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1.
Nat Commun ; 12(1): 5216, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471137

RESUMO

Bacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.


Assuntos
Bactérias/metabolismo , Biomarcadores/metabolismo , Técnicas Biossensoriais , Proteínas de Transporte/metabolismo , Patologia Molecular/métodos , Bactérias/genética , Ácidos e Sais Biliares/sangue , Técnicas Biossensoriais/métodos , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Transplante de Fígado , Engenharia Metabólica/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência , Vibrio , Vibrioses/diagnóstico
2.
Planta ; 254(4): 71, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34505938

RESUMO

MAIN CONCLUSION: Plant class IV ACBPs diverged with the split of monocots and eudicots. Difference in the subcellular localization supported the functional variation of plant class IV ACBP. Acyl-CoA-binding proteins (ACBPs) are divided into class I-IV in plants. Class IV ACBPs are kelch motif containing proteins that are specific to plants. The currently known subcellular localizations of plant class IV ACBPs are either in the cytosol (Arabidopsis) or in the peroxisomes (rice). However, it is not clear whether peroxisomal localization of class IV ACBP is a shared character that distinguishes eudicots and monocots. Here, the phylogeny of class IV ACBPs from 73 plant species and subcellular localization of class IV ACBPs from six monocots and eudicots were conducted. Phylogenetic analysis of 112 orthologues revealed that monocot class IV ACBPs were basal to the monophyletic clade formed by eudicots and basal angiosperm. Transient expression of GFP fusions in onion epidermal cells demonstrated that monocot maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) and eudicot poplar (Populus trichocarpa) all contained at least one peroxisomal localized class IV ACBP, while orthologues from cucumber (Cucumis sativus L.) and soybean (Glycine max) were all cytosolic. Combining the location of Arabidopsis and rice class IV ACBPs, it indicates that maintaining at least one peroxisomal class IV ACBP could be a shared feature within the tested monocots, while cytosolic class IV ACBPs would be preferred in the tested eudicots. Furthermore, the interaction between OsACBP6 and peroxisomal ATP-binding cassette (ABC) transporter provided clues for the functional mechanism of OsACBP6.


Assuntos
Arabidopsis , Inibidor da Ligação a Diazepam , Arabidopsis/metabolismo , Proteínas de Transporte/genética , Coenzima A , Inibidor da Ligação a Diazepam/genética , Inibidor da Ligação a Diazepam/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445203

RESUMO

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Genes Fúngicos , Doenças das Plantas/microbiologia , Ácido Salicílico/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Triticum/microbiologia , Proteínas de Transporte/genética , Proteínas Fúngicas/genética , Fusarium/genética
4.
Biomed Res Int ; 2021: 1978434, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337001

RESUMO

Lung cancer is one of the most serious leading cancers with high incidence globally. Identifying molecular markers is key for disease diagnosis and treatment. Coal dust might be important triggering factors in disease development. Here, we first performed RNA-seq-based screening in coal dust treated and nontreated RAW264.7 cell lines. PHLDB2 was found to be the top differentially expressed gene. By retrieving TCGA lung cancer dataset, we observed that PHLDB2 showed upregulations in males and smoker patients. Patients with lower PHLDB2 expression survived longer than those with higher expressions. Furthermore, PHLDB2 was negatively correlated with EMT makers, and a total of 2.74% mutation rate were observed in 1,059 patients. This finding highlights the critical role of PHLDB2 in lung cancer development. PHLDB2 might be a molecular maker for disease diagnosis or treatment.


Assuntos
Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Carvão Mineral , Poeira , Detecção Precoce de Câncer , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , RNA-Seq , Animais , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Morte Celular/genética , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Células RAW 264.7 , Análise de Sobrevida
5.
Molecules ; 26(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34443333

RESUMO

The latest data link the chronic consumption of large amounts of fructose present in food with the generation of hypertension and disturbances in carbohydrate and lipid metabolism, which promote the development of obesity, non-alcoholic fatty liver disease, insulin resistance, and type 2 diabetes. This effect is possible after fructose is absorbed by the small intestine cells and, to a lesser extent, by hepatocytes. Fructose transport is dependent on proteins from the family of glucose transporters (GLUTs), among which GLUT5 selectively absorbs fructose from the intestine. In this study, we examined the effect of four phenolic-rich extracts obtained from A. graveolens, B. juncea, and M. chamomilla on fructose uptake by Caco-2 cells. Extracts from B. juncea and M. chamomilla most effectively reduced fluorescent fructose analogue (NBDF) accumulation in Caco-2, as well as downregulated GLUT5 protein levels. These preparations were able to decrease the mRNA level of genes encoding transcription factors regulating GLUT5 expression-thioredoxin-interacting protein (TXNIP) and carbohydrate-responsive element-binding protein (ChREBP). Active extracts contained large amounts of apigenin and flavonols. The molecular docking simulation suggested that some of identified phenolic constituents can play an important role in the inhibition of GLUT5-mediated fructose transport.


