RESUMO
Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.
Assuntos
Lesão Pulmonar , Necroptose , Infecções por Orthomyxoviridae , Inibidores de Proteínas Quinases , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Feminino , Humanos , Masculino , Camundongos , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/virologia , Células Epiteliais Alveolares/metabolismo , Vírus da Influenza A/classificação , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Lesão Pulmonar/complicações , Lesão Pulmonar/patologia , Lesão Pulmonar/prevenção & controle , Lesão Pulmonar/virologia , Camundongos Endogâmicos C57BL , Necroptose/efeitos dos fármacos , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Síndrome do Desconforto Respiratório/complicações , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/prevenção & controle , Síndrome do Desconforto Respiratório/virologiaRESUMO
BACKGROUND: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. METHODS: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. RESULTS: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. CONCLUSIONS: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Predisposição Genética para Doença , Replicação do DNA , Mutação em Linhagem Germinativa , Células GerminativasRESUMO
BACKGROUND: Alpha 1 Antitrypsin (AAT) is a key serum proteinase inhibitor encoded by SERPINA1. Sequence variants of the gene can cause Alpha 1 Antitrypsin Deficiency (AATD), a condition associated with lung and liver disease. The majority of AATD cases are caused by the 'Z' and 'S' variants - single-nucleotide variations (SNVs) that result in amino acid substitutions of E342K and E264V. However, SERPINA1 is highly polymorphic, with numerous potentially clinically relevant variants reported. Novel variants continue to be discovered, and without reports of pathogenicity, it can be difficult for clinicians to determine the best course of treatment. METHODS: We assessed the utility of next-generation sequencing (NGS) and predictive computational analysis to guide the diagnosis of patients suspected of having AATD. Blood samples on serum separator cards were submitted to the DNA1 Advanced Screening Program (Biocerna LLC, Fulton, Maryland, USA) by physicians whose patients were suspected of having AATD. Laboratory analyses included quantification of serum AAT levels, qualitative analysis by isoelectric focusing, and targeted genotyping and NGS of the SERPINA1 gene. Molecular modeling software UCSF Chimera (University College of San Francisco, CA) was used to visualize the positions of amino acid changes as a result of rare/novel SNVs. Predictive software was used to assess the potential pathogenicity of these variants; methods included a support vector machine (SVM) program, PolyPhen-2 (Harvard University, Cambridge, MA), and FoldX (Centre for Genomic Regulation, Barcelona, Spain). RESULTS: Samples from 23 patients were analyzed; 21 rare/novel sequence variants were identified by NGS, including splice variants (n = 2), base pair deletions (n = 1), stop codon insertions (n = 2), and SNVs (n = 16). Computational modeling of protein structures caused by the novel SNVs showed that 8 were probably deleterious, and two were possibly deleterious. For the majority of probably/possibly deleterious SNVs (I50N, P289S, M385T, M221T, D341V, V210E, P369H, V333M and A142D), the mechanism is probably via disruption of the packed hydrophobic core of AAT. Several deleterious variants occurred in combination with more common deficiency alleles, resulting in very low AAT levels. CONCLUSIONS: NGS and computational modeling are useful tools that can facilitate earlier, more precise diagnosis, and consideration for AAT therapy in AATD.
