RESUMO
Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.
Assuntos
Artemisininas/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Receptores de GABA-A/metabolismo , Transdução de Sinais , Animais , Artemeter , Artemisininas/administração & dosagem , Proteínas de Transporte/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Ratos , Análise de Célula Única , Fatores de Transcrição/metabolismo , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismoRESUMO
Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.
Assuntos
Imunidade Inata , Interferons/fisiologia , Mapeamento de Interação de Proteínas , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Expressão Gênica , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/genética , Espectrometria de Massas , Receptores CCR4/metabolismo , Receptores de Peptídeos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismoRESUMO
The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , Proteômica , SARS-CoV-2/patogenicidade , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Animais , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Conjuntos de Dados como Assunto , Avaliação Pré-Clínica de Medicamentos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteoma/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Viroporinas/metabolismoRESUMO
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Inflamação/tratamento farmacológico , Metiltransferases/metabolismo , Camundongos , Capuzes de RNA/metabolismo , RNA Viral/genética , Ribose , Proteínas não Estruturais Virais/genéticaRESUMO
Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Proteômica , Zika virus/patogenicidade , Animais , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno/genética , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genética , Zika virus/metabolismoRESUMO
There is a significant unmet need for clinical reflex tests that increase the specificity of prostate-specific antigen blood testing, the longstanding but imperfect tool for prostate cancer diagnosis. Towards this endpoint, we present the results from a discovery study that identifies new prostate-specific antigen reflex markers in a large-scale patient serum cohort using differentiating technologies for deep proteomic interrogation. We detect known prostate cancer blood markers as well as novel candidates. Through bioinformatic pathway enrichment and network analysis, we reveal associations of differentially abundant proteins with cytoskeletal, metabolic, and ribosomal activities, all of which have been previously associated with prostate cancer progression. Additionally, optimized machine learning classifier analysis reveals proteomic signatures capable of detecting the disease prior to biopsy, performing on par with an accepted clinical risk calculator benchmark.
Assuntos
Biomarcadores Tumorais , Neoplasias da Próstata , Proteômica , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/sangue , Biomarcadores Tumorais/sangue , Proteômica/métodos , Espectrometria de Mobilidade Iônica/métodos , Antígeno Prostático Específico/sangue , Idoso , Aprendizado de Máquina , Pessoa de Meia-IdadeRESUMO
Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.
Assuntos
Infecções por Orthomyxoviridae/imunologia , Proteínas de Ligação ao Cap de RNA/imunologia , Proteínas de Ligação ao Cap de RNA/metabolismo , Animais , Vírus da Influenza A/imunologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/metabolismo , Biossíntese de Proteínas/fisiologiaRESUMO
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Aminoquinolinas/farmacologia , Animais , Antineoplásicos/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Crizotinibe , Cristalização , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Reparo do DNA , Enzimas Reparadoras do DNA/biossíntese , Enzimas Reparadoras do DNA/química , Modelos Animais de Doenças , Feminino , Homeostase/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Nucleotídeos/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteômica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Pirazóis/química , Piridinas/química , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genéticaRESUMO
RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.
Assuntos
RNA Helicases DEAD-box/metabolismo , Vírus da Coriomeningite Linfocítica/enzimologia , Modelos Moleculares , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Sistemas CRISPR-Cas , Biologia Computacional , Cruzamentos Genéticos , RNA Helicases DEAD-box/química , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/veterinária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Organismos Livres de Patógenos Específicos , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.1-8 In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.9-11 We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.
Assuntos
Proteínas de Membrana/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Antibacterianos/farmacologia , Biotinilação , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatografia Líquida/métodos , Humanos , Espectrometria de Massas/métodos , Proteínas de Membrana/genética , Proteoma/genética , Reprodutibilidade dos Testes , Tunicamicina/farmacologiaRESUMO
Secretion of type I interferon (IFN) is the first cellular reaction to invading pathogens. Despite the protective function of these cytokines, an excessive response to their action can contribute to serious pathologies, such as autoimmune diseases. Transcripts of most cytokines contain adenylate-uridylate (A/U)-rich elements (AREs) that make them highly unstable. RNA-binding proteins (RBPs) are mediators of the regulatory mechanisms that determine the fate of mRNAs containing AREs. Here, we applied an affinity proteomic approach and identified lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR) as the predominant RBP of the IFN-ß mRNA ARE. Reduced expression or chemical inhibition of HuR severely hampered the type I IFN response in various cell lines and fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients. These results define a role for HuR as a potent modulator of the type I IFN response. Taken together, HuR could be used as therapeutic target for diseases where type I IFN production is exaggerated.