Assuntos
Dieta , Frutose/metabolismo , Transportador de Glucose Tipo 5/metabolismo , Fenóis/análise , Extratos Vegetais/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proteínas de Transporte/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/genética
6.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445467

RESUMO

Ribosome-binding protein 1 (RRBP1) is a potential oncogene in several cancer types. However, the correlation between RRBP1 expression and the prognosis of patients with upper tract urothelial carcinoma (UTUC) remains unclear. In this study, we identified that RRBP1 is associated with carcinogenesis and metastasis in UTUC using a methylation profiling microarray. High correlations between RRBP1 and cancer stages, nodal metastasis status, molecular subtypes, and prognosis in bladder urothelial cancer (BLCA) were found. Aberrant DNA methylation in the gene body region of RRBP1 was determined in UTUC tissues by methylation-specific PCR. RRBP1 expression was significantly increased in UTUC tissues and cell lines, as determined by real-time PCR and immunohistochemistry. RRBP1 depletion significantly reduced BFTC909 cell growth induced by specific shRNA. On the other hand, molecular subtype analysis showed that the expression of RRBP1 was associated with genes related to cell proliferation, epithelial-mesenchymal transition, and basal markers. A patient-derived organoid model was established to analyze patients' responses to different drugs. The expression of RRBP1 was related to chemoresistance. Taken together, these results provide the first evidence that RRBP1 gene body hypomethylation predicts RRBP1 high expression in UTUC. The data highlight the importance of RRBP1 in UTUC malignancy and chemotherapeutic tolerance.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Camundongos , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 12(1): 4178, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234147

RESUMO

Mosaic loss of chromosome Y (LOY) in leukocytes is the most common form of clonal mosaicism, caused by dysregulation in cell-cycle and DNA damage response pathways. Previous genetic studies have focussed on identifying common variants associated with LOY, which we now extend to rarer, protein-coding variation using exome sequences from 82,277 male UK Biobank participants. We find that loss of function of two genes-CHEK2 and GIGYF1-reach exome-wide significance. Rare alleles in GIGYF1 have not previously been implicated in any complex trait, but here loss-of-function carriers exhibit six-fold higher susceptibility to LOY (OR = 5.99 [3.04-11.81], p = 1.3 × 10-10). These same alleles are also associated with adverse metabolic health, including higher susceptibility to Type 2 Diabetes (OR = 6.10 [3.51-10.61], p = 1.8 × 10-12), 4 kg higher fat mass (p = 1.3 × 10-4), 2.32 nmol/L lower serum IGF1 levels (p = 1.5 × 10-4) and 4.5 kg lower handgrip strength (p = 4.7 × 10-7) consistent with proposed GIGYF1 enhancement of insulin and IGF-1 receptor signalling. These associations are mirrored by a common variant nearby associated with the expression of GIGYF1. Our observations highlight a potential direct connection between clonal mosaicism and metabolic health.


Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Y/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Mosaicismo , Adulto , Idoso , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Insulina/metabolismo , Leucócitos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genética , Sequenciamento Completo do Exoma
8.
FASEB J ; 35(8): e21800, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34324733

RESUMO

Hemophilia A and B are congenital bleeding disorders caused by a deficiency in pro-coagulant factor VIII or IX that is treated by downregulation of antithrombin. However, the molecular mechanisms that regulate antithrombin expression remain poorly understood. Here, we identified Cullin 2 and USP2 (ubiquitin-specific peptidase-2) as novel regulators of antithrombin expression that act by modulating antithrombin ubiquitination. Inhibition of the proteasome caused accumulation of antithrombin and its ubiquitinated forms in HepG2 and SMMC7721 cells. Notably, inhibition of neddylation with MLN4924 suppressed both ubiquitination and degradation of antithrombin, which is recapitulated by silencing of the neddylation enzymes, NAE1, UBA3, and UBE2M, with small interfering RNA (siRNA). We identified Cullin 2 as the interaction partner of antithrombin, and siRNA-mediated Cullin 2 knockdown reduced antithrombin ubiquitination and increased antithrombin protein. We further found that USP2 interacted with antithrombin and regulated antithrombin expression, showing that overexpression of USP2 inhibits the ubiquitination and proteasomal clearance of antithrombin, whereas pharmacological inhibition or siRNA-mediated knockdown of USP2 downregulates antithrombin. Collectively, these results suggest that Cullin 2 E3 ubiquitin ligase and USP2 coordinately regulate antithrombin ubiquitination and degradation. Thus, targeting Cullin 2 and USP2 could be a potential strategy for treatment of hemophilia.


