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1.
Genes Dev ; 33(13-14): 857-870, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31147388

RESUMO

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/fisiologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , RNA Nucleolar Pequeno/biossíntese , Animais , RNA Nuclear Pequeno/metabolismo
2.
Genes Dev ; 33(15-16): 1031-1047, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31196865

RESUMO

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.


Assuntos
Aneuploidia , Citosol/metabolismo , Compensação de Dosagem (Genética)/fisiologia , Agregados Proteicos/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Agregação Patológica de Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
BMC Bioinformatics ; 20(1): 300, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159772

RESUMO

BACKGROUND: Although a considerable number of proteins operate as multiprotein complexes and not on their own, organism-wide studies so far are only able to quantify individual proteins or protein-coding genes in a condition-specific manner for a sizeable number of samples, but not their assemblies. Consequently, there exist large amounts of transcriptomic data and an increasing amount of data on proteome abundance, but quantitative knowledge on complexomes is missing. This deficiency impedes the applicability of the powerful tool of differential analysis in the realm of macromolecular complexes. Here, we present a pipeline for differential analysis of protein complexes based on predicted or manually assigned complexes and inferred complex abundances, which can be easily applied on a whole-genome scale. RESULTS: We observed for simulated data that results obtained by our complex abundance estimation algorithm were in better agreement with the ground truth and physicochemically more reasonable compared to previous efforts that used linear programming while running in a fraction of the time. The practical usability of the method was assessed in the context of transcription factor complexes in human monocyte and lymphoblastoid samples. We demonstrated that our new method is robust against false-positive detection and reports deregulated complexomes that can only be partially explained by differential analysis of individual protein-coding genes. Furthermore we showed that deregulated complexes identified by the tool potentially harbor significant yet unused information content. CONCLUSIONS: CompleXChange allows to analyze deregulation of the protein complexome on a whole-genome scale by integrating a plethora of input data that is already available. A platform-independent Java binary, a user guide with example data and the source code are freely available at https://sourceforge.net/projects/complexchange/ .


Assuntos
Complexos Multiproteicos/metabolismo , Software , Benchmarking , Bases de Dados de Proteínas , Feminino , Humanos , Programação Linear , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
4.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035650

RESUMO

Citrate is a key intermediate of the tricarboxylic acid cycle and acts as an allosteric signal to regulate the production of cellular ATP. An elevated cytosolic citrate concentration inhibits growth in several types of human cancer cells; however, the underlying mechanism by which citrate induces the growth arrest of cancer cells remains unclear. The results of this study showed that treatment of human pharyngeal squamous carcinoma (PSC) cells with a growth-suppressive concentration of citrate caused cell cycle arrest at the G2/M phase. A coimmunoprecipitation study demonstrated that citrate-induced cell cycle arrest in the G2/M phase was associated with stabilizing the formation of cyclin B1-phospho (p)-cyclin-dependent kinase 1 (CDK1) (Thr 161) complexes. The citrate-induced increased levels of cyclin B1 and G2/M phase arrest were suppressed by the caspase-3 inhibitor Ac-DEVD-CMK and caspase-3 cleavage of mutant p21 (D112N). Ectopic expression of the constitutively active form of protein kinase B (Akt1) could overcome the induction of p21 cleavage, cyclin B1-p-CDK1 (Thr 161) complexes, and G2/M phase arrest by citrate. p85α-phosphatase and tensin homolog deleted from chromosome 10 (PTEN) complex-mediated inactivation of Akt was required for citrate-induced G2/M phase cell cycle arrest because PTEN short hairpin RNA or a PTEN inhibitor (SF1670) blocked the suppression of Akt Ser 473 phosphorylation and the induction of cyclin B1-p-CDK1 (Thr 161) complexes and G2/M phase arrest by citrate. In conclusion, citrate induces G2/M phase arrest in PSC cells by inducing the formation of p85α-PTEN complexes to attenuate Akt-mediated signaling, thereby causing the formation of cyclin B1-p-CDK1 (Thr 161) complexes.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Ácido Cítrico/farmacologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Faríngeas/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Humanos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Neoplasias Faríngeas/genética , Fosforilação , Transdução de Sinais
5.
Acta Crystallogr D Struct Biol ; 75(Pt 5): 467-474, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063149

RESUMO

Coat proteins mediate vesicular transport between intracellular compartments, which is essential for the distribution of molecules within the eukaryotic cell. The global arrangement of coat proteins on the membrane is key to their function, and cryo-electron tomography and subtomogram averaging have been used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer performed on in vitro reconstitutions or in situ is summarized. These studies have answered long-standing questions regarding the mechanisms of membrane binding, polymerization and assembly regulation of coat proteins.