Assuntos
Variação Genética , Modelos Moleculares , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Pennsylvania , Conformação Proteica em alfa-Hélice , Splicing de RNA , Análise de Sequência de Proteína , Virulência/genética , alfa 1-Antitripsina/sangue , Deficiência de alfa 1-Antitripsina/diagnósticoRESUMO
Mutations in the cystathionine ß-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The p.G307S mutation is the most frequent cause of CBS deficiency in Ireland, which has the highest prevalence of CBS deficiency in Europe. Individuals homozygous for this mutation tend to be severely affected and are pyridoxine nonresponsive, but the molecular basis for the strong effects of this mutation is unclear. Here, we characterized a transgenic mouse model lacking endogenous Cbs and expressing human p.G307S CBS protein from a zinc-inducible metallothionein promoter (Tg-G307S Cbs-/-). Unlike mice expressing other mutant CBS alleles, the Tg-G307S transgene could not efficiently rescue neonatal lethality of Cbs-/- in a C57BL/6J background. In a C3H/HeJ background, zinc-induced Tg-G307S Cbs-/- mice expressed high levels of p.G307S CBS in the liver, and this protein variant forms multimers, similarly to mice expressing WT human CBS. However, the p.G307S enzyme had no detectable residual activity. Moreover, treating mice with proteasome inhibitors failed to significantly increase CBS-specific activity. These findings indicated that the G307S substitution likely affects catalytic function as opposed to causing a folding defect. Using molecular dynamics simulation techniques, we found that the G307S substitution likely impairs catalytic function by limiting the ability of the tyrosine at position 308 to assume the proper conformational state(s) required for the formation of the pyridoxal-cystathionine intermediate. These results indicate that the p.G307S CBS is stable but enzymatically inert and therefore unlikely to respond to chaperone-based therapy.
Assuntos
Cistationina beta-Sintase/genética , Mutação , Substituição de Aminoácidos , Animais , Catálise , Cistationina beta-Sintase/química , Cistationina beta-Sintase/metabolismo , Homocistinúria/genética , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Proteassoma/farmacologia , Conformação Proteica , Estabilidade Proteica , Piridoxina/farmacologiaRESUMO
Retroviral integrases are reported to form alternate dimer assemblies like the core-core dimer and reaching dimer. The core-core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit's core and C-terminal domains (CTD), as well as CTD-CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16-18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core-core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core-core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer < HIV reaching dimer < composite core-core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.
Assuntos
Vírus do Sarcoma Aviário/química , Integrase de HIV/química , HIV-1/química , Simulação de Dinâmica Molecular , Multimerização Proteica , Motivos de Aminoácidos , Vírus do Sarcoma Aviário/enzimologia , Domínio Catalítico , Cristalografia por Raios X , HIV-1/enzimologia , Cinética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , TermodinâmicaRESUMO
In CASP11, the organizers sought to bring the biological inferences from predicted structures to the fore. To accomplish this, we assessed the models for their ability to perform quantifiable tasks related to biological function. First, for 10 targets that were probable homodimers, we measured the accuracy of docking the models into homodimers as a function of GDT-TS of the monomers, which produced characteristic L-shaped plots. At low GDT-TS, none of the models could be docked correctly as homodimers. Above GDT-TS of â¼60%, some models formed correct homodimers in one of the largest docked clusters, while many other models at the same values of GDT-TS did not. Docking was more successful when many of the templates shared the same homodimer. Second, we docked a ligand from an experimental structure into each of the models of one of the targets. Docking to the models with two different programs produced poor ligand RMSDs with the experimental structure. Measures that evaluated similarity of contacts were reasonable for some of the models, although there was not a significant correlation with model accuracy. Finally, we assessed whether models would be useful in predicting the phenotypes of missense mutations in three human targets by comparing features calculated from the models with those calculated from the experimental structures. The models were successful in reproducing accessible surface areas but there was little correlation of model accuracy with calculation of FoldX evaluation of the change in free energy between the wild-type and the mutant. Proteins 2016; 84(Suppl 1):370-391. © 2016 Wiley Periodicals, Inc.
Assuntos
Amidoidrolases/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteína gp120 do Envelope de HIV/química , Fator de Crescimento de Hepatócito/química , Modelos Estatísticos , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Sítios de Ligação , Biologia Computacional/métodos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Ligantes , Mutação de Sentido Incorreto , Fenótipo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Collaborations between the Wlodawer and Skalka laboratories have covered a period of almost 30 years. During that time our groups have co-authored 18 publications, including several much cited journal articles, book chapters, and scholarly reviews. It has therefore been most rewarding for us to share enthusiasm, insights, and expertise with our Frederick colleagues over the years, and also to enjoy lasting friendships.
