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1.
Adv Exp Med Biol ; 1185: 395-400, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884644

RESUMO

Mattapallil et al. described that vendor lines for C57BL/6 N mice may carry the rd8 mutation that leads to an ocular phenotype, which could be mistaken for an induced retinal degeneration. This mouse strain is widely used in ophthalmic research as a background for modeling retinal degeneration. In the process of studying Cln3Δex7/8 knock-in mice on a C57BL/6 N background, we became aware of this issue. The aim of this study thus was to use electroretinography to investigate the age-dependent functional loss in Cln3+/+ rd8-/rd8- mice and compare it to C57BL/6 J mice.The scotopic and photopic amplitudes of the a-wave and b-wave decrease significantly in mutant mice with increasing age, and the implicit time is prolonged. Especially the oscillatory potentials arising from inner retinal interaction seem to be notably affected by the rd8 mutation. Surprisingly, the amplitudes in young C57BL/6 J mice were lower than those measured in C57BL/6 N at any time point.Our results indicate that the rd8 mutation present in C57BL/6 N mice affects the function of the inner and outer retina. This is surprising given that the major retinal morphological alterations due to the rd8 mutation are found in the outer retina.We conclude that the rd8 mutation does affect the retinal function in Cln3+/+ rd8-/rd8- mice in a variable manner. Epigenetic factors and modifying genes lead to a phenotype shift in these mice. Interpreting the results of previous studies in mutant mice on C57BL/6 N background is challenging as comparing results obtained in independent studies or on other mouse backgrounds may be misleading. Using littermates as controls remains the only valid option.


Assuntos
Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Degeneração Retiniana/genética , Envelhecimento , Animais , Modelos Animais de Doenças , Eletrorretinografia , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Retina/fisiopatologia
2.
Nat Neurosci ; 22(12): 1966-1974, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768050

RESUMO

To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , Exoma/genética , Predisposição Genética para Doença/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Estudos de Casos e Controles , Feminino , Variação Genética/genética , Humanos , Masculino
3.
Invest Ophthalmol Vis Sci ; 60(14): 4820-4829, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747682

RESUMO

Purpose: This study was aimed at investigating the association of long noncoding RNA (lncRNA)-related single nucleotide polymorphisms (SNPs) with Vogt-Koyanagi-Harada (VKH) disease. Methods: LncRNA-related SNPs were selected by multi-omics analysis. Genotyping, expression of lncRNA and mRNA, cell proliferation, and cytokine production were tested by MassARRAY System, real-time PCR, CCK8, and ELISA. Results: A significant association with VKH was found for lnc-TOR3A-1:1/rs3829794, which is located in a non-HLA region (CC genotype: Bonferroni corrected P values [PC] = 2.98 × 10-8, odds ratio [OR] = 0.62; TT genotype: PC = 1.64 × 10-8, OR = 1.57; C allele: PC = 1.39 × 10-12, OR = 0.71). Additionally, an association was found for four lncRNA SNPs located in the HLA region. Functional experiments in rs3829794 genotyped individuals showed decreased ABL2 (ABL proto-oncogene 2, nonreceptor tyrosine kinase) expression, decreased proliferation of anti-CD3 plus anti-CD28-stimulated peripheral blood mononuclear cells (PBMCs), and an increased production of IL-10 in CC carriers compared to TT carriers (P = 0.0073, P = 0.0011, and P = 0.002, respectively). Conclusions: Our study identified five new loci associated with VKH susceptibility and identified a functional variant (lnc-TOR3A-1:1/rs3829794) that confers risk for VKH, which is possibly mediated by modulating gene expression, proliferation of lymphocytes, and regulation of anti-inflammatory cytokine production.