Assuntos
Antitrombinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Proteínas Culina/genética , Regulação da Expressão Gênica , Interferência de RNA , Ubiquitina Tiolesterase/genética , Ubiquitinação
9.
J Int Med Res ; 49(7): 3000605211032807, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34311595

RESUMO

OBJECTIVE: Lung cancer (LC) is one of the most prevalent malignant tumors worldwide. As a subtype of LC, lung squamous cell carcinoma (LUSC) has a 5-year survival rate of less than 15%. In this study, we aimed to evaluate the prognostic value of a glycolysis-related gene signature in LUSC patients. METHODS: We obtained RNA-Seq data from The Cancer Genome Atlas (TCGA) database. Prognosis-related genes were screened out by Gene Set Enrichment Analysis (GSEA) and Cox proportional regression models. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to verify the mRNA expression levels in relevant tissues. RESULTS: We found that sperm-associated antigen 4 (SPAG4) overexpression was an independent risk factor for overall survival (OS) in LUSC. Patients with high-risk scores had higher mortality rates than those with low-risk scores. Moreover, by using RT-qPCR, we validated that SPAG4 mRNA was overexpressed in LUSC tissue samples compared with their paired para-cancerous histological normal tissues. CONCLUSIONS: Analysis of aberrantly overexpressed SPAG4 may provide a further useful approach to complement existing methods and predict prognosis in LUSC patients.


Assuntos
Carcinoma de Células Escamosas , Proteínas de Transporte/genética , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Prognóstico
10.
Nucleic Acids Res ; 49(13): 7318-7329, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197604

RESUMO

Integrating omics data with quantification of biological traits provides unparalleled opportunities for discovery of genetic regulators by in silico inference. However, current approaches to analyze genetic-perturbation screens are limited by their reliance on annotation libraries for prioritization of hits and subsequent targeted experimentation. Here, we present iTARGEX (identification of Trait-Associated Regulatory Genes via mixture regression using EXpectation maximization), an association framework with no requirement of a priori knowledge of gene function. After creating this tool, we used it to test associations between gene expression profiles and two biological traits in single-gene deletion budding yeast mutants, including transcription homeostasis during S phase and global protein turnover. For each trait, we discovered novel regulators without prior functional annotations. The functional effects of the novel candidates were then validated experimentally, providing solid evidence for their roles in the respective traits. Hence, we conclude that iTARGEX can reliably identify novel factors involved in given biological traits. As such, it is capable of converting genome-wide observations into causal gene function predictions. Further application of iTARGEX in other contexts is expected to facilitate the discovery of new regulators and provide observations for novel mechanistic hypotheses regarding different biological traits and phenotypes.


Assuntos
Perfilação da Expressão Gênica , Genes Reguladores , Proteólise , Fase S/genética , Software , Transcrição Genética , Proteínas de Transporte/genética , Biologia Computacional/métodos , Replicação do DNA , Deleção de Genes , Homeostase , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Arterioscler Thromb Vasc Biol ; 41(9): 2483-2493, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34320838

RESUMO

Objective: Despite considerable research, the goal of finding nonsurgical remedies against thoracic aortic aneurysm and acute aortic dissection remains elusive. We sought to identify a novel aortic PK (protein kinase) that can be pharmacologically targeted to mitigate aneurysmal disease in a well-established mouse model of early-onset progressively severe Marfan syndrome (MFS). Approach and Results: Computational analyses of transcriptomic data derived from the ascending aorta of MFS mice predicted a probable association between thoracic aortic aneurysm and acute aortic dissection development and the multifunctional, stress-activated HIPK2 (homeodomain-interacting protein kinase 2). Consistent with this prediction, Hipk2 gene inactivation significantly extended the survival of MFS mice by slowing aneurysm growth and delaying transmural rupture. HIPK2 also ranked among the top predicted PKs in computational analyses of DEGs (differentially expressed genes) in the dilated aorta of 3 MFS patients, which strengthened the clinical relevance of the experimental finding. Additional in silico analyses of the human and mouse data sets identified the TGF (transforming growth factor)-ß/Smad3 signaling pathway as a potential target of HIPK2 in the MFS aorta. Chronic treatment of MFS mice with an allosteric inhibitor of HIPK2-mediated stimulation of Smad3 signaling validated this prediction by mitigating thoracic aortic aneurysm and acute aortic dissection pathology and partially improving aortic material stiffness. Conclusions: HIPK2 is a previously unrecognized determinant of aneurysmal disease and an attractive new target for antithoracic aortic aneurysm and acute aortic dissection multidrug therapy.