Assuntos
Bioquímica/história , Cristalografia/história , Proteínas dos Retroviridae/química , Retroviridae/enzimologia , Cristalografia/métodos , História do Século XX , História do Século XXI , Conformação Proteica , Proteínas dos Retroviridae/metabolismo , Estados UnidosRESUMO
Risk assessment for prostate cancer is challenging due to its genetic heterogeneity. In this study, our goal was to develop an operational framework to select and evaluate gene variants that may contribute to familial prostate cancer risk. Drawing on orthogonal sources, we developed a candidate list of genes relevant to prostate cancer, then analyzed germline exomes from 12 case-only prostate cancer patients from high-risk families to identify patterns of protein-damaging gene variants. We described an average of 5 potentially disruptive variants in each individual and annotated them in the context of public databases representing human variation. Novel damaging variants were found in several genes of relevance to prostate cancer. Almost all patients had variants associated with defects in DNA damage response. Many also had variants linked to androgen signaling. Treatment of primary T-lymphocytes from these prostate cancer patients versus controls with DNA damaging agents showed elevated levels of the DNA double strand break (DSB) marker γH2AX (p < 0.05), supporting the idea of an underlying defect in DNA repair. This work suggests the value of focusing on underlying defects in DNA damage in familial prostate cancer risk assessment and demonstrates an operational framework for exome sequencing in case-only prostate cancer genetic evaluation.
Assuntos
Reparo do DNA/genética , Predisposição Genética para Doença/genética , Mutação , Neoplasias da Próstata/genética , Adulto , Idoso , Antineoplásicos Fitogênicos/farmacologia , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Etoposídeo/farmacologia , Exoma/genética , Saúde da Família , Histonas/metabolismo , Humanos , Mutação INDEL , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/patologia , Medição de Risco , Fatores de Risco , Análise de Sequência de DNA , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla , Variação Genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Bases de Dados Genéticas , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exoma , Feminino , Frequência do Gene , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Instabilidade Genômica , Células HCT116 , Hereditariedade , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênicos/farmacologia , Fenótipo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , RecQ Helicases/genética , RecQ Helicases/metabolismo , Análise de Sequência de DNA , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção , Regulação para Cima , Helicase da Síndrome de WernerRESUMO
BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) before cystectomy is the standard of care for muscle-invasive bladder cancer (MIBC), with 25-50% of patients expected to achieve a pathologic response. Validated biomarkers predictive of response are currently lacking. OBJECTIVE: To discover and validate biomarkers predictive of response to NAC for MIBC. DESIGN, SETTING, AND PARTICIPANTS: Pretreatment MIBC samples prospectively collected from patients treated in two separate clinical trials of cisplatin-based NAC provided the discovery and validation sets. DNA from pretreatment tumor tissue was sequenced for all coding exons of 287 cancer-related genes and was analyzed for base substitutions, indels, copy number alterations, and selected rearrangements in a Clinical Laboratory Improvements Amendments-certified laboratory. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The mean number of variants and variant status for each gene were correlated with response. Variant data from the discovery cohort were used to create a classification tree to discriminate responders from nonresponders. The resulting decision rule was then tested in the independent validation set. RESULTS AND LIMITATIONS: Patients with a pathologic complete response had more alterations than those with residual tumor in both the discovery (p=0.024) and validation (p=0.018) sets. In the discovery set, alteration in one or more of the three DNA repair genes ATM, RB1, and FANCC predicted pathologic response (p<0.001; 87% sensitivity, 100% specificity) and better overall survival (p=0.007). This test remained predictive for pathologic response in the validation set (p=0.033), with a trend towards better overall survival (p=0.055). These results require further validation in additional sample sets. CONCLUSIONS: Genomic alterations in the DNA repair-associated genes ATM, RB1, and FANCC predict response and clinical benefit after cisplatin-based chemotherapy for MIBC. The results suggest that defective DNA repair renders tumors sensitive to cisplatin. PATIENT SUMMARY: Chemotherapy given before bladder removal (cystectomy) improves the chance of cure for some but not all patients with muscle-invasive bladder cancer. We found a set of genetic mutations that when present in tumor tissue predict benefit from neoadjuvant chemotherapy, suggesting that testing before chemotherapy may help in selecting patients for whom this approach is recommended.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Reparo do DNA , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica , Prognóstico , Estudos Prospectivos , Neoplasias da Bexiga Urinária/patologiaRESUMO
The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell's DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus. We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions.