Assuntos
Adenosina Trifosfatases/genética , Predisposição Genética para Doença/genética , Antígenos de Histocompatibilidade Menor/genética , Chaperonas Moleculares/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética , Síndrome Uveomeningoencefálica/genética , Adulto , Estudos de Casos e Controles , Proliferação de Células/fisiologia , Células Cultivadas , Citocinas/metabolismo , Replicação do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome Uveomeningoencefálica/metabolismo , Síndrome Uveomeningoencefálica/patologia
4.
Georgian Med News ; (294): 41-45, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31687947

RESUMO

The aim of this study was to identify susceptibility variants of CACNA1A, POMP, TMEM136, AGPAT1, RBMS3, and SEMA6A genes for Exfoliation Syndrome (XFS) and Exfoliation Glaucoma (XFG) by a case-control association study approach among Georgian population. Self-reported Georgian subjects were recruited between 2015 and 2017 at a specialized ophthalmic center. Patients underwent detailed ophthalmic examination to diagnose or exclude Exfoliation Syndrome and Exfoliation Glaucoma. Patients underwent peripheral blood sampling. Genome-Wide Association Study (GWAS) was performed using Illumina OmniExpress Microarray (USA). One hundred and thirty-two XFS patients (including XFG-affected individuals) and 199 healthy subjects were included into the study. Six genes CACNA1A rs4926244, POMP rs7329408, TMEM136 rs11827818, AGPAT1 rs3130283, RBMS3 rs12490863 and SEMA6A rs10072088 variants were identified. The A alleles of SEMA6A and POMP genes are likely the risk factors of disease development in Georgians with p=0.001; OR= 1.8, 95% CI 1.2676 to 2.6973 and p=0.001; OR=1.6, 95% CI 0.9931 to 2.5634, respectively. SEMA6A homozygotes have 4 times greater risk compared to normal individuals, with p<0.004; OR=4.0, 95% CI 1.1531 to 13.9903. The G allele of CACNA1A in homozygous state increases the risk up to 3-fold with p<0.05, OR=3.15, 95% CI 0.9275 to 10.6658. The A alleles of SEMA6A and POMP increased XFG susceptibility more than 3 times (p=0.04; OR= 3.4; 95% CI: 1.2676 to 2.6973 and p=0.02; OR= 2.7; 95% CI: 0.9931 to 2.5634, respectively). Three high-risk genes have been identified in connection to XFS in Georgian population. Two genes are relevant to XFG. Three other previously described genes are not associated with the disease development.


Assuntos
Síndrome de Exfoliação/genética , Marcadores Genéticos , Predisposição Genética para Doença , Chaperonas Moleculares/genética , Semaforinas/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Síndrome de Exfoliação/patologia , Matriz Extracelular/patologia , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo Genético
5.
Life Sci ; 239: 117035, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31697952

RESUMO

AIMS: The purpose of this study was to investigate the role of long non-coding RNA taurine-upregulated gene 1 (TUG1) in colon cancer (Cc) and related molecular mechanisms. MATERIALS AND METHODS: RT-qPCR, Western blot and immunohistochemistry were used to detect the expression of related proteins. BrdU and Transwell assays were used to detect cell proliferation and invasion, respectively. Immunofluorescence was used to detect the expression of Vimentin. KEY FINDINGS: TUG1 expression was up-regulated in CaCO-2, SW620 and HT-29 cells, while miR-26a-5p was down-regulated. Bioinformatics analysis showed that miR-26a-5p was the target of TUG1, and the targeting relationship was further confirmed by dual-luciferase report analysis. Besides, matrix metalloproteinases-14 (MMP-14) was a target of mir-26a-5p. Knockdown of TUG1 by shRNA (sh-TUG1) inhibited MMP-14 expression. Functional analysis showed that sh-TUG1 significantly inhibited Cc cell proliferation, invasion and epithelial-mesenchymal transformation (EMT). Notably, miR-26a-5p inhibitor reversed the promotion of Cc caused by sh-TUG1. Mechanically, the overexpression of TUG1 significantly up-regulated the levels of MMP-14, VEGF, p-p38 mitogen-activated protein kinase (p-p38 MAPK) and p-HSP27 (heat shock protein 27), and promoted the proliferation, invasion and EMT of Cc cells. However, MAPK pathway inhibitor SB203580 has shown the opposite effect. Additionally, animal studies have shown that sh-TUG1 inhibited tumor growth and motility in vivo in the same way. SIGNIFICANCE: This study demonstrated that TUG1 accelerates the development of colon cancer by regulating miR-26a-5p/MMP14/p38 MAPK/Hsp27 axis in vitro and in vivo. Therefore, TUG1 provides a new direction for the treatment of Cc.