Assuntos
Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte Biológico , Membrana Celular/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Proteínas de Transporte Vesicular/ultraestrutura
6.
Nat Immunol ; 20(6): 677-686, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110312

RESUMO

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.


Assuntos
Células Secretoras de Insulina/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Animais , Proliferação de Células , Ciclina D2/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Humanos , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Obesidade/tratamento farmacológico , Parabiose , Ligação Proteica , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
7.
Essays Biochem ; 63(1): 133-145, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30967477

RESUMO

The organization and regulation of genomic DNA as nuclear chromatin is necessary for proper DNA function inside living eukaryotic cells. While this has been extensively explored, no true consensus is currently reached regarding the exact mechanism of chromatin organization. The traditional view has assumed that the DNA is packaged into a hierarchy of structures inside the nucleus based on the regular 30-nm chromatin fiber. This is currently being challenged by the fluid-like model of the chromatin which views the chromatin as a dynamic structure based on the irregular 10-nm fiber. In this review, we focus on the recent progress in chromatin structure elucidation highlighting the paradigm shift in chromatin folding mechanism from the classical textbook perspective of the regularly folded chromatin to the more dynamic fluid-like perspective.


Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína
8.
Life Sci ; 229: 13-20, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30953643

RESUMO

AIM: HIV-1 Nef downregulates surface MHC-I to protect the infected cells from CTLs-mediated killing. Although MHC-I downregulation has been extensively studied, the Nef-dependent assembly of the multi-protein complex and subsequent pathways activation has not yet been well explored. The present study is aimed for the identification of Nef-mediated sequential recruitment of cellular proteins that constitute the functional multi-protein complex, required for the downregulation of MHC-I. MAIN METHODS: Different Cellular protein complexes were identified by co-immunoprecipitation in Nef or NefE4A mutant-expressing Jurkat T, and THP-1 cells followed by exposure to Nef-specific peptides 24 h post infection. The MHC-I downregulation was analyzed by confocal microscopy and flow cytometry. KEY FINDINGS: We found the association of Nef with PACS-2, GCC185, PI3K, AP-1, SFK, and MHC-I proteins that probably constitute a functional multi-protein complex. Furthermore, the immunoprecipitations with PACS-2 and GCC185 in the presence or absence of Nef, Nef E4A mutant and Nef with CP-inhibitor divide the functional complex of Nef into Nef-dependent (AP-1 and PI3K) and GCC185-dependent complex (MHC-I and SFK). The molecular mechanisms for activation of cellular pathways have been deciphered on the basis of these interactions that are brought in close proximity through Nef-GCC185 interaction. Knockdown of GCC185 using siRNA in Jurkat T cells showed a direct relationship between the assembly of functional multi-protein complex and MHC-I accumulation at GCC185. SIGNIFICANCE: Overall, our study elucidates that GCC185 is a focal point for the assembly of the Nef-mediated multi-protein complex at TGN.


Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Proteínas da Matriz do Complexo de Golgi/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Complexos Multiproteicos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Rede trans-Golgi/metabolismo , Endocitose , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Jurkat , Transporte Proteico
9.
MBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914516

RESUMO

Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG's function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG's function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention.IMPORTANCE Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention.


Assuntos
Distroglicanas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vírus Lassa/fisiologia , Complexos Multiproteicos/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Linhagem Celular , Humanos
10.
Proc Natl Acad Sci U S A ; 116(14): 7043-7052, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894498

RESUMO

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.


