RESUMEN
Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability. SIGNIFICANCE STATEMENT: Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.
Asunto(s)
Citocromo P-450 CYP3A , Hepatocitos , Células Cultivadas , Cromatografía Liquida/métodos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Hepatocitos/metabolismo , Humanos , Espectrometría de Masas , ARN Mensajero/metabolismoRESUMEN
The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109-110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Citomegalovirus/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Virales/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc4 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Citomegalovirus/genética , Células HEK293 , Células HeLa , Humanos , Fosforilación , Unión Proteica , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Virales/genéticaRESUMEN
Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.
Asunto(s)
Infecciones por Citomegalovirus/genética , Citomegalovirus/patogenicidad , Proteínas Inmediatas-Precoces/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , ARN Viral/genética , Proteínas Virales/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Inmunoprecipitación , Luciferasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/antagonistas & inhibidores , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genética , Replicación ViralRESUMEN
Tau protein is a key target of interest in developing therapeutics for neurodegenerative diseases. Here, we sought to develop a method that quantifies extracellular tau protein concentrations in human cerebrospinal fluid (CSF) without antibody-based enrichment strategies. We demonstrate that the fit-for-purpose validated method in Alzheimer's Disease CSF is limited to quasi quantitative measures of tau surrogate peptides. We also provide evidence that CSF total Tau measures by LC-MS are feasible in the presence of monoclonal therapeutic antibodies in human CSF. Our Tau LC-MS/MS method is a translational bioanalytical tool for assaying target engagement and pharmacodynamics for anti-tau antibody drug development campaigns.
Asunto(s)
Enfermedad de Alzheimer , Proteínas tau , Enfermedad de Alzheimer/líquido cefalorraquídeo , Anticuerpos Monoclonales , Cromatografía Liquida/métodos , Humanos , Espectrometría de Masas en Tándem/métodos , Proteínas tau/líquido cefalorraquídeoRESUMEN
Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T cells. We also show MS(1) evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS(2) fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry. This article is part of a Special Issue entitled: Protein Species. BIOLOGICAL SIGNIFICANCE: Characterizing transcription factor proteoforms in human cells is of high value to the field of molecular biology; many agree that post-translational modifications and combinations thereof play a critical role in modulating transcription factor activity. Thus, measuring these modifications promises increased understanding of molecular mechanisms governing the regulation of complex gene expression outcomes. To date, comprehensive characterization of transcription factor proteoforms within human cells has eluded measurement, owing primarily-with regard to top down mass spectrometry-to large protein size and low relative abundance. Here, we utilized HaloTag technology and recombinant protein expression to overcome these limitations and show top down mass spectrometry characterization of proteoforms of the 60kDa NF-kB protein, p65. By optimizing the analytical procedure (i.e. purification, MS(1), and MS(2)), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci.
Asunto(s)
Espectrometría de Masas , Multimerización de Proteína , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismo , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Infection with the ß-herpesvirus human cytomegalovirus (HCMV) is lifelong, causing limited disease in healthy adults, but life threatening in immunocompromised individuals. The viral kinase pUL97, a functional ortholog of cellular cyclin-dependent kinases (CDKs), is critical for HCMV replication and a target for antiviral drug development. Upon kinase inhibition, drug-resistant strains emerge with mutations in UL27, an HCMV gene of unknown function. Using a proteomics approach, we discovered that pUL27 is necessary and sufficient to degrade Tip60, a host acetyltransferase and interacting partner of HIV Tat. Consistent with this, the expression of Tat restored antiviral inhibition of an otherwise resistant HCMV strain. The functional consequence of Tip60 degradation was the induction of the CDK inhibitor p21(Waf1/Cip1) and cell-cycle arrest, representing changes necessary for the antiviral effects of pUL97 inhibition. Consequently, either increasing p21(Waf1/Cip1) expression or decreasing Tip60 levels improved the antiviral activity of the HCMV kinase inhibitor maribavir.