RESUMO
Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.
Assuntos
Proteínas Sanguíneas/genética , Genômica , Proteoma/genética , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Masculino , Mutação de Sentido Incorreto/genética , Mieloblastina/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Proteínas Proto-Oncogênicas/genética , Locos de Características Quantitativas/genética , Vasculite/genética , alfa 1-Antitripsina/genéticaRESUMO
Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.
Assuntos
Aminoácidos/química , Imunoconjugados/química , Linhagem Celular , Sistema Livre de Células , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Humanos , Imunoconjugados/farmacologia , Espectrometria de Massas em TandemRESUMO
Protein engineering projects often amass numerous raw DNA sequences, but no readily available software combines sequence processing and activity correlation required for efficient lead identification. XLibraryDisplay is an open source program integrated into Microsoft Excel for Windows that automates batch sequence processing via a simple step-by-step, menu-driven graphical user interface. XLibraryDisplay accepts any DNA template which is used as a basis for trimming, filtering, translating, and aligning hundreds to thousands of sequences (raw, FASTA, or Phred PHD file formats). Key steps for library characterization through lead discovery are available including library composition analysis, filtering by experimental data, graphing and correlating to experimental data, alignment to structural data extracted from PDB files, and generation of PyMOL visualization scripts. Though larger data sets can be handled, the program is best suited for analyzing approximately 10 000 or fewer leads or naïve clones which have been characterized using Sanger sequencing and other experimental approaches. XLibraryDisplay can be downloaded for free from sourceforge.net/projects/xlibrarydisplay/ .
Assuntos
Engenharia de Proteínas/instrumentação , Análise de Sequência de DNA/métodos , Interface Usuário-Computador , Sequência de Bases , Processamento Eletrônico de Dados , Biblioteca Gênica , Humanos , Internet , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Alinhamento de Sequência , Análise de Sequência de DNA/estatística & dados numéricosRESUMO
The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.
Assuntos
Translocador 1 do Nucleotídeo Adenina/fisiologia , Apoptose/fisiologia , Ciclo Celular/fisiologia , Fase G2/fisiologia , Produtos do Gene vpr/fisiologia , Proteína X Associada a bcl-2/fisiologia , Translocador 1 do Nucleotídeo Adenina/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos T CD4-Positivos/citologia , Caspases/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Regulação para Baixo , Regulação Viral da Expressão Gênica , Produtos do Gene vpr/genética , HIV-1/genética , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutagênicos/farmacologia , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteína X Associada a bcl-2/genética , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.
Assuntos
Produtos do Gene vpr/fisiologia , HIV-1/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Fase G2 , Produtos do Gene vpr/isolamento & purificação , HIV-1/efeitos dos fármacos , Células HeLa/citologia , Células HeLa/fisiologia , Células HeLa/virologia , Humanos , Rim , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/genética , RNA Viral/genética , Transfecção , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Eukaryotic cells have evolved a complex mechanism for sensing DNA damage during genome replication. Activation of this pathway prevents entry into mitosis to allow for either DNA repair or, in the event of irreparable damage, commitment to apoptosis. Under conditions of replication stress, the damage signal is initiated by the ataxia-telangiectasia-mutated and Rad3-related kinase ATR. We recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) gene product viral protein R (Vpr) arrests infected cells in the G(2) phase via the activation of ATR. In the present study, we show that the activation of ATR by Vpr is analogous to activation by certain genotoxic agents, both mechanistically and in its downstream consequences. Specifically, we show a requirement for Rad17 and Hus1 to induce G(2) arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1. These results demonstrate that G(2) arrest mediated by the HIV-1 gene product Vpr utilizes the cellular signaling pathway whose physiological function is to recognize replication stress. These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4(+)-lymphocyte cell cycle and apoptosis induction in the progressive CD4(+)-lymphocyte depletion characteristic of HIV-1 pathogenesis.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fase G2 , Produtos do Gene vpr/metabolismo , HIV-1/fisiologia , Histonas/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Antígenos CD4/metabolismo , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Produtos do Gene vpr/genética , Histonas/genética , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas de Schizosaccharomyces pombe , Timo/metabolismo , Timo/patologia , Proteínas Supressoras de Tumor , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Cullin-RING ligases (CRLs) compose the largest class of E3 ubiquitin ligases. CRLs are modular, multisubunit enzymes, comprising interchangeable substrate receptors dedicated to particular Cullin-RING catalytic cores. Recent structural studies have revealed numerous ways in which CRL E3 ligase activities are controlled, including multimodal E3 ligase activation by covalent attachment of the ubiquitin-like protein NEDD8, inhibition of CRL assembly/activity by CAND1, and several mechanisms of regulated substrate recruitment. These features highlight the potential for CRL activities to be tuned in responses to diverse cellular cues, and for modulating CRL functions through small-molecule agonists or antagonists. As the second installment of a two-review series, this article focuses on recent structural studies advancing our knowledge of how CRL activities are regulated.
