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1.
Cell ; 181(3): 688-701.e16, 2020 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-32315618

RESUMEN

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.


Asunto(s)
Proteína Fosfatasa 2/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activadores de Enzimas/metabolismo , Células HEK293 , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteína Fosfatasa 2/química , Subunidades de Proteína
2.
Clin Chem ; 67(7): 977-986, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-33720347

RESUMEN

BACKGROUND: Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results. METHODS: Over a 2-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis. RESULTS: There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] vs 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] vs 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6. CONCLUSION: Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.


Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , Inmunoglobulina G/sangre , Nucleocápside/inmunología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Área Bajo la Curva , COVID-19/virología , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Juego de Reactivos para Diagnóstico , Estudios Retrospectivos , SARS-CoV-2/aislamiento & purificación
3.
PLoS Comput Biol ; 15(2): e1006678, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30811403

RESUMEN

We present CoPhosK to predict kinase-substrate associations for phosphopeptide substrates detected by mass spectrometry (MS). The tool utilizes a Naïve Bayes framework with priors of known kinase-substrate associations (KSAs) to generate its predictions. Through the mining of MS data for the collective dynamic signatures of the kinases' substrates revealed by correlation analysis of phosphopeptide intensity data, the tool infers KSAs in the data for the considerable body of substrates lacking such annotations. We benchmarked the tool against existing approaches for predicting KSAs that rely on static information (e.g. sequences, structures and interactions) using publically available MS data, including breast, colon, and ovarian cancer models. The benchmarking reveals that co-phosphorylation analysis can significantly improve prediction performance when static information is available (about 35% of sites) while providing reliable predictions for the remainder, thus tripling the KSAs available from the experimental MS data providing to a comprehensive and reliable characterization of the landscape of kinase-substrate interactions well beyond current limitations.


Asunto(s)
Biología Computacional/métodos , Proteínas Quinasas/fisiología , Especificidad por Sustrato/fisiología , Teorema de Bayes , Sitios de Unión , Bases de Datos de Proteínas , Humanos , Espectrometría de Masas , Fosforilación/fisiología , Fosfotransferasas/fisiología , Unión Proteica , Mapeo de Interacción de Proteínas , Proteoma , Análisis de Secuencia de Proteína , Programas Informáticos
4.
Retrovirology ; 15(1): 44, 2018 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-29970186

RESUMEN

BACKGROUND: Viral reprogramming of host cells enhances replication and is initiated by viral interaction with the cell surface. Upon human immunodeficiency virus (HIV) binding to CD4+ T cells, a signal transduction cascade is initiated that reorganizes the actin cytoskeleton, activates transcription factors, and alters mRNA splicing pathways. METHODS: We used a quantitative mass spectrometry-based phosphoproteomic approach to investigate signal transduction cascades initiated by CCR5-tropic HIV, which accounts for virtually all transmitted viruses and the vast majority of viruses worldwide. RESULTS: CCR5-HIV signaling induced significant reprogramming of the actin cytoskeleton and mRNA splicing pathways, as previously described. In addition, CCR5-HIV signaling induced profound changes to the mRNA transcription, processing, translation, and post-translational modifications pathways, indicating that virtually every stage of protein production is affected. Furthermore, we identified two kinases regulated by CCR5-HIV signaling-p70-S6K1 (RPS6KB1) and MK2 (MAPKAPK2)-that were also required for optimal HIV infection of CD4+ T cells. These kinases regulate protein translation and cytoskeletal architecture, respectively, reinforcing the importance of these pathways in viral replication. Additionally, we found that blockade of CCR5 signaling by maraviroc had relatively modest effects on CCR5-HIV signaling, in agreement with reports that signaling by CCR5 is dispensable for HIV infection but in contrast to the critical effects of CXCR4 on cortical actin reorganization. CONCLUSIONS: These results demonstrate that CCR5-tropic HIV induces significant reprogramming of host CD4+ T cell protein production pathways and identifies two novel kinases induced upon viral binding to the cell surface that are critical for HIV replication in host cells.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores CCR5/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Linfocitos T CD4-Positivos/inmunología , Citoesqueleto/metabolismo , Infecciones por VIH/inmunología , Interacciones Huésped-Patógeno , Humanos , Memoria Inmunológica , Fosfoproteínas/metabolismo , Proteómica/métodos , Receptores CXCR4/metabolismo , Tropismo Viral , Replicación Viral
5.
Bioinformatics ; 33(21): 3489-3491, 2017 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-28655153

RESUMEN

Summary: Computational characterization of differential kinase activity from phosphoproteomics datasets is critical for correctly inferring cellular circuitry and how signaling cascades are altered in drug treatment and/or disease. Kinase-Substrate Enrichment Analysis (KSEA) offers a powerful approach to estimating changes in a kinase's activity based on the collective phosphorylation changes of its identified substrates. However, KSEA has been limited to programmers who are able to implement the algorithms. Thus, to make it accessible to the larger scientific community, we present a web-based application of this method: the KSEA App. Overall, we expect that this tool will offer a quick and user-friendly way of generating kinase activity estimates from high-throughput phosphoproteomics datasets. Availability and Implementation: the KSEA App is a free online tool: casecpb.shinyapps.io/ksea/. The source code is on GitHub: github.com/casecpb/KSEA/. The application is also available as the R package "KSEAapp" on CRAN: CRAN.R-project.org/package=KSEAapp/. Contact: mark.chance@case.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

