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1.
Life Sci Alliance ; 5(4)2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35022246

RESUMO

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme's turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

2.
Cell Death Dis ; 12(11): 1040, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725334

RESUMO

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

3.
Nucleic Acids Res ; 49(15): 8866-8885, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329466

RESUMO

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Espalhamento a Baixo Ângulo , Fatores de Transcrição/ultraestrutura , Difração de Raios X
4.
Cell Host Microbe ; 29(8): 1316-1332.e12, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34237247

RESUMO

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

5.
FASEB J ; 35(7): e21691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118085

RESUMO

Amyloid ß peptide (Aß) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of Aß. Deficiency of p38α-MAPK in neurons increases lysosomal degradation of BACE1 and decreases Aß deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38α-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38α-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38α-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38α-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38α-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38α-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38α-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/fisiologia , Ácido Aspártico Endopeptidases/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Presenilina-1/fisiologia , Terminações Pré-Sinápticas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Transporte Axonal , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas de Transporte Vesicular/genética
6.
Sci Adv ; 7(26)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34172453

RESUMO

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.

7.
Cell Rep ; 35(6): 109100, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33979607

RESUMO

RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins. Furthermore, we identify the cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo. We identify mRNAs bound to and regulated by Cpeb4 in cardiomyocytes. Cpeb4 regulates cardiac remodeling by differential expression of transcription factors. Among Cpeb4 target mRNAs, two zinc finger transcription factors (Zeb1 and Zbtb20) are discovered. We show that Cpeb4 regulates the expression of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as a critical regulator of cardiomyocyte function by differential binding to specific mRNAs in response to pathological growth stimulation.

8.
iScience ; 24(4): 102389, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33981976

RESUMO

Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

9.
EMBO Rep ; 22(6): e52626, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34009726

RESUMO

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.


Assuntos
Proteômica , Espectrometria de Massas
10.
Mol Syst Biol ; 17(2): e10188, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33590968

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a system-wide scale so that potential host-directed therapies can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.


Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Estabilidade Proteica , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Antivirais/farmacologia , COVID-19/virologia , Humanos , Proteoma , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Temperatura , Replicação Viral/efeitos dos fármacos
11.
Mol Syst Biol ; 16(10): e9500, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33022891

RESUMO

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry-based proteomics to characterize aggregation and disaggregation in human cells after non-lethal heat shock. Aggregation-prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point, and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions were associated with faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared with control condition. Therefore, our results provide a rich resource of heat stress-related protein solubility data and can foster further studies related to protein aggregation diseases.


Assuntos
Núcleo Celular/metabolismo , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Linhagem Celular , Núcleo Celular/genética , Sobrevivência Celular/genética , Imunofluorescência , Histonas/metabolismo , Humanos , Espectrometria de Massas , Peso Molecular , Biossíntese de Proteínas/genética , Dobramento de Proteína , Proteoma/genética , Solubilidade
12.
Nucleic Acids Res ; 48(9): 4725-4740, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32313943

RESUMO

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo , Biossíntese de Proteínas , Estabilidade de RNA
13.
Immunity ; 52(2): 404-416.e5, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32049054

RESUMO

Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.


Assuntos
Mastócitos/citologia , Mastócitos/imunologia , Animais , Biomarcadores/metabolismo , Degranulação Celular , Linhagem da Célula , Células Cultivadas , Tecido Conjuntivo/imunologia , Humanos , Imunoterapia , Mastócitos/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Neuroimunomodulação , Proteoglicanas/biossíntese , Proteoma , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/imunologia , Receptores de Neuropeptídeos/metabolismo , Pele/imunologia
14.
Sci Rep ; 10(1): 1159, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980684

RESUMO

Apolipoprotein A-Ib (ApoA-Ib) is a high molecular weight form of Apolipoprotein A-I (ApoA-I) found specifically in the urine of kidney-transplanted patients with recurrent idiopathic focal segmental glomerulosclerosis (FSGS). To determine the nature of the modification present in ApoA-Ib, we sequenced the whole APOA1 gene in ApoA-Ib positive and negative patients, and we also studied the protein primary structure using mass spectrometry. No genetic variations in the APOA1 gene were found in the ApoA-Ib positive patients that could explain the increase in its molecular mass. The mass spectrometry analysis revealed three extra amino acids at the N-Terminal end of ApoA-Ib that were not present in the standard plasmatic form of ApoA-I. These amino acids corresponded to half of the propeptide sequence of the immature form of ApoA-I (proApoA-I) indicating that ApoA-Ib is a misprocessed form of proApoA-I. The description of ApoA-Ib could be relevant not only because it can allow the automated analysis of this biomarker in the clinical practice but also because it has the potential to shed light into the molecular mechanisms that cause idiopathic FSGS, which is currently unknown.