Assuntos
Citomegalovirus/fisiologia , Células Epiteliais/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Tropismo Viral/fisiologia , Células A549 , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Virais/genética
11.
Nature ; 568(7750): 127-130, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30867591

RESUMO

The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner1-3. LAT1, an antiporter of the amino acid-polyamine-organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as L-3,4-dihydroxyphenylalanine across membranes2-6. Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs7-11. LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)-a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane8,9. Despite extensive cell-based characterization of the LAT1-4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown12-19. Here we report the cryo-electron microscopy structures of human LAT1-4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1-4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease.


Assuntos
Microscopia Crioeletrônica , Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/ultraestrutura , Transportador 1 de Aminoácidos Neutros Grandes/química , Transportador 1 de Aminoácidos Neutros Grandes/ultraestrutura , Aminoácidos/metabolismo , Sítios de Ligação , Transporte Biológico , Dissulfetos/química , Dissulfetos/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/antagonistas & inibidores , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Modelos Moleculares , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica
12.
Mol Cell Biochem ; 457(1-2): 105-118, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30895499

RESUMO

Chlorogenic acid (CGA) exists as multiple isomers (e.g., 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in foods such as coffee beverages, fruits and vegetables. This study aimed to investigate relative activities of these six different CGA isomers to modify redox biology in inflamed Caco-2 cells that involved Nrf2 signaling. Caco-2 cells were pre-treated with individual CGA isomers to assess the relative effectiveness to mitigate oxidative stress. Isomer-specific capacity of different CGA isomers for direct free radical scavenging activity and potential endogenous control of oxidative stress were determined using chemical assays and cell-based experiments, respectively. Molecular dynamics simulations of the CGA and Keap1-Nrf2 complex were performed to predict CGA structure-specific interactions. Results demonstrated that dicaffeoylquinic acid (diCQA including 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) isomers had greater (p < 0.05) affinity to ameliorate oxidative stress through direct free radical scavenging activity. This observation corresponded to greater (p < 0.05) capacity to activate Nrf2 signaling compared to caffeoylquinic acid (CQA including 3-CQA, 4-CQA, and 5-CQA) isomers in inflamed differentiated Caco-2 cells. Simulations revealed that differences between the ability of CQA and diCQA to interact with the Keap1-Nrf2 complex may be due to differences in relative orientation within this complex. The observed CGA isomer-specific affinity for CQA to activate Nrf2 signaling was confirmed by nuclear translocation of Nrf2 induced by CGA and greater (p < 0.05) upregulation of genes related to Nrf2 expression.


Assuntos
Ácido Clorogênico , Proteína 1 Associada a ECH Semelhante a Kelch , Simulação de Dinâmica Molecular , Complexos Multiproteicos , Fator 2 Relacionado a NF-E2 , Transdução de Sinais/efeitos dos fármacos , Células CACO-2 , Ácido Clorogênico/química , Ácido Clorogênico/farmacologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo
13.
Int J Mol Sci ; 20(3)2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30744200

RESUMO

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are structurally and functionally distinct proteins containing specific domains and motifs that enable the proteins to bind certain nucleotide sequences, particularly those found in human telomeres. In human malignant cells (HMCs), hnRNP-A1-the most studied hnRNP-is an abundant multifunctional protein that interacts with telomeric DNA and affects telomerase function. In addition, it is believed that other hnRNPs in HMCs may also be involved in the maintenance of telomere length. Accordingly, these proteins are considered possible participants in the processes associated with HMC immortalization. In our review, we discuss the results of studies on different hnRNPs that may be crucial to solving molecular oncological problems and relevant to further investigations of these proteins in HMCs.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Família Multigênica , Complexos Multiproteicos/metabolismo , Ligação Proteica , Telomerase/metabolismo
14.
Genes Dev ; 33(5-6): 276-281, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30804226

RESUMO

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.


Assuntos
Adenosina Trifosfatases/metabolismo , Anáfase/fisiologia , DNA Topoisomerases Tipo II/metabolismo , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Tanquirases/metabolismo , Aneuploidia , Segregação de Cromossomos , Dano ao DNA/genética , DNA Ribossômico/genética , Humanos , Saccharomyces cerevisiae/genética
15.
Nat Cell Biol ; 21(3): 359-371, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804505

RESUMO

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.