6.
Proteomics ; 17(22)2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28961369

RESUMEN

Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS-related signaling are attenuated for A549 compared to H358, while the effects on cell cycle-related kinases are noticeably more prominent in A549. Network-based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteómica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Espectrometría de Masas , Fosforilación , Transducción de Señal
7.
bioRxiv ; 2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38352401

RESUMEN

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

8.
J Clin Virol ; 145: 105020, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34736075

RESUMEN

BACKGROUND: Our institution utilizes an antigen/antibody screening test followed by a confirmatory antibody assay for preliminary positive results. Given the low prevalence for HIV infections in our institution's county, we suspect that a substantial portion of the reactive screens are false positives. OBJECTIVES: We aimed to characterize the false positivity rate of the HIV screening test performed at Stanford Health Care. In parallel, we modified our reporting workflow to release both the screening and confirmatory results simultaneously to mitigate the stress of a presumptive positive test. STUDY DESIGN: We reviewed 45,296 eligible HIV screen specimens that underwent the Abbott ARCHITECT™ 4th generation HIV antigen/antibody combination assay between August 5, 2016 and March 16, 2021. Final sample signal/cutoff (S/CO) ratios ≥ 1 were deemed positive, which triggers a reflex order for the confirmatory Bio-Rad Geenius™ HIV 1/2 Supplemental Assay. Additional chart review was performed for positive screen cases with negative or indeterminate confirmatory results. RESULTS: Our institution demonstrated a 0.28% (128/45,296) positive screen rate, with 12.5% (16/128) of these samples confirmed as false positives based on a negative HIV-1 RNA test. Median S/CO ratios of true positive screens were significantly higher than those with negative or indeterminate confirmatory tests (602.27vs 2.98; p = 0.0000323). We implemented a new synchronized reporting system for positive screens, which co-releases screen and confirmatory reports without compromise in the overall turnaround time. CONCLUSIONS: Our study demonstrates a relatively high percentage of false positive screens. Subsequently, by providing a more complete picture up front, our new reporting pipeline may reduce anxiety of a stand-alone positive screen and optimize downstream clinical decision-making.


Asunto(s)
Infecciones por VIH , VIH-1 , Algoritmos , Anticuerpos Anti-VIH , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , VIH-2 , Humanos , Tamizaje Masivo , Prevalencia , Sensibilidad y Especificidad , Flujo de Trabajo
9.
Oncogene ; 39(3): 703-717, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31541192

RESUMEN

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/ß), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.


Asunto(s)
Resistencia a Antineoplásicos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 2/genética , Proteínas Recombinantes/genética , Sustitución de Aminoácidos , Arginina/genética , Proteínas de Unión a Calmodulina/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Proteínas de la Membrana/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Neoplasias/genética , Proteínas del Tejido Nervioso/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Fosfatasa 2/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Transfección , Tirosina/genética , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Cancer Res ; 78(8): 2065-2080, 2018 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-29358171

RESUMEN

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.


Asunto(s)
Activadores de Enzimas/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Proteína Fosfatasa 2C/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Línea Celular Tumoral , Activadores de Enzimas/farmacología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones SCID , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2C/metabolismo , Proteómica , ARN Mensajero/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología
11.
Methods Mol Biol ; 1666: 539-556, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28980264

RESUMEN

In this chapter, we introduce interaction networks by describing how they are generated, where they are stored, and how they are shared. We focus on publicly available interaction networks and describe a simple way of utilizing these resources. This chapter features two case studies, both of which utilize Cytoscape, an open source and user-friendly network visualization and analysis tool. In the first example, we demonstrate the basic functionalities of Cytoscape by building an interaction network from a publicly available database, analyzing its topological features, and performing gene ontology enrichment. For the second section, we constructed a network from scratch starting with an experimental gene expression dataset. From there, we implement more advanced visual annotations of the network and perform subnetwork enrichment. The methods described are applicable to larger networks that can be collected from various resources.


Asunto(s)
Redes Reguladoras de Genes , Programas Informáticos , Animales , Bases de Datos Genéticas , Ontología de Genes , Genómica/métodos , Humanos , Internet , Mapas de Interacción de Proteínas
13.
J Clin Invest ; 127(6): 2081-2090, 2017 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-28504649

RESUMEN

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.


Asunto(s)
Antineoplásicos/farmacología , Activadores de Enzimas/farmacología , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Activación Enzimática , Activadores de Enzimas/química , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Unión Proteica , Proteína Fosfatasa 2/química , Transducción de Señal , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto
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