc and p46Shc isoforms (p52Shc/46Shc-KO) and carried out histological analysis of skeletal and cardiac muscle of parental and genetically modified rats with impaired gait. p52Shc/46Shc-KO rats demonstrate severe functional abnormalities associated with impaired gait. Our analysis of p52Shc/46Shc-KO rat axons and myelin sheets in cross-sections of the sciatic nerve revealed the presence of significant anomalies. Based on the lack of skeletal muscle fiber atrophy and the presence of sciatic nerve abnormalities, we suggest that the impaired gait in p52Shc/46Shc-KO rats might be due to the sensory feedback from active muscle to the brain locomotor centers. The lack of dystrophin in some heart muscle fibers reflects damage due to dilated cardiomyopathy. Since rats with only p52Shc knockout do not display the phenotype of p52Shc/p46Shc-KO, abnormal locomotion is likely to be caused by p46Shc deletion. Our data suggest a previously unknown role of 46Shc actions and signaling in regulation of gait.


Assuntos
Cardiomiopatia Dilatada/genética , Marcha/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Cardiomiopatia Dilatada/patologia , Técnicas de Inativação de Genes , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Isoformas de Proteínas/genética , Ratos Transgênicos , Nervo Isquiático/patologia
10.
Methods Enzymol ; 654: 345-364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120721

RESUMO

Alternative splicing of RNA transcripts allows a single gene to generate multiple products and is a key means of generating functionally diverse voltage-gated ion channels. Splicing can be regulated according to cell type, cell state, and stage of development to produce a bespoke complement of protein isoforms. Characterizing the identities of full-length transcript isoforms is essential in order to fully understand a gene's expression and function. However, the repertoire of transcript isoforms is not well characterized for most genes. Long read nanopore sequencing allows full-length isoforms to be sequenced, therefore identifying full-length transcripts. Using this approach, we recently discovered that the human CACNA1C gene gives rise to a far greater repertoire of splice isoforms than previously appreciated. Here we provide a detailed overview of the technical approach we used to achieve this. The method described in this chapter combines long read nanopore sequencing with PCR targeting to selectively sequence transcripts of a specific gene of interest.


Assuntos
Processamento Alternativo , Splicing de RNA , Perfilação da Expressão Gênica , Humanos , Canais Iônicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
11.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068576

RESUMO

Apolipoprotein E (ApoE) is a protein that plays an important role in the transport of fatty acids and cholesterol and in cellular signaling. On the surface of the cells, ApoE lipoparticles bind to low density lipoprotein receptors (LDLR) that mediate the uptake of the lipids and downstream signaling events. There are three alleles of the human ApoE gene. Presence of ApoE4 allele is a major risk factor for developing Alzheimer's disease (AD) and other disorders late in life, but the mechanisms responsible for biological differences between different ApoE isoforms are not well understood. We here propose that the differences between ApoE isoforms can be explained by differences in the pH-dependence of the association between ApoE3 and ApoE4 isoforms and LDL-A repeats of LDLR. As a result, the following endocytosis ApoE3-associated LDLRs are recycled back to the plasma membrane but ApoE4-containing LDLR complexes are trapped in late endosomes and targeted for degradation. The proposed mechanism is predicted to lead to a reduction in steady-state surface levels of LDLRs and impaired cellular signaling in ApoE4-expressing cells. We hope that this proposal will stimulate experimental research in this direction that allows the testing of our hypothesis.


Assuntos
Apolipoproteína E3/genética , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Receptores de LDL/genética , Alelos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Endocitose/genética , Regulação da Expressão Gênica , Humanos , Isoformas de Proteínas/genética , Transdução de Sinais
12.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068748

RESUMO

Estrogen receptor beta (ERß) plays a critical role in granulosa cell (GC) functions. The existence of four human ERß splice isoforms in the ovary suggests their differential implication in 17ß-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERß isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERß isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERß isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERß1, ERß2, ERß4, and ERß5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERß4 and ERß5 isoforms in these cells. In addition, we demonstrated that overexpression of ERß1 and ERß4 in HGrC1 cells increased cell apoptosis by 225% while ERß5 or ERß2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERß isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERß isoforms.


Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Células da Granulosa/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Ovário/efeitos dos fármacos , Isoformas de Proteínas/genética , RNA Mensageiro/genética
13.
Nat Commun ; 12(1): 3416, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099706

RESUMO

APOE and Trem2 are major genetic risk factors for Alzheimer's disease (AD), but how they affect microglia response to Aß remains unclear. Here we report an APOE isoform-specific phospholipid signature with correlation between human APOEε3/3 and APOEε4/4 AD brain and lipoproteins from astrocyte conditioned media of APOE3 and APOE4 mice. Using preclinical AD mouse models, we show that APOE3 lipoproteins, unlike APOE4, induce faster microglial migration towards injected Aß, facilitate Aß uptake, and ameliorate Aß effects on cognition. Bulk and single-cell RNA-seq demonstrate that, compared to APOE4, cortical infusion of APOE3 lipoproteins upregulates a higher proportion of genes linked to an activated microglia response, and this trend is augmented by TREM2 deficiency. In vitro, lack of TREM2 decreases Aß uptake by APOE4-treated microglia only, suggesting TREM2-APOE interaction. Our study elucidates phenotypic and transcriptional differences in microglial response to Aß mediated by APOE3 or APOE4 lipoproteins in preclinical models of AD.