Assuntos
Carcinogênese/genética , Neoplasias do Colo/genética , Proteínas de Choque Térmico/biossíntese , Sistema de Sinalização das MAP Quinases/genética , Metaloproteinase 14 da Matriz/biossíntese , MicroRNAs/biossíntese , Chaperonas Moleculares/biossíntese , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Biologia Computacional , Transição Epitelial-Mesenquimal/genética , Proteínas de Choque Térmico/genética , Humanos , Masculino , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Chaperonas Moleculares/genética , Regulação para Cima
6.
BMC Evol Biol ; 19(1): 205, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694524

RESUMO

BACKGROUND: Antarctic fishes of the Notothenioidei suborder constitutively upregulate multiple inducible chaperones, a highly derived adaptation that preserves proteostasis in extreme cold, and represent a system for studying the evolution of gene frontloading. We screened for Hsf1-binding sites, as Hsf1 is a master transcription factor of the heat shock response, and highly-conserved non-coding elements within proximal promoters of chaperone genes across 10 Antarctic notothens, 2 subpolar notothens, and 17 perciform fishes. We employed phylogenetic models of molecular evolution to determine whether (i) changes in motifs associated with Hsf1-binding and/or (ii) relaxed purifying selection or exaptation at ancestral cis-regulatory elements coincided with the evolution of chaperone frontloading in Antarctic notothens. RESULTS: Antarctic notothens exhibited significantly fewer Hsf1-binding sites per bp at chaperone promoters than subpolar notothens and Serranoidei, the most closely-related suborder to Notothenioidei included in this study. 90% of chaperone promoters exhibited accelerated substitution rates among Antarctic notothens relative to other perciformes. The proportion of bases undergoing accelerated evolution (i) was significantly greater in Antarctic notothens than in subpolar notothens and Perciformes in 70% of chaperone genes and (ii) increased among bases that were more conserved among perciformes. Lastly, we detected evidence of relaxed purifying selection and exaptation acting on ancestrally conserved cis-regulatory elements in the Antarctic notothen lineage and its major branches. CONCLUSION: A large degree of turnover has occurred in Notothenioidei at chaperone promoter regions that are conserved among perciform fishes following adaptation to the cooling of the Southern Ocean. Additionally, derived reductions in Hsf1-binding site frequency suggest cis-regulatory modifications to the classical heat shock response. Of note, turnover events within chaperone promoters were less frequent in the ancestral node of Antarctic notothens relative to younger Antarctic lineages. This suggests that cis-regulatory divergence at chaperone promoters may be greater between Antarctic notothen lineages than between subpolar and Antarctic clades. These findings demonstrate that strong selective forces have acted upon cis-regulatory elements of chaperone genes among Antarctic notothens.


Assuntos
Evolução Molecular , Proteínas de Peixes/genética , Chaperonas Moleculares/genética , Perciformes/genética , Perciformes/fisiologia , Regiões Promotoras Genéticas , Adaptação Fisiológica , Animais , Regiões Antárticas , Regulação da Expressão Gênica , Resposta ao Choque Térmico , Filogenia
7.
Nat Commun ; 10(1): 4781, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636255

RESUMO

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Cristalização , Técnicas In Vitro , Insetos , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de Proteína
8.
PLoS Pathog ; 15(9): e1008065, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557263

RESUMO

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.