Assuntos
Integrases/metabolismo , Infecções por Retroviridae/virologia , Retroviridae/enzimologia , Proteínas Virais/metabolismo , Animais , Humanos , Integrases/química , Integrases/genética , Modelos Moleculares , Retroviridae/genética , Retroviridae/fisiologia , Proteínas Virais/química , Proteínas Virais/genética , Integração ViralRESUMO
STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.
Assuntos
Canais de Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Moléculas de Adesão Celular/química , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteína ORAI1 , Homologia de Sequência de Aminoácidos , Molécula 1 de Interação Estromal , Molécula 2 de Interação EstromalRESUMO
The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis.
Assuntos
Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Heterocromatina/química , Humanos , Interfase , Microscopia Confocal , Mitose , Lâmina Nuclear/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.
Assuntos
Heparitina Sulfato/metabolismo , Células Matadoras Naturais/imunologia , Receptores KIR2DL4/metabolismo , Sulfotransferases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Linhagem Celular , Cricetulus , Endocitose , Células HEK293 , Heparina/metabolismo , Humanos , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Receptores KIR2DL4/genética , Receptores KIR2DL4/imunologia , Transdução de Sinais/imunologia , Sindecana-4/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.
Assuntos
DNA/metabolismo , Integrase de HIV/química , Integrase de HIV/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Substituição de Aminoácidos , Biocatálise/efeitos dos fármacos , Dicroísmo Circular , Reagentes de Ligações Cruzadas/química , Ácido Edético/química , Ácido Edético/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Integrase de HIV/genética , Modelos Moleculares , Mutação , Ligação Proteica , Espalhamento a Baixo Ângulo , Cloreto de Sódio/química , Cloreto de Sódio/farmacologia , Especificidade por Substrato , Ureia/química , Ureia/farmacologia , Difração de Raios XRESUMO
The stress-induced HSP70 is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in nontransformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here, we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70 and requires the C-terminal helical "lid" of this protein (amino acids 573-616) to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure-activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B-cell lymphoma, compared with the parent compound (P = 0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the anaphase promoting complex/cyclosome (APC/C) in cell-free extracts, and induce G2-M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anticancer compound with several notable mechanisms of action.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Choque Térmico HSP72/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Sulfonamidas/administração & dosagem , Animais , Simulação por Computador , Regulação Leucêmica da Expressão Gênica , Instabilidade Genômica/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.
Assuntos
Vírus do Sarcoma Aviário/enzimologia , Pareamento de Bases , DNA Viral/química , Integrase de HIV/metabolismo , HIV-1/enzimologia , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Sequência de Bases , Domínio Catalítico , Coenzimas/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , HIV-1/genética , HIV-1/metabolismo , Metais/metabolismo , Reprodutibilidade dos TestesRESUMO
We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.
Assuntos
Integrases/química , Retroviridae/enzimologia , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Bases de Dados de Proteínas , Dimerização , Integrase de HIV/química , Luz , Espectrometria de Massas/métodos , Conformação Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Espalhamento a Baixo Ângulo , Raios XRESUMO
BACKGROUND: HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag. RESULTS: A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg++- and Mn++-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn++ than Mg++. Although the pH optima for these two enzymes are similar with Mn++, they differ significantly in the presence of Mg++, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn++. CONCLUSION: A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10-30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.