Assuntos
Doença de Alzheimer/patologia , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Encéfalo/patologia , Microglia/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apolipoproteína E3/administração & dosagem , Apolipoproteína E3/genética , Apolipoproteína E4/administração & dosagem , Apolipoproteína E4/genética , Encéfalo/citologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fosfolipídeos/metabolismo , Presenilina-1/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
14.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070744

RESUMO

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.


Assuntos
Encéfalo/metabolismo , Canais de Cloreto/genética , Células Intersticiais do Testículo/metabolismo , Neurônios/metabolismo , Doença de Pelizaeus-Merzbacher/genética , Proteostase/genética , Animais , Benzoquinonas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células CHO , Canais de Cloreto/deficiência , Cricetulus , Modelos Animais de Doenças , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Doença de Pelizaeus-Merzbacher/tratamento farmacológico , Doença de Pelizaeus-Merzbacher/metabolismo , Doença de Pelizaeus-Merzbacher/patologia , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo
15.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070875

RESUMO

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.


Assuntos
Subunidades Proteicas/química , Fator 2 Associado a Receptor de TNF/química , Tirosina/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Termodinâmica , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
16.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071104

RESUMO

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.


Assuntos
Colina O-Acetiltransferase/biossíntese , Neurônios Colinérgicos/metabolismo , Gânglios Espinais/citologia , Proteínas do Tecido Nervoso/biossíntese , Células Receptoras Sensoriais/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/biossíntese , Acetilcolina/metabolismo , Processamento Alternativo , Animais , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurogênese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/citologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
17.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073823

RESUMO

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase C/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
18.
BMC Bioinformatics ; 22(Suppl 10): 271, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34058988

RESUMO

BACKGROUND: Translational regulation is one important aspect of gene expression regulation. Dysregulation of translation results in abnormal cell physiology and leads to diseases. Ribosome profiling (RP), also called ribo-seq, is a powerful experimental technique to study translational regulation. It can capture a snapshot of translation by deep sequencing of ribosome-protected mRNA fragments. Many ribosome profiling data processing tools have been developed. However, almost all tools analyze ribosome profiling data at the gene level. Since different isoforms of a gene may produce different proteins with distinct biological functions, it is advantageous to analyze ribosome profiling data at the isoform level. To meet this need, previously we developed a pipeline to analyze 610 public human ribosome profiling data at the isoform level and constructed HRPDviewer database. RESULTS: To allow other researchers to use our pipeline as well, here we implement our pipeline as an easy-to-use software tool called RPiso. Compared to Ribomap (a widely used tool which provides isoform-level ribosome profiling analyses), our RPiso (1) estimates isoform abundance more accurately, (2) supports analyses on more species, and (3) provides a web-based viewer for interactively visualizing ribosome profiling data on the selected mRNA isoforms. CONCLUSIONS: In this study, we developed RPiso software tool ( http://cosbi7.ee.ncku.edu.tw/RPiso/ ) to provide isoform-level ribosome profiling analyses. RPiso is very easy to install and execute. RPiso also provides a web-based viewer for interactively visualizing ribosome profiling data on the selected mRNA isoforms. We believe that RPiso is a useful tool for researchers to analyze and visualize their own ribosome profiling data at the isoform level.


Assuntos
Biossíntese de Proteínas , Ribossomos , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Software
19.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065232

RESUMO

Tau protein is largely responsible for tauopathies, including Alzheimer's disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3ß, whose activity is inhibited by the cellular prion protein (PrPC), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrPC and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrPC and the implication of GSK3ß in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrPC ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrPC expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrPC levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3ß activity downstream from PrPC in this phenomenon. Our results indicate that PrPC plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3ß.


Assuntos
Regulação para Baixo/genética , Éxons/genética , Glicogênio Sintase Quinase 3 beta/genética , Príons/genética , Proteínas tau/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo/genética , Doença de Alzheimer/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Corpos de Inclusão/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/patologia , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Tauopatias/genética
20.
Hum Genet ; 140(8): 1121-1141, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34169326

RESUMO

Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.


Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/biossíntese , Osteoblastos/metabolismo , Osteogênese Imperfeita/metabolismo , Pró-Colágeno/biossíntese , Processamento de Proteína Pós-Traducional , Osso e Ossos/patologia , Colágeno Tipo I/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Osteoblastos/patologia , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Pró-Colágeno/genética , Biossíntese de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Transporte Proteico , Índice de Gravidade de Doença
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