Assuntos
Proteínas de Protozoários/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidade
9.
Nucleic Acids Res ; 47(19): 10212-10234, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31538203

RESUMO

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.


Assuntos
Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mapas de Interação de Proteínas/genética , Proteína Supressora de Tumor p53/genética , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Transdução de Sinais/genética
10.
J Biochem ; 166(6): 529-535, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504613

RESUMO

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.


Assuntos
Bacteriófago mu/química , Chaperonas Moleculares/química , Sequência de Aminoácidos , Bacteriófago mu/genética , Bacteriófago mu/isolamento & purificação , Sítios de Ligação , Cristalização , Lipopolissacarídeos/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Alinhamento de Sequência
11.
Nucleic Acids Res ; 47(18): 9888-9901, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504775

RESUMO

tRNA-derived RNA fragments (tRFs) have emerged as a new class of functional RNAs implicated in cancer, metabolic and neurological disorders, and viral infection. Yet our understanding of their biogenesis and functions remains limited. In the present study, through analysis of small RNA profile we have identified a distinct set of tRFs derived from pre-tRNA 3' trailers in the hepatocellular carcinoma cell line Huh7. Among those tRFs, tRF_U3_1, which is a 19-nucleotide-long chr10.tRNA2-Ser(TGA)-derived trailer, was expressed most abundantly in both Huh7 and cancerous liver tissues, being present primarily in the cytoplasm. We show that genetic loss of tRF_U3_1 does not affect cell growth and it is not involved in Ago2-mediated gene silencing. Using La/SSB knockout Huh7 cell lines, we demonstrate that this nuclear-cytoplasmic shuttling protein directly binds to the 3' U-tail of tRF_U3_1 and other abundantly expressed trailers and plays a critical role in their stable cytoplasmic accumulation. The pre-tRNA trailer-derived tRFs capable of sequestering the limiting amounts of La/SSB in the cytoplasm rendered cells resistant to various RNA viruses, which usurp La/SSB with RNA chaperone activity for their gene expression. Collectively, our results establish the trailer-derived tRF-La/SSB interface, regulating viral gene expression.


Assuntos
Proliferação de Células/genética , Citoplasma/genética , Precursores de RNA/genética , RNA de Transferência/genética , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/genética , Humanos , Chaperonas Moleculares/genética
12.
Nat Commun ; 10(1): 4046, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492860

RESUMO

Nucleosomes containing the histone H3 variant CENP-A are the epigenetic mark of centromeres, the kinetochore assembly sites required for chromosome segregation. HJURP is the CENP-A chaperone, which associates with Mis18α, Mis18ß, and M18BP1 to target centromeres and deposit new CENP-A. How these proteins interact to promote CENP-A deposition remains poorly understood. Here we show that two repeats in human HJURP proposed to be functionally distinct are in fact interchangeable and bind concomitantly to the 4:2:2 Mis18α:Mis18ß:M18BP1 complex without dissociating it. HJURP binds CENP-A:H4 dimers, and therefore assembly of CENP-A:H4 tetramers must be performed by two Mis18αß:M18BP1:HJURP complexes, or by the same complex in consecutive rounds. The Mis18α N-terminal tails blockade two identical HJURP-repeat binding sites near the Mis18αß C-terminal helices. These were identified by photo-cross-linking experiments and mutated to separate Mis18 from HJURP centromere recruitment. Our results identify molecular underpinnings of eukaryotic chromosome inheritance and shed light on how centromeres license CENP-A deposition.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Histonas/química , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos
13.
PLoS Genet ; 15(8): e1008328, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404065

RESUMO

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5' untranslated regions (5'UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5'UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5'UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.