Assuntos
Aneurisma Dissecante/prevenção & controle , Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Fibrilina-1/genética , Síndrome de Marfan/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Remodelação Vascular/efeitos dos fármacos , Adulto , Aneurisma Dissecante/enzimologia , Aneurisma Dissecante/genética , Aneurisma Dissecante/patologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/enzimologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dilatação Patológica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Síndrome de Marfan/complicações , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Proteína Smad3/metabolismo
12.
Eur J Med Genet ; 64(9): 104285, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34229114

RESUMO

Recently, an autosomal recessive disorder including the triad of microcephaly, infantile epileptic encephalopathy, and permanent neonatal diabetes syndrome (MEDS, OMIM#614231) has emerged as a new distinguishing syndrome. Eight cases of whom seven from Arab countries, have been reported in association with biallelic variants in the IER3IP1 gene (Immediate early response-3 interacting protein-1). Here, we describe a Tunisian boy who presented with permanent neonatal diabetes, microcephaly, generalized seizures and hypovirilized external genitalia consisting of a small genitalia and unilateral cryptorchidism. Chromosomal analysis indicated a 46, XY karyotype in all metaphases. Exome sequencing identified a homozygous missense variant (c.62 T > G; p. Val21Gly) in the IER3IP1 gene, that is predicted to alter the protein structure within the hydrophobic/transmembrane. This variant was previously reported in two cases associated with MEDS. This is the first reported case of MEDS in Tunisia. Our report focuses on the IER3IP1 related phenotypic spectrum and assumes abnormal genitalia as part of the syndrome. Consequently, we recommend to perform hormonal testing on this topic to understand the effect of the IER3IP1 variant on the male genital pathway.


Assuntos
Proteínas de Transporte/genética , Criptorquidismo/patologia , Diabetes Mellitus/patologia , Epilepsia/patologia , Doenças do Recém-Nascido/patologia , Proteínas de Membrana/genética , Transtornos Psicomotores/patologia , Proteínas de Transporte/química , Criptorquidismo/genética , Diabetes Mellitus/genética , Epilepsia/genética , Humanos , Recém-Nascido , Doenças do Recém-Nascido/genética , Cariótipo , Masculino , Proteínas de Membrana/química , Mutação de Sentido Incorreto , Domínios Proteicos , Transtornos Psicomotores/genética , Síndrome
13.
Nat Commun ; 12(1): 4373, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272385

RESUMO

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Animais , Camptotecina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , RNA Interferente Pequeno , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação
14.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203883

RESUMO

Variants of the TTLL5 gene, which encodes tubulin tyrosine ligase-like family member five, are a rare cause of cone dystrophy (COD) or cone-rod dystrophy (CORD). To date, only a few TTLL5 patients have been clinically and genetically described. In this study, we report five patients harbouring biallelic variants of TTLL5. Four adult patients presented either COD or CORD with onset in the late teenage years. The youngest patient had a phenotype of early onset severe retinal dystrophy (EOSRD). Genetic analysis was performed by targeted next generation sequencing of gene panels and assessment of copy number variants (CNV). We identified eight variants, of which six were novel, including two large multiexon deletions in patients with COD or CORD, while the EOSRD patient harboured the novel homozygous p.(Trp640*) variant and three distinct USH2A variants, which might explain the observed rod involvement. Our study highlights the role of TTLL5 in COD/CORD and the importance of large deletions. These findings suggest that COD or CORD patients lacking variants in known genes may harbour CNVs to be discovered in TTLL5, previously undetected by classical sequencing methods. In addition, variable phenotypes in TTLL5-associated patients might be due to the presence of additional gene defects.