Assuntos
Apolipoproteína A-I/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Especificidade de Anticorpos , Apolipoproteína A-I/genética , Apolipoproteína A-I/imunologia , Apolipoproteína A-I/urina , Biomarcadores , Western Blotting , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Peso Molecular , Polimorfismo de Nucleotídeo Único , Precursores de Proteínas/metabolismo , Recidiva
15.
Circ Res ; 125(4): 431-448, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31284834

RESUMO

RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.


Assuntos
Miócitos Cardíacos/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , Disfunção Ventricular/genética , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Hemodinâmica , Masculino , Camundongos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/química , Estresse Fisiológico , Disfunção Ventricular/metabolismo
16.
Cell Oncol (Dordr) ; 42(2): 173-196, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30756254

RESUMO

PURPOSE: Previous analyses of the tumor microenvironment (TME) have resulted in a concept that tumor progression may depend on interactions between cancer cells and its surrounding stroma. An important aspect of these interactions is the ability of cancer cells to modulate stroma behavior, and vice versa, through the action of a variety of soluble mediators. Here, we aimed to identify soluble factors present in the TME of colorectal cancer cells that may affect relevant pathways through secretome profiling. METHODS: To partially recapitulate the TME and its architecture, we co-cultured colorectal cancer cells (SW480, TC) with stromal fibroblasts (MRC-5, F) as 3D-spheroids. Subsequent characterization of both homotypic (TC) and heterotypic (TC + F) spheroid secretomes was performed using label-free liquid chromatography-mass spectrometry (LC-MS). RESULTS: Through bioinformatic analysis using the NCI-Pathway Interaction Database (NCI-PID) we found that the HIF-1 signaling pathway was most highly enriched among the proteins whose secretion was enhanced in the heterotypic spheroids. Previously, we found that HIF-1 may be associated with resistance of colorectal cancer cells to photodynamic therapy (PDT), an antitumor therapy that combines photosensitizing agents, O2 and light to create a harmful photochemical reaction. Here, we found that the presence of fibroblasts considerably diminished the sensitivity of colorectal cancer cells to photodynamic activity. Although the biological significance of the HIF-1 pathway of secretomes was decreased after photosensitization, this decrease was partially reversed in heterotypic 3D-spheroids. HIF-1 pathway modulation by both PDT and stromal fibroblasts was confirmed through expression assessment of the HIF-target VEGF, as well as through HIF transcriptional activity assessment. CONCLUSION: Collectively, our results delineate a potential mechanism by which stromal fibroblasts may enhance colorectal cancer cell survival and photodynamic treatment resistance via HIF-1 pathway modulation.


Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fotoquimioterapia , Proteoma/metabolismo , Proteômica/métodos , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Humanos , Transdução de Sinais , Microambiente Tumoral
17.
PLoS One ; 14(1): e0211180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682149

RESUMO

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we aimed to identify new lumenal interaction partners of the Sec61 channel by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/genética , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/genética , Proteínas Repressoras/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
Nat Commun ; 9(1): 4408, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30352994

RESUMO

Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.


Assuntos
Mapas de Interação de Proteínas , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Genoma , Humanos , Células Jurkat , Poli A/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , RNA Ribossômico/metabolismo , Serina-Treonina Quinases TOR/metabolismo
19.
Nat Commun ; 9(1): 2183, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29855469

RESUMO

The previously published version of this Article contained an error in Figure 1. In panel d, the Arabidopsis SERRATE protein was incorrectly labelled 'Human SERRATE' and should have been labelled 'SERRATE'. The error has been corrected in both the PDF and HTML versions of the Article.

20.
Nat Commun ; 9(1): 1701, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703953

RESUMO

ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC), it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here, we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC-ARS2-NCBP3 form a ternary complex that is mutually exclusive with CBC-ARS-PHAX (involved in snRNA export). Our results support that mutually exclusive higher-order CBC-ARS2 complexes are critical in determining Pol II transcript fate.


Assuntos
Proteínas Nucleares/química , Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Transcrição Genética/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cristalografia por Raios X , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/fisiologia , Domínios Proteicos , RNA Polimerase II/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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