Assuntos
Proteínas Arqueais/metabolismo , Mathanococcus/fisiologia , Chaperonas Moleculares/metabolismo , RNA Arqueal/metabolismo , Regiões 5' não Traduzidas/genética , Proteínas Arqueais/genética , Genoma Arqueal/genética , Chaperonas Moleculares/genética , Transcrição Genética , Transcriptoma/genética
14.
Mol Carcinog ; 58(11): 2052-2064, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31397499

RESUMO

Recent studies have indicated that using statins to inhibit the mevalonate pathway induces mutant p53 degradation by impairing the interaction of mutant p53 with DnaJ subfamily A member 1 (DNAJA1). However, the role of the C-terminus of DNAJA1 with a CAAX box for farnesylation in the binding, folding, and translocation of client proteins such as mutant p53 is not known. In the present study, we used a genetically engineered mouse model of pancreatic carcinoma and showed that atorvastatin significantly increased animal survival and inhibited pancreatic carcinogenesis. There was a dramatic decrease in mutant p53 protein accumulation in the pancreatic acini, pancreas intraepithelial neoplasia lesions, and adenocarcinoma. Supplementation with farnesyl pyrophosphate, a substrate for protein farnesylation, rescued atorvastatin-induced mutant p53 degradation in pancreatic cancer cells. Tipifarnib, a farnesyltransferase inhibitor, mirrored atorvastatin's effects on mutant p53, degraded mutant p53 in a dose-dependent manner, and converted farnesylated DNAJA1 into unfarnesylated DNAJA1. Farnesyltransferase gene knockdown also significantly promoted mutant p53 degradation. Coimmunoprecipitation either by an anti-DNAJA1 or p53 antibody confirmed the direct interaction of mutant p53 and DNAJA1 and higher doses of atorvastatin treatments converted more farnesylated DNAJA1 into unfarnesylated DNAJA1 with much less mutant p53 pulled down by DNAJA1. Strikingly, C394S mutant DNAJA1, in which the cysteine of the CAAX box was mutated to serine, was no longer able to be farnesylated and lost the ability to maintain mutant p53 stabilization. Our results show that farnesylated DNAJA1 is a crucial chaperone in maintaining mutant p53 stabilization and targeting farnesylated DNAJA1 by atorvastatin will be critical for inhibiting p53 mutant cancer.


Assuntos
Atorvastatina/farmacologia , Proteínas de Choque Térmico HSP40/genética , Neoplasias Pancreáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Chaperonas Moleculares/genética , Proteínas Mutantes/genética , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prenilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinolonas/farmacologia
15.
EMBO J ; 38(19): e96659, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454099

RESUMO

Loss of the histone H3.3-specific chaperone component ATRX or its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres (ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRX or DAXX depletion gradually induces telomere DNA replication dysfunction that activates not only homology-directed DNA repair responses but also cell cycle checkpoint control. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomere length. Combined ATAC-seq and telomere chromatin immunoprecipitation studies reveal that ATRX loss provokes progressive telomere decondensation that culminates in the inception of persistent telomere replication dysfunction. We further show that endogenous telomerase activity cannot overcome telomere dysfunction induced by ATRX loss, leaving telomere repair-based ALT as the only viable mechanism for telomere maintenance during immortalization. Together, these findings implicate ALT activation as an adaptive response to ATRX/DAXX loss-induced telomere replication dysfunction.


Assuntos
Proteínas Correpressoras/genética , Chaperonas Moleculares/genética , Homeostase do Telômero , Telômero/metabolismo , Proteína Nuclear Ligada ao X/genética , Linhagem Celular , Reparo do DNA , Deleção de Genes , Células HEK293 , Humanos , Telomerase/metabolismo
16.
Nat Commun ; 10(1): 3262, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332180

RESUMO

TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.


Assuntos
Adenosina Trifosfatases/química , Modelos Moleculares , Chaperonas Moleculares/química , Polímeros/química , Conformação Proteica , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Polimerização , Polímeros/metabolismo
17.
Nat Protoc ; 14(8): 2279-2317, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332354

RESUMO

A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor's nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8-10 working days, and initial data analysis can be completed within 1-2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.