Assuntos
Proteínas de Transporte/genética , Distrofias de Cones e Bastonetes/genética , Oftalmopatias Hereditárias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação/genética , Distrofias Retinianas/genética , Adulto , Idoso , Criança , Pontos de Quebra do Cromossomo , Simulação por Computador , Distrofias de Cones e Bastonetes/fisiopatologia , Variações do Número de Cópias de DNA/genética , Eletrorretinografia , Oftalmopatias Hereditárias/fisiopatologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Distrofias Retinianas/fisiopatologia
15.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204919

RESUMO

Defects in transcriptional and cell cycle regulation have emerged as novel pathophysiological mechanisms in congenital neuromuscular disease with the recent identification of mutations in the TRIP4 and ASCC1 genes, encoding, respectively, ASC-1 and ASCC1, two subunits of the ASC-1 (Activating Signal Cointegrator-1) complex. This complex is a poorly known transcriptional coregulator involved in transcriptional, post-transcriptional or translational activities. Inherited defects in components of the ASC-1 complex have been associated with several autosomal recessive phenotypes, including severe and mild forms of striated muscle disease (congenital myopathy with or without myocardial involvement), but also cases diagnosed of motor neuron disease (spinal muscular atrophy). Additionally, antenatal bone fractures were present in the reported patients with ASCC1 mutations. Functional studies revealed that the ASC-1 subunit is a novel regulator of cell cycle, proliferation and growth in muscle and non-muscular cells. In this review, we summarize and discuss the available data on the clinical and histopathological phenotypes associated with inherited defects of the ASC-1 complex proteins, the known genotype-phenotype correlations, the ASC-1 pathophysiological role, the puzzling question of motoneuron versus primary muscle involvement and potential future research avenues, illustrating the study of rare monogenic disorders as an interesting model paradigm to understand major physiological processes.


Assuntos
Proteínas de Transporte/genética , Anormalidades Congênitas/genética , Doenças Neuromusculares/genética , Fatores de Transcrição/genética , Anormalidades Congênitas/patologia , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Complexos Multiproteicos/genética , Mutação , Doenças Neuromusculares/patologia
16.
Nat Commun ; 12(1): 4050, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193871

RESUMO

The investigation of genetic forms of juvenile neurodegeneration could shed light on the causative mechanisms of neuronal loss. Schinzel-Giedion syndrome (SGS) is a fatal developmental syndrome caused by mutations in the SETBP1 gene, inducing the accumulation of its protein product. SGS features multi-organ involvement with severe intellectual and physical deficits due, at least in part, to early neurodegeneration. Here we introduce a human SGS model that displays disease-relevant phenotypes. We show that SGS neural progenitors exhibit aberrant proliferation, deregulation of oncogenes and suppressors, unresolved DNA damage, and resistance to apoptosis. Mechanistically, we demonstrate that high SETBP1 levels inhibit P53 function through the stabilization of SET, which in turn hinders P53 acetylation. We find that the inheritance of unresolved DNA damage in SGS neurons triggers the neurodegenerative process that can be alleviated either by PARP-1 inhibition or by NAD + supplementation. These results implicate that neuronal death in SGS originates from developmental alterations mainly in safeguarding cell identity and homeostasis.


Assuntos
Anormalidades Múltiplas/patologia , Proteínas de Transporte/metabolismo , Anormalidades Craniofaciais/patologia , Dano ao DNA , Deformidades Congênitas da Mão/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Deficiência Intelectual/patologia , Mutação , Unhas Malformadas/patologia , Células-Tronco Neurais/patologia , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Deformidades Congênitas da Mão/genética , Deformidades Congênitas da Mão/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Unhas Malformadas/genética , Unhas Malformadas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Organoides
17.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208277

RESUMO

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteoma/análise , Proteoma/metabolismo , RNA Mensageiro/genética
18.
Nat Commun ; 12(1): 4380, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34282138

RESUMO

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.


Assuntos
Proteínas de Transporte/metabolismo , Células Germinativas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis , Proteínas de Transporte/química , Proteínas de Transporte/genética , Adesão Celular , Fusão Celular , Chlamydomonas/metabolismo , Fertilização/fisiologia , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/metabolismo , Proteínas Recombinantes , Especificidade da Espécie
19.
Eur J Med Res ; 26(1): 75, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256840

