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1.
Mol Cell ; 82(21): 4001-4017.e7, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-36265488

RESUMEN

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.


Asunto(s)
ARN Largo no Codificante , Homeostasis del Telómero , Cromatina/genética , Proteómica , Telómero/genética , Telómero/metabolismo , ARN Largo no Codificante/genética , Homeostasis
2.
Mol Cell Proteomics ; 20: 100111, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34126241

RESUMEN

Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anticancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade, and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 monoallelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale monoallelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC-binding pocket diversity in the training data and extend allelic coverage in under profiled populations. To improve generalizability, SHERPA systematically integrates 128 monoallelic and 384 multiallelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution, and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44-fold improvement of positive predictive value compared with existing tools when evaluated on independent monoallelic datasets and a 1.15-fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.


Asunto(s)
Antígenos de Neoplasias , Complejo Mayor de Histocompatibilidad , Modelos Teóricos , Péptidos , Algoritmos , Presentación de Antígeno , Línea Celular , Humanos , Proteoma , Transcriptoma
3.
Mol Cell Proteomics ; 19(9): 1436-1449, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32541000

RESUMEN

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WTversus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.


Asunto(s)
Síndrome de Andersen/metabolismo , Miocitos Cardíacos/metabolismo , Placofilinas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Mapas de Interacción de Proteínas , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Síndrome de Andersen/genética , Síndrome de Andersen/fisiopatología , Cromatografía Liquida , Desmosomas/efectos de los fármacos , Desmosomas/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Chaperonas Moleculares/metabolismo , Mutación , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , Mapas de Interacción de Proteínas/genética , Mapas de Interacción de Proteínas/fisiología , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Somatomedinas/metabolismo , Espectrometría de Masas en Tándem , Utrofina/metabolismo
4.
Proc Natl Acad Sci U S A ; 115(33): E7748-E7757, 2018 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-30065114

RESUMEN

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Eritrocitos/metabolismo , Complejos Multiproteicos/biosíntesis , Proteínas de Transporte Vesicular/biosíntesis , Anemia Diseritropoyética Congénita/genética , Anemia Diseritropoyética Congénita/metabolismo , Médula Ósea/metabolismo , Médula Ósea/patología , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Eritrocitos/patología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Especificidad de la Especie , Proteínas de Transporte Vesicular/genética
5.
Nat Methods ; 14(5): 513-520, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28394336

RESUMEN

There is a need to better understand and handle the 'dark matter' of proteomics-the vast diversity of post-translational and chemical modifications that are unaccounted in a typical mass spectrometry-based analysis and thus remain unidentified. We present a fragment-ion indexing method, and its implementation in peptide identification tool MSFragger, that enables a more than 100-fold improvement in speed over most existing proteome database search tools. Using several large proteomic data sets, we demonstrate how MSFragger empowers the open database search concept for comprehensive identification of peptides and all their modified forms, uncovering dramatic differences in modification rates across experimental samples and conditions. We further illustrate its utility using protein-RNA cross-linked peptide data and using affinity purification experiments where we observe, on average, a 300% increase in the number of identified spectra for enriched proteins. We also discuss the benefits of open searching for improved false discovery rate estimation in proteomics.


Asunto(s)
Biología Computacional/métodos , Fragmentos de Péptidos/química , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Biología Computacional/instrumentación , Bases de Datos de Proteínas , Células HEK293 , Humanos , Procesamiento Proteico-Postraduccional , Proteómica/instrumentación
8.
PLoS Genet ; 11(10): e1005564, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26447709

RESUMEN

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.


Asunto(s)
Hifa/genética , Fosfotransferasas/biosíntesis , Ribonucleoproteínas/biosíntesis , Saccharomyces cerevisiae/genética , Candida albicans/genética , Regulación Fúngica de la Expresión Génica , Hifa/crecimiento & desarrollo , Fenotipo , Fosforilación , Fosfotransferasas/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal
9.
PLoS Genet ; 10(3): e1004183, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24603354

RESUMEN

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.


Asunto(s)
Glucosa/metabolismo , Hifa/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Diferenciación Celular/genética , Hifa/crecimiento & desarrollo , Nitrógeno/metabolismo , Fosforilación , Saccharomyces cerevisiae
10.
Nat Methods ; 10(8): 730-6, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23921808

RESUMEN

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.


Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/análisis , Proteómica/métodos , Bases de Datos Factuales , Humanos
11.
Br J Haematol ; 170(1): 66-79, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25824111

RESUMEN

Toward our goal of personalized medicine, we comprehensively profiled pre-treatment malignant plasma cells from multiple myeloma patients and prospectively identified pathways predictive of favourable response to bortezomib-based treatment regimens. We utilized two complementary quantitative proteomics platforms to identify differentially-regulated proteins indicative of at least a very good partial response (VGPR) or complete response/near complete response (CR/nCR) to two treatment regimens containing either bortezomib, liposomal doxorubicin and dexamethasone (VDD), or lenalidomide, bortezomib and dexamethasone (RVD). Our results suggest enrichment of 'universal response' pathways that are common to both treatment regimens and are probable predictors of favourable response to bortezomib, including a subset of endoplasmic reticulum stress pathways. The data also implicate pathways unique to each regimen that may predict sensitivity to DNA-damaging agents, such as mitochondrial dysfunction, and immunomodulatory drugs, which was associated with acute phase response signalling. Overall, we identified patterns of tumour characteristics that may predict response to bortezomib-based regimens and their components. These results provide a rationale for further evaluation of the protein profiles identified herein for targeted selection of anti-myeloma therapy to increase the likelihood of improved treatment outcome of patients with newly-diagnosed myeloma.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Adulto , Anciano , Ácidos Borónicos/administración & dosificación , Bortezomib , Dexametasona/administración & dosificación , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Humanos , Lenalidomida , Persona de Mediana Edad , Mieloma Múltiple/metabolismo , Polietilenglicoles/administración & dosificación , Medicina de Precisión/métodos , Proteómica/métodos , Pirazinas/administración & dosificación , Talidomida/administración & dosificación , Talidomida/análogos & derivados
12.
Nat Methods ; 8(1): 70-3, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21131968

RESUMEN

We present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification-mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data.


Asunto(s)
Cromatografía de Afinidad/métodos , Biología Computacional , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Simulación por Computador , Espectrometría de Masas , Probabilidad , Unión Proteica , Proteínas/aislamiento & purificación
13.
Nat Commun ; 13(1): 1925, 2022 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-35414054

RESUMEN

Human leukocyte antigen loss of heterozygosity (HLA LOH) allows cancer cells to escape immune recognition by deleting HLA alleles, causing the suppressed presentation of tumor neoantigens. Despite its importance in immunotherapy response, few methods exist to detect HLA LOH, and their accuracy is not well understood. Here, we develop DASH (Deletion of Allele-Specific HLAs), a machine learning-based algorithm to detect HLA LOH from paired tumor-normal sequencing data. With cell line mixtures, we demonstrate increased sensitivity compared to previously published tools. Moreover, our patient-specific digital PCR validation approach provides a sensitive, robust orthogonal approach that could be used for clinical validation. Using DASH on 610 patients across 15 tumor types, we find that 18% of patients have HLA LOH. Moreover, we show inflated HLA LOH rates compared to genome-wide LOH and correlations between CD274 (encodes PD-L1) expression and microsatellite instability status, suggesting the HLA LOH is a key immune resistance strategy.


Asunto(s)
Pérdida de Heterocigocidad , Neoplasias , Algoritmos , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II , Humanos , Pérdida de Heterocigocidad/genética , Aprendizaje Automático , Repeticiones de Microsatélite/genética , Neoplasias/genética
14.
Clin Cancer Res ; 27(15): 4265-4276, 2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-34341053

RESUMEN

PURPOSE: While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers, such as tumor mutational burden (TMB). EXPERIMENTAL DESIGN: Tumors from a cohort of patients with late-stage melanoma (n = 51) were profiled using an immune-enhanced exome and transcriptome platform. We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB. RESULTS: Our neoantigen burden score, which integrates both exome and transcriptome features, more significantly stratified responders and nonresponders (P = 0.016) than TMB alone (P = 0.049). Extension of this model to include immune-related resistance mechanisms affecting the antigen presentation machinery, such as HLA allele-specific LOH, resulted in a composite neoantigen presentation score (NEOPS) that demonstrated further increased association with therapy response (P = 0.002). CONCLUSIONS: NEOPS proved the statistically strongest biomarker compared with all single-gene biomarkers, expression signatures, and TMB biomarkers evaluated in this cohort. Subsequent confirmation of these findings in an independent cohort of patients (n = 110) suggests that NEOPS is a robust, novel biomarker of ICB response in melanoma.