Assuntos
Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos , RNA Mensageiro , Ribossomos , Saccharomyces cerevisiae , Biblioteca Gênica , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
18.
Mol Med Rep ; 20(3): 2410-2418, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322176

RESUMO

The aim of the present study was to investigate the effect of bortezomib on heat shock protein 27 (HSP27) in multiple myeloma (MM) and provide a potential new target for clinical treatment. Peripheral blood was collected from 50 normal subjects and 50 patients with newly diagnosed MM and the expression of HSP27 was detected by ELISA. The changes of HSP27 after conventional vincristine, doxorubicin and dexamethasone (VAD) chemotherapy, and bortezomib plus VAD were compared. The effect of bortezomib on U266 cell proliferation and apoptosis was detected using a Cell Counting Kit­8 assay and Annexin V­FITC/propidium iodide double staining with flow cytometry. The content of HSP27 following bortezomib treatment was determined by ELISA. Western blot analysis and reverse transcription­quantitative PCR were used to detect the mRNA and protein expression of HSP27, Bax and Bcl­2. HSP27 expression was increased in patients with MM compared with healthy control subjects, and the expression was increased as the cancer progressed (P<0.05). Compared with the VAD chemotherapy group, the bortezomib plus VAD chemotherapy regimen significantly inhibited the expression of HSP27 (P<0.05), and the content of HSP27 was decreased in patients in which treatment was effective compared to those patients that exhibited disease progression (P<0.05). The efficacy of the treatment regimes was not associated with age or gender. Compared with the control group, bortezomib or OGX­427 (HSP27 inhibitor) treatment inhibited U266 cell proliferation, promoted U266 cell apoptosis (P<0.05) and significantly decreased HSP27 expression (P<0.05). Furthermore, the expression of HSP27 and Bcl­2 was significantly decreased, while the expression of Bax was increased by bortezomib and OGX­427 (P<0.05). There was no significant difference between the bortezomib and OGX­427 group in the in vitro analysis. HSP27 was positively correlated with Bcl­2 expression and negatively correlated with Bax expression in U266 cells. In conclusion, bortezomib promotes the apoptosis of MM cells, potentially by downregulating the expression of HSP27, providing a potential novel target for the clinical treatment of multiple myeloma.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib/uso terapêutico , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico/análise , Humanos , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/análise , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia
19.
Nat Commun ; 10(1): 3084, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300652

RESUMO

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.


Assuntos
Proteínas do Domínio Armadillo/ultraestrutura , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Bovinos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X
20.
J Agric Food Chem ; 67(30): 8393-8401, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31291721

RESUMO

The ginsenoside 20-O-ß-glucopyranosyl-20(S)-protopanaxadiol or compound K is an essential ingredient in functional food, cosmetics, and traditional medicines. However, no study has reported the complete conversion of all protopanaxadiol (PPD)-type ginsenosides from ginseng extract into compound K using whole-cell conversion. To increase the production of compound K from ginseng extract using whole recombinant cells, the ß-glucosidase enzyme from Caldicellulosiruptor bescii was coexpressed with a chaperone expression system (pGro7), and the cells expressing the coexpression system were permeabilized with ethylenediaminetetraacetic acid. The permeabilized cells carrying the chaperone coexpression system showed a 2.6-fold increase in productivity and yield as compared with nontreated cells, and completely converted all PPD-type ginsenosides from ginseng root extract into compound K with the highest productivity among the results reported so far. Our results will contribute to the industrial biological production of compound K.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Firmicutes/enzimologia , Ginsenosídeos/metabolismo , Chaperonas Moleculares/genética , Sapogeninas/metabolismo , beta-Glucosidase/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Escherichia coli/química , Firmicutes/genética , Engenharia Genética , Ginsenosídeos/química , Chaperonas Moleculares/metabolismo , Panax/química , Sapogeninas/química , beta-Glucosidase/metabolismo
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