RESUMO

BACKGROUND: The aim of this study was to evaluate the expression of four up/down-regulated inflammatory miRNAs and their mRNA targets in the serum samples of COVID-19 patients with different grades. Also, we investigated the relative expression of these miRNAs and mRNAs during hospitalization. METHODS: In this cross-sectional study, 5 mL of blood sample were taken from COVID-19 patients with different grades and during hospitalization from several health centers of Yazd, Tehran, and Zahedan province of Iran from December 20, 2020 to March 2, 2021. The relative expression of miRNAs and mRNAs was evaluated by q-PCR. RESULTS: We found that the relative expression of hsa-miR-31-3p, hsa-miR-29a-3p, and hsa-miR-126-3p was significantly decreased and the relative expression of their mRNA targets (ZMYM5, COL5A3, and CAMSAP1) was significantly increased with the increase of disease grade. Conversely, the relative expression of hsa-miR-17-3p was significantly increased and its mRNA target (DICER1) was significantly decreased with the increase of disease grade. This pattern was exactly seen during hospitalization of COVID-19 patients who did not respond to treatment. In COVID-19 patients who responded to treatment, the expression of selected miRNAs and their mRNA targets returned to the normal level. A negative significant correlation was seen between (1) the expression of hsa-miR-31-3p and ZMYM5, (2) hsa-miR-29a-3p and COL5A3, (3) hsa-miR-126-3p and CAMSAP1, and (4) hsa-miR-17-3p and DICER1 in COVID-19 patients with any grade (P < 0.05) and during hospitalization. CONCLUSIONS: In this study, we gained a more accurate understanding of the expression of up/down-regulated inflammatory miRNAs in the blood of COVID-19 patients. The obtained data may help us in the diagnosis and prognosis of COVID-19. TRIAL REGISTRATION: The ethics committee of Zahedan University of Medical Sciences, Zahedan, Iran. (Ethical Code: IR.ZAUMS.REC.1399.316) was registered for this project.


Assuntos
COVID-19/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , COVID-19/sangue , COVID-19/virologia , Proteínas de Transporte/genética , Colágeno/genética , Estudos Transversais , RNA Helicases DEAD-box/genética , Hospitalização/estatística & dados numéricos , Humanos , Irã (Geográfico) , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Ribonuclease III/genética , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença
20.
Am J Physiol Heart Circ Physiol ; 321(2): H259-H274, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34085839

RESUMO

Underlying molecular mechanisms for the development of diabetic cardiomyopathy remain to be determined. Long-term exposure to hyperglycemia causes oxidative stress, which leads to cardiomyocyte dysfunction. Previous studies established the importance of thioredoxin-interacting protein (Txnip) in cellular redox homeostasis and glucose metabolism. Txnip is a highly glucose-responsive molecule that interacts with the catalytic center of reduced thioredoxin and inhibits the antioxidant function of thioredoxin. Here, we show that the molecular interaction between Txnip and thioredoxin plays a pivotal role in the regulation of redox balance in the diabetic myocardium. High glucose increased Txnip expression, decreased thioredoxin activities, and caused oxidative stress in cells. The Txnip-thioredoxin complex was detected in cells with overexpressing wild-type Txnip but not Txnip cysteine 247 to serine (C247S) mutant that disrupts the intermolecular disulfide bridge. Then, diabetes was induced in cardiomyocyte-specific Txnip C247S knock-in mice and their littermate control animals by injections of streptozotocin (STZ). Prolonged hyperglycemia upregulated myocardial Txnip expression in both genotypes. The absence of Txnip's inhibition of thioredoxin in Txnip C247S mutant hearts promoted mitochondrial antioxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage by diabetes. Stress hemodynamic analysis uncovered that Txnip C247S knock-in hearts have a greater left ventricular contractile reserve than wild-type hearts under STZ-induced diabetic conditions. These results provide novel evidence that Txnip serves as a regulator of hyperglycemia-induced cardiomyocyte toxicities through direct inhibition of thioredoxin and identify the single cysteine residue in Txnip as a therapeutic target for diabetic injuries.NEW & NORTEWORTHY Thioredoxin-interacting protein (Txnip) has been of great interest as a molecular mechanism to mediate diabetic organ damage. Here, we provide novel evidence that a single mutation of Txnip confers a defense mechanism against myocardial oxidative stress in streptozotocin-induced diabetic mice. The results demonstrate the importance of Txnip as a cysteine-containing redox protein that regulates antioxidant thioredoxin via disulfide bond-switching mechanism and identify the cysteine in Txnip as a therapeutic target for diabetic cardiomyopathy.


Assuntos
Proteínas de Transporte/genética , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/genética , Tiorredoxinas/metabolismo , Função Ventricular Esquerda/genética , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Técnicas de Introdução de Genes , Glucose/farmacologia , Células HEK293 , Humanos , Preparação de Coração Isolado , Camundongos , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Tiorredoxinas/genética
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