RESUMO
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteoma/genética , Biologia Computacional/métodos , Células HCT116/metabolismo , Células HEK293/metabolismo , Humanos , Espectrometria de Massas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteoma/metabolismo , Proteômica/métodosRESUMO
All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here, we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ≈50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute timescale and then co-assemble from center to periphery in a â¼1 h-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily applicable beyond the NPC, making it possible to analyze the intracellular dynamics of a variety of multiprotein assemblies.
Assuntos
Substâncias Macromoleculares/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Coloração e Rotulagem , Bioensaio , Cinética , Modelos Biológicos , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/químicaRESUMO
Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.
Assuntos
Dano ao DNA , Reparo do DNA , Fatores de Transcrição de Domínio TEA , Humanos , Carcinogênese/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismoRESUMO
Understanding RNA-protein interactions is crucial for deciphering the cellular functions and molecular mechanisms of regulatory RNAs. Consequently, there is a constant need to develop innovative and cost-effective methods to uncover such interactions. We developed a simple and cost-effective technique called Multiple Oligo assisted RNA Pulldown via Hybridization (MORPH) to identify proteins interacting with a specific RNA. MORPH employs a tiling array of antisense oligos (ASOs) to efficiently capture the RNA of interest along with proteins associated with it. Unlike existing techniques that rely on multiple individually biotinylated oligos spanning the entire RNA length, MORPH stands out by utilizing a single biotinylated oligo to capture all the ASOs. To evaluate MORPH's efficacy, we applied this technique combined with mass spectrometry to identify proteins interacting with lncRNA NEAT1, which has previously been studied using various methods. Our results demonstrate that despite being a simple and inexpensive procedure, MORPH performs on par with existing methods.Abbreviations: ASO, Antisense oligo; lncRNA, long non-coding RNA; MORPH, Multiple Oligo assisted RNA Pulldown via Hybridization.
Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Hibridização de Ácido Nucleico , Espectrometria de Massas/métodos , Proteínas/genéticaRESUMO
The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.
Assuntos
Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Células HEK293 , Proteômica/métodos , Ligação Proteica , Proteínas de Choque Térmico HSP70/metabolismo , Estabilidade Proteica , Dobramento de ProteínaRESUMO
The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide a comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 221 unique high-confidence interactors, of which the vast majority represent novel SLX4-binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and several emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , DNA/genética , Recombinação Homóloga , Mamíferos/genética , Mamíferos/metabolismo , Recombinases/química , Recombinases/genética , Recombinases/metabolismoRESUMO
Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.
Assuntos
Fator 2 Relacionado a NF-E2 , Peptídeos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/química , Espectrometria de Massas/métodos , Cromatografia de AfinidadeRESUMO
Interactomics is a branch of systems biology that deals with the study of protein-protein interactions and how these interactions influence phenotypes. Identifying the interactomes involved during host-pathogen interaction events may bring us a step closer to deciphering the molecular mechanisms underlying plant defence. Here, we conducted a systematic review of plant interactomics studies over the last two decades and found that while a substantial progress has been made in the field, plant-pathogen interactomics remains a less-travelled route. As an effort to facilitate the progress in this field, we provide here a comprehensive research pipeline for an in planta plant-pathogen interactomics study that encompasses the in silico prediction step to the validation step, unconfined to model plants. We also highlight four challenges in plant-pathogen interactomics with plausible solution(s) for each.
Assuntos
Interações Hospedeiro-Patógeno , Plantas , Plantas/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genéticaRESUMO
The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.
Assuntos
Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the Escherichia coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of nine different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergents. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies. Data are available via ProteomeXchange with identifier PXD032315.
Assuntos
Proteínas de Escherichia coli , Proteômica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana , Proteoma/análise , Proteômica/métodosRESUMO
Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed.
Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Proteoma/metabolismo , Animais , Humanos , Proteoma/análiseRESUMO
Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1â µM to 1490â µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.
Assuntos
Diabetes Mellitus Tipo 2 , Inibidores da Dipeptidil Peptidase IV , Fosfato de Sitagliptina , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Diabetes Mellitus Tipo 2/induzido quimicamente , Inibidores da Dipeptidil Peptidase IV/farmacologia , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Fosfato de Sitagliptina/farmacologiaRESUMO
The epidermal growth factor receptor (EGFR) signal modulates cell proliferation, migration, and survival. Aberrant activation of EGFR constitutes the major cause of various cancers. Receptor ubiquitination and degradation mediated by CBL proteins play negative regulatory roles and control the intensity and duration of the signaling. With the construction of stable cell lines inducibly expressing FLAG-tagged CBL or CBLB, we identified 102 and 82 stable interacting proteins of CBL and CBLB, respectively, through the affinity purification followed by mass spectrometry (AP-MS) approach. Time-resolved profiling at six different time points combined with functional annotations of the temporal interactomes provides insights into the dynamic assembly of signal proteins upon EGFR signaling activation. Comparison between the interactomes of CBL and CBLB indicates their redundant but also complementary functions. Importantly, we validated the stable association of EPS15L1 and ITSN2 and temporal association of TNK2 to both CBL and CBLB through biochemical assays. Collectively, these results offer a useful resource for CBL and CBLB interactomes and highlight their prominent and diverse roles in the EGFR signaling network.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fator de Crescimento Epidérmico , Proteínas Proto-Oncogênicas c-cbl , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular , Linhagem Celular , Fator de Crescimento Epidérmico/metabolismo , Humanos , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.
Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa 2 de Fator de Ligação ao Core , Fator de Transcrição MSX1 , Mioblastos , Animais , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Técnicas de Inativação de Genes , Fator de Transcrição MSX1/química , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Fator de Transcrição MSX1/fisiologia , Camundongos , Mioblastos/citologia , Mioblastos/metabolismoRESUMO
The protein arginine methyltransferase family (PRMT) is known as being the catalytic driving force for arginine methylation. This specific type of post translational modification is extensively used in biological processes, and therefore is highly relevant in the pathology of a profusion of diseases. Since altered PRMT expression or deregulation has been shown to contribute to a vast range of those diseases including cancer, their study is of great interest. Although an increasing number of substrates are being discovered for each PRMT, large scale proteomic methods can be used to identify novel interactors/substrates, further elucidating the role that PRMTs perform in physiological or disease states. Here, we describe the use of affinity purification (AP) coupled with stable isotope labeling with amino acids in cell culture (SILAC) quantitative mass spectrometry (MS) to identify protein interactors and substrates of PRMTs. We also explore the possibility of exploiting the fact most PRMTs display lower dissociation rates with their hypomethylated substrates as a strategy to increase the proportion of substrates identified in AP/MS studies.
Assuntos
Cromatografia de Afinidade/métodos , Inibidores Enzimáticos/química , Espectrometria de Massas/métodos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteômica/métodos , Aminoácidos/metabolismo , Arginina/análise , Arginina/química , Arginina/metabolismo , Expressão Gênica , Histonas/química , Histonas/metabolismo , Humanos , Marcação por Isótopo , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas RecombinantesRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Sistemas de Liberação de Medicamentos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.
Assuntos
Chaperonas Moleculares , Proteômica , Humanos , Chaperonas Moleculares/genética , Fases de Leitura AbertaRESUMO
Manipulation of host cellular processes by translocated bacterial effectors is key to the success of bacterial pathogens and some symbionts. Therefore, a comprehensive understanding of effectors is of critical importance to understand infection biology. It has become increasingly clear that the identification of host protein targets contributes invaluable knowledge to the characterization of effector function during pathogenesis. Recent advances in mapping protein-protein interaction networks by means of mass spectrometry-based interactomics have enabled the identification of host targets at large-scale. In this review, we highlight mass spectrometry-driven proteomics strategies and recent advances to elucidate type-III secretion system effector-host protein-protein interactions. Furthermore, we highlight approaches for defining spatial and temporal effector-host interactions, and discuss possible avenues for studying natively delivered effectors in the context of infection. Overall, the knowledge gained when unravelling effector complexation with host factors will provide novel opportunities to control infectious disease outcomes.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/metabolismo , Fenômenos Fisiológicos Bacterianos , Interações Hospedeiro-Patógeno/fisiologia , Mapeamento de Interação de Proteínas , Proteômica , Sistemas de Secreção Tipo III/metabolismo , Humanos , Espectrometria de MassasRESUMO
The Ku heterodimer, composed of Ku70 and Ku80, is best characterized for its role in repairing double-stranded DNA breaks but is also known to participate in other regulatory processes. Despite our understanding of Ku protein interplay during DNA repair, the extent of Ku's protein interactions in other processes has never been fully determined. Using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS) with wild-type Ku70, we identified candidate proteins that interact with the Ku heterodimer in HEK293 cells, in the absence of exogenously induced DNA damage. BioID analysis identified approximately 250 nuclear proteins, appearing in at least two replicates, including known Ku-interacting factors such as MRE11A, WRN, and NCOA6. Meanwhile, AP-MS analysis identified approximately 50 candidate proteins. Of the novel protein interactors identified, many were involved in functions already suspected to involve Ku such as transcriptional regulation, DNA replication, and DNA repair, while several others suggest that Ku may be involved in additional functions such as RNA metabolism, chromatin-remodeling, and microtubule dynamics. Using a combination of BioID and AP-MS, this is the first report that comprehensively characterizes the Ku protein interaction landscape, revealing new cellular processes and protein complexes involving the Ku complex.