Assuntos
Polaridade Celular , Transição Epitelial-Mesenquimal , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CACO-2 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Proteína Quinase C/metabolismo , Proteólise , Interferência de RNA , Fatores de Transcrição da Família Snail/genética , Transplante Heterólogo
16.
Methods Mol Biol ; 1929: 437-445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710289

RESUMO

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated, Ca2+-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2.


Assuntos
Anexina A2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas S100/metabolismo , Corpos de Weibel-Palade/metabolismo , Cálcio , Membrana Celular/metabolismo , Exocitose , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia de Interferência , Complexos Multiproteicos/metabolismo
17.
Methods Mol Biol ; 1949: 69-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790250

RESUMO

Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This lipid exchange occurs by non-vesicular trafficking pathways at MCSs between mitochondria and plastids. By a biochemical approach, a mitochondrial lipoprotein super-complex called MTL (Mitochondrial Transmembrane Lipoprotein complex) involved in mitochondria lipid trafficking has been identified in Arabidopsis thaliana. This protocol describes the method to isolate the MTL complex and to study the implication of a component of this complex (AtMic60) in mitochondria lipid trafficking.


Assuntos
Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Transporte Biológico , Cromatografia Líquida , Lipoproteínas/isolamento & purificação , Espectrometria de Massas , Proteínas de Membrana/isolamento & purificação , Solubilidade
18.
Dev Biol ; 446(2): 193-205, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30599151

RESUMO

Proper germ cell sex determination in Caenorhabditis nematodes requires a network of RNA-binding proteins (RBPs) and their target mRNAs. In some species, changes in this network enabled limited XX spermatogenesis, and thus self-fertility. In C. elegans, one of these selfing species, the global sex-determining gene tra-2 is regulated in germ cells by a conserved RBP, GLD-1, via the 3' untranslated region (3'UTR) of its transcript. A C. elegans-specific GLD-1 cofactor, FOG-2, is also required for hermaphrodite sperm fate, but how it modifies GLD-1 function is unknown. Germline feminization in gld-1 and fog-2 null mutants has been interpreted as due to cell-autonomous elevation of TRA-2 translation. Consistent with the proposed role of FOG-2 in translational control, the abundance of nearly all GLD-1 target mRNAs (including tra-2) is unchanged in fog-2 mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss of gld-1 function elevates germline TRA-2 expression to detectable levels, but loss of fog-2 function does not. A simple quantitative model of tra-2 activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly, fog-2 and gld-1 activity enable the sperm fate even when GLD-1 cannot bind to the tra-2 3' UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites within tra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as "hyper-repressors" of tra-2 activity. These variants bind RNA more weakly in vitro than does wild-type GLD-1. These results indicate that gld-1 and fog-2 regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in the C. elegans hermaphrodite is far from complete.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Organismos Hermafroditas/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Organismos Hermafroditas/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Genéticos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo
19.
J Exp Clin Cancer Res ; 38(1): 13, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626422

RESUMO

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks. METHODS: Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage. RESULTS: Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. CONCLUSIONS: IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Ácidos e Sais Biliares/metabolismo , Dano ao DNA , Proteína Quinase Ativada por DNA/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ácidos e Sais Biliares/farmacologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico
20.
Methods Mol Biol ; 1880: 341-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610709

RESUMO

Mass spectrometry (MS)-based identification and characterization of protein complexes is becoming a prerequisite for in-depth biochemical analyses of intracellular processes. Here, we describe two state-of-the-art MS-based approaches to characterize protein-protein interactions and multi-protein complexes involved in autophagy in mammalian cells. The combination of affinity purification (AP)-MS, which identifies binary protein-protein interactions, with size-exclusion chromatography (SEC)-protein correlation profiling (PCP), which helps monitor protein complex assemblies, is a powerful tool to acquire a full overview of the interlinkage and regulation of novel multi-protein complexes that might play a role in autophagy.


Assuntos
Autofagia/fisiologia , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Massas em Tandem/métodos , Autofagia/efeitos dos fármacos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Mapeamento de Interação de Proteínas/instrumentação , Multimerização Proteica/fisiologia , Espectrometria de Massas em Tandem/instrumentação
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