Assuntos
Proteínas ELAV/imunologia , Interferon Tipo I/biossíntese , Interferon beta/genética , Elementos Ricos em Adenilato e Uridilato , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Sequência de Bases , Proteínas ELAV/antagonistas & inibidores , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1 , Células HeLa , Humanos , Indutores de Interferon/farmacologia , Dados de Sequência Molecular , Poli I-C/farmacologia , Multimerização Proteica , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Membrana Sinovial/imunologiaRESUMO
The imprinted Airn macro long non-coding (lnc) RNA is an established example of a cis-silencing lncRNA. Airn expression is necessary to initiate paternal-specific silencing of the Igf2r gene, which is followed by gain of a somatic DNA methylation imprint on the silent Igf2r promoter. However, the developmental requirements for Airn initiation of Igf2r silencing and the role of Airn or DNA methylation in maintaining stable Igf2r repression have not been investigated. Here, we use inducible systems to control Airn expression during mouse embryonic stem cell (ESC) differentiation. By turning Airn expression off during ESC differentiation, we show that continuous Airn expression is needed to maintain Igf2r silencing, but only until the paternal Igf2r promoter is methylated. By conditionally turning Airn expression on, we show that Airn initiation of Igf2r silencing is not limited to one developmental 'window of opportunity' and can be maintained in the absence of DNA methylation. Together, this study shows that Airn expression is both necessary and sufficient to silence Igf2r throughout ESC differentiation and that the somatic methylation imprint, although not required to initiate or maintain silencing, adds a secondary layer of repressive epigenetic information.
Assuntos
Inativação Gênica , Impressão Genômica/genética , RNA Longo não Codificante/genética , Receptor IGF Tipo 2/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inativação Gênica/fisiologia , Genes Reporter , Impressão Genômica/fisiologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification-mass spectrometry (AP-MS) is a highly desirable step in the quest to understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation-related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.
Assuntos
Laboratórios/normas , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Complexos Multiproteicos/química , Linhagem Celular , Cromatografia de Afinidade , Bases de Dados Factuais , Humanos , Integrinas , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Filogenia , Mapas de Interação de Proteínas , Reprodutibilidade dos TestesRESUMO
We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Furanos , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Alzheimer's disease (AD) and related dementias (ADRD) is a complex disease with multiple pathophysiological drivers that determine clinical symptomology and disease progression. These diseases develop insidiously over time, through many pathways and disease mechanisms and continue to have a huge societal impact for affected individuals and their families. While emerging blood-based biomarkers, such as plasma p-tau181 and p-tau217, accurately detect Alzheimer neuropthology and are associated with faster cognitive decline, the full extension of plasma proteomic changes in ADRD remains unknown. Earlier detection and better classification of the different subtypes may provide opportunities for earlier, more targeted interventions, and perhaps a higher likelihood of successful therapeutic development. In this study, we aim to leverage unbiased mass spectrometry proteomics to identify novel, blood-based biomarkers associated with cognitive decline. 1,786 plasma samples from 1,005 patients were collected over 12 years from partcipants in the Massachusetts Alzheimer's Disease Research Center Longitudinal Cohort Study. Patient metadata includes demographics, final diagnoses, and clinical dementia rating (CDR) scores taken concurrently. The Proteograph™ Product Suite (Seer, Inc.) and liquid-chromatography mass-spectrometry (LC-MS) analysis were used to process the plasma samples in this cohort and generate unbiased proteomics data. Data-independent acquisition (DIA) mass spectrometry results yielded 36,259 peptides and 4,007 protein groups. Linear mixed effects models revealed 138 differentially abundant proteins between AD and healthy controls. Machine learning classification models for AD diagnosis identified potential candidate biomarkers including MBP, BGLAP, and APoD. Cox regression models were created to determine the association of proteins with disease progression and suggest CLNS1A, CRISPLD2, and GOLPH3 as targets of further investigation as potential biomarkers. The Proteograph workflow provided deep, unbiased coverage of the plasma proteome at a speed that enabled a cohort study of almost 1,800 samples, which is the largest, deep, unbiased proteomics study of ADRD conducted to date.
RESUMO
Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/isolamento & purificação , Mapeamento de Interação de Proteínas/métodos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína alfa Estimuladora de Ligação a CCAAT/química , Linhagem Celular , Cromatografia de Afinidade , Cromatografia de Fase Reversa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Ligação Proteica , Mapas de Interação de Proteínas , Ratos , Estreptavidina/biossíntese , Estreptavidina/química , Estreptavidina/isolamento & purificaçãoRESUMO
While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.
Assuntos
Compostos de Anilina/química , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Nitrilas/química , Proteínas Tirosina Quinases/isolamento & purificação , Pirimidinas/química , Quinolinas/química , Tiazóis/química , Animais , Biópsia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Cromatografia de Afinidade , Dasatinibe , Humanos , Células K562 , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Anotação de Sequência Molecular , Terapia de Alvo Molecular , Transplante de Neoplasias , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/química , ProteômicaAssuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Transtornos Mieloproliferativos/genética , Análise Mutacional de DNA , Família , Feminino , Humanos , Masculino , Transtornos Mieloproliferativos/diagnóstico , Linhagem , Ubiquitina-Proteína LigasesRESUMO
Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes. Here, we isolate ER membranes from ZIKV-infected cells and determine their proteome. Forty-six host cell factors are enriched in ZIKV remodeled membranes, several of these having a role in redox and methylation pathways. Four proteins are characterized in detail: thioredoxin reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and increasing stability of viral transmembrane proteins; adenosylhomocysteinase (AHCY) indirectly promoting m6A methylation of ZIKV RNA by decreasing the level of S- adenosyl homocysteine and thus, immune evasion. These results highlight the involvement of redox and methylation enzymes in the ZIKV life cycle and their accumulation at virally remodeled ER membranes.