Asunto(s)
Resistencia a Antineoplásicos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Modelos Inmunológicos , Predicción , Humanos , Resultado del Tratamiento
15.
J Clin Invest ; 129(12): 5568-5583, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31557132

RESUMEN

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/terapia , Terapia Genética , Glicina-ARNt Ligasa/genética , Interferencia de ARN , Alelos , Animales , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Mutación
16.
Oncotarget ; 9(31): 22123-22136, 2018 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-29774127

RESUMEN

The Polymerase Associated Factor 1 complex (PAF1c) is an epigenetic co-modifying complex that directly contacts RNA polymerase II (RNAPII) and several epigenetic regulating proteins. Mutations, overexpression and loss of expression of subunits of the PAF1c are observed in various forms of cancer suggesting proper regulation is needed for cellular development. However, the biochemical interactions with the PAF1c that allow dynamic gene regulation are unclear. We and others have shown that the PAF1c makes a direct interaction with MLL fusion proteins, which are potent oncogenic drivers of acute myeloid leukemia (AML). This interaction is critical for the maintenance of MLL translocation driven AML by targeting MLL fusion proteins to the target genes Meis1 and Hoxa9. Here, we use a proteomics approach to identify protein-protein interactions with the PAF1c subunit CDC73 that regulate the function of the PAF1c. We identified a novel interaction with a histone H3 lysine 9 (H3K9) methyltransferase protein, SETDB1. This interaction is stabilized with a mutant CDC73 that is incapable of supporting AML cell growth. Importantly, transcription of Meis1 and Hoxa9 is reduced and promoter H3K9 trimethylation (H3K9me3) increased by overexpression of SETDB1 or stabilization of the PAF1c-SETDB1 interaction in AML cells. These findings were corroborated in human AML patients where increased SETDB1 expression was associated with reduced HOXA9 and MEIS1. To our knowledge, this is the first proteomics approach to search for CDC73 protein-protein interactions in AML, and demonstrates that the PAF1c may play a role in H3K9me3-mediated transcriptional repression in AML.

17.
JCI Insight ; 3(11)2018 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-29875314

RESUMEN

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.


Asunto(s)
Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Animales Recién Nacidos , Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Proteínas del Choque Térmico HSC70/genética , Haploinsuficiencia , Humanos , Miocardio/patología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , Proteostasis/genética , Ratas , Sarcómeros/patología , Tabique Interventricular/patología
18.
Nat Med ; 21(4): 344-52, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25822367

RESUMEN

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.


Asunto(s)
Resistencia a Antineoplásicos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Androgénicos/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias de la Próstata , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Transducción de Señal , Resultado del Tratamiento
19.
Genetics ; 193(4): 1297-310, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23410832

RESUMEN

The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This growth transition has been studied extensively as a model signaling system with similarity to processes of hyphal development that are linked with virulence in related fungal pathogens. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. Here, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. This cohort includes 79 genes lacking previous phenotypic characterization. Pathway enrichment analysis of the gene set identifies networks mediating mitogen-activated protein kinase (MAPK) signaling and cell cycle progression. In particular, overexpression screening suggests that nuclear export of the osmoresponsive MAPK Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. Through epistasis and deletion studies, we also identified genetic relationships with the G2 cyclin Clb2p and phenotypes in filamentation induced by S-phase arrest. In sum, this work presents a unique and informative resource toward understanding the breadth of genes and pathways that collectively constitute the molecular basis of filamentation.


Asunto(s)
Redes Reguladoras de Genes , Genoma Fúngico , Hifa/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Ciclina B/genética , Ciclina B/metabolismo , Epistasis Genética , Eliminación de Gen , Hifa/genética , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenotipo , Fase S/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética
20.
Curr Protoc Bioinformatics ; Chapter 8: 8.15.1-8.15.23, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22948729

RESUMEN

Significance Analysis of INTeractome (SAINT) is a software package for scoring protein-protein interactions based on label-free quantitative proteomics data (e.g., spectral count or intensity) in affinity purification-mass spectrometry (AP-MS) experiments. SAINT allows bench scientists to select bona fide interactions and remove nonspecific interactions in an unbiased manner. However, there is no 'one-size-fits-all' statistical model for every dataset, since the experimental design varies across studies. Key variables include the number of baits, the number of biological replicates per bait, and control purifications. Here we give a detailed account of input data format, control data, selection of high-confidence interactions, and visualization of filtered data. We explain additional options for customizing the statistical model for optimal filtering in specific datasets. We also discuss a graphical user interface of SAINT in connection to the LIMS system ProHits, which can be installed as a virtual machine on Mac OS X or Windows computers.


Asunto(s)
Proteínas/química , Proteómica/métodos , Sitios de Unión , Cromatografía de Afinidad/métodos , Bases de Datos de Proteínas , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Programas Informáticos
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