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1.
Eur J Immunol ; 51(9): 2210-2217, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34145909

RESUMO

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an inhibitory receptor with a hitherto unknown ligand, and is expressed on human monocytes and neutrophils. SIRL-1 inhibits myeloid effector functions such as reactive oxygen species (ROS) production. In this study, we identify S100 proteins as SIRL-1 ligands. S100 proteins are composed of two calcium-binding domains. Various S100 proteins are damage-associated molecular patterns (DAMPs) released from damaged cells, after which they initiate inflammation by ligating activating receptors on immune cells. We now show that the inhibitory SIRL-1 recognizes individual calcium-binding domains of all tested S100 proteins. Blocking SIRL-1 on human neutrophils enhanced S100 protein S100A6-induced ROS production, showing that S100A6 suppresses neutrophil ROS production via SIRL-1. Taken together, SIRL-1 is an inhibitory receptor recognizing the S100 protein family of DAMPs. This may help limit tissue damage induced by activated neutrophils.


Assuntos
Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/imunologia , Proteínas S100/imunologia , Alarminas/imunologia , Humanos , Inflamação/imunologia , Monócitos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Transdução de Sinais/imunologia
2.
Nat Struct Mol Biol ; 25(12): 1119-1127, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30510221

RESUMO

TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.


Assuntos
Chaperonina com TCP-1/fisiologia , Modelos Moleculares , Fator de Transcrição TFIID/química , Chaperonina com TCP-1/metabolismo , Cristalografia por Raios X , Células HeLa , Humanos , Espectrometria de Massas , Domínios Proteicos , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica
3.
Proc Natl Acad Sci U S A ; 115(17): E3996-E4005, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632210

RESUMO

Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.


Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/metabolismo , Animais , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteína Wnt3A/genética
4.
BMC Biol ; 14: 66, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27506200

RESUMO

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.


Assuntos
Proteínas de Caenorhabditis elegans/isolamento & purificação , Caenorhabditis elegans/metabolismo , Guanilato Quinases/metabolismo , Especificidade de Órgãos , Mapeamento de Interação de Proteínas/métodos , Sequência de Aminoácidos , Animais , Biotinilação , Proteínas de Caenorhabditis elegans/metabolismo , Imunofluorescência , Complexos Multiproteicos/isolamento & purificação , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Reprodutibilidade dos Testes
5.
Nat Struct Mol Biol ; 23(4): 324-32, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26974125

RESUMO

Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.


Assuntos
Proteína Axina/genética , Proteína Axina/metabolismo , Neoplasias/genética , Mutação Puntual , Agregados Proteicos , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Proteína Axina/análise , Proteína Axina/ultraestrutura , Linhagem Celular , Drosophila/química , Drosophila/genética , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Neoplasias/patologia , Conformação Proteica , Mapas de Interação de Proteínas , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios X
6.
Nature ; 488(7413): 665-9, 2012 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-22895187

RESUMO

LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endocitose , Proteínas Oncogênicas/metabolismo , Receptores Wnt/metabolismo , Células-Tronco/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Adenoma/metabolismo , Adenoma/patologia , Animais , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Organoides/citologia , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Wnt/antagonistas & inibidores , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
7.
Cell ; 149(6): 1245-56, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22682247

RESUMO

Degradation of cytosolic ß-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that ß-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound ß-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses ß-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-ß-catenin. Subsequently, newly synthesized ß-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.


Assuntos
Proteína Axina/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Dados de Sequência Molecular , Mutação , Peptídeos/análise , Peptídeos/química , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , beta Catenina/genética
8.
Mol Syst Biol ; 7: 550, 2011 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-22108792

RESUMO

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.


Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes/metabolismo , Proteoma/análise , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Proteoma/genética , Proteoma/metabolismo
9.
Nature ; 476(7360): 293-7, 2011 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-21727895

RESUMO

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores Frizzled/metabolismo , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Regeneração , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A
10.
Mol Cell Proteomics ; 10(10): M110.006452, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21715320

RESUMO

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 µg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 µg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.


Assuntos
Basófilos/enzimologia , Fracionamento Químico , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Fosfopeptídeos/análise , Fosfotransferases/química , Aminoácidos Básicos/análise , Resinas de Troca de Cátion/química , Gentisatos/química , Células HeLa , Humanos , Hidrocarbonetos Fluorados/química , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Ligação Proteica , Titânio/química , Ácido Trifluoracético/química
11.
Mol Cell Proteomics ; 10(10): O111.008474, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21705516

RESUMO

In quantitative proteomics stable isotope labeling has progressed from cultured cells toward the total incorporation of labeled atoms or amino acids into whole multicellular organisms. For instance, the recently introduced (13)C(6)-lysine labeled SILAC mouse allows accurate comparison of protein expression directly in tissue. In this model, only lysine, but not arginine, residues are isotope labeled, as the latter may cause complications to the quantification by in vivo conversion of arginine to proline. The sole labeling of lysines discourages the use of trypsin, as not all peptides will be quantifiable. Therefore, in the initial work Lys-C was used for digestion. Here, we demonstrate that the lysine-directed protease metalloendopeptidase Lys-N is an excellent alternative. As lysine directed peptides generally yield longer and higher charged peptides, alongside the more traditional collision induced dissociation we also implemented electron transfer dissociation in a quantitative stable isotope labeling with amino acid in cell culture workflow for the first time. The utility of these two complementary approaches is highlighted by investigating the differences in protein expression between the left and right ventricle of a mouse heart. Using Lys-N and electron transfer dissociation yielded coverage to a depth of 3749 proteins, which is similar as earlier investigations into the murine heart proteome. In addition, this strategy yields quantitative information on ∼ 2000 proteins with a median coverage of four peptides per protein in a single strong cation exchange-liquid chromatography-MS experiment, revealing that the left and right ventricle proteomes are very similar qualitatively as well as quantitatively.


Assuntos
Ventrículos do Coração/metabolismo , Metaloendopeptidases/química , Proteoma/metabolismo , Proteômica/métodos , Animais , Isótopos de Carbono/química , Transporte de Elétrons , Feminino , Regulação da Expressão Gênica , Ventrículos do Coração/química , Marcação por Isótopo , Lisina/química , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Peptídeos/análise , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/análise
12.
J Proteomics ; 74(10): 2204-9, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21616183

RESUMO

High-resolution mass spectrometry and the use of stable isotopes have greatly improved our ability to quantify proteomes. Typically, the relative abundance of peptides is estimated by identifying the isotopic clusters and by comparing the peak intensities of peptide pairs. However, when the mass shift between the labeled peptides is small, there can be the possibility for overlap of the isotopic clusters which will hamper quantification accuracy with a typical upwards bias for the heavier peptide. Here, we investigated the impact of the overlapping peak issue with respect to dimethyl based quantification and we confirmed there can be need for correction. In addition, we present a tool that can correct overlapping issues when they arise which is based on modeling isotopic distributions. We demonstrate that our approach leads to improved accuracy and precision of protein quantification.


Assuntos
Marcação por Isótopo/métodos , Peptídeos/análise , Proteômica/métodos , Algoritmos , Células HeLa , Humanos , Isótopos , Metilação , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
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