Assuntos
Proteínas Culina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Simulação por Computador , Proteínas Culina/metabolismo , Inibidores Enzimáticos/metabolismo , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Ubiquitinas/metabolismoRESUMO
The cullin-RING ubiquitin ligases (CRLs) are the largest family of multi-subunit E3 ligases in eukaryotes, which ubiquitinate protein substrates in numerous cellular pathways. CRLs share a common arched scaffold and a RING domain catalytic subunit, but use different adaptors and substrate receptors to assemble unique E3 machineries. In comparison to the first CRL structure, recent findings have revealed increased complexity in the overall architecture and assembly mode of CRLs, including multi-domain organization, inter-domain flexibility, and subunit dimerization. These features highlight the capacity of CRLs to catalyze protein ubiquitination under distinct cellular contexts and in response to diverse signals. As the first installment of a two-review series, this article will focus on recent advances in our understanding of CRL assembly mechanisms.
Assuntos
Ubiquitina-Proteína Ligases/química , Animais , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Humanos , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically, induction of G2 arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G2 arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35 of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding to Vpr is dispensable for the ability of Vpr to induce G2 arrest. Vpr expression in CypA-/- cells leads to induction of G2 arrest in a manner that is indistinguishable from that in CypA+ cells. CsA abolished CypA-Vpr binding but had no effect on induction of G2 arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr.
Assuntos
Ciclofilina A/fisiologia , Fase G2 , Produtos do Gene vpr/fisiologia , Linhagem Celular , Ciclofilina A/antagonistas & inibidores , Humanos , FenótipoRESUMO
The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.
Assuntos
Replicação do DNA , Produtos do Gene vpr/fisiologia , HIV-1/patogenicidade , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Células Cultivadas , Efeito Citopatogênico Viral , DNA Viral/biossíntese , DNA Viral/genética , Fase G2 , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Interferência de RNA , Transdução de Sinais , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of gamma-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/efeitos dos fármacos , Produtos do Gene vpr/farmacologia , HIV-1/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Produtos do Gene vpr/genética , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/genética , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Integration into the host cell DNA is an essential part of the retroviral life cycle and is required for the productive replication of a retrovirus. Retroviral integration involves cleavage of the host DNA and insertion of the viral DNA, forming an integration intermediate that contains two gaps, each with a viral 5' flap. The flaps are then removed, and the gap is filled by as yet unidentified nuclease and polymerase activities. It is thought that repair of these gaps flanking the site of retroviral integration is achieved by host DNA repair machinery. The ATM and Rad3-related protein (ATR) is a member of the phosphatidylinositol 3 kinase-related family of protein kinases that play a major role in sensing and triggering repair of DNA lesions in mammalian cells. In an effort to examine the role of ATR in retroviral integration, we used RNA interference to selectively downregulate ATR and measured integration efficiency. In addition, we examined the possible role that Vpr may play in enhancing integration and, in particular, whether activation of ATR by Vpr (Roshal et al., J. Biol. Chem. 278:25879-25886, 2003) will favor human immunodeficiency virus type 1 integration. We conclude that cells in which ATR has been depleted are competent for retroviral integration. We also conclude that the presence of Vpr as a virion-bound protein does not enhance integration of a lentivirus vector in dividing cells.