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The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aß42 and Aß40 across all approaches. However, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.
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The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase ß (Polß), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polß. Required for Polß foci formation, TRIP12 regulates Polß involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Polß affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Polß-directed BER, optimizes DNA repair pathway choice at complex lesion sites.
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INTRODUCTION: The reliability of plasma Alzheimer's disease (AD) biomarkers can be compromised by protease-induced degradation. This limits the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). This study conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. METHODS: We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 hours. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0h or 24h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA or P100 tubes, followed by storage at RT for 0h or 24h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. RESULTS: Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aß42 and Aß40 across all approaches. Additionally, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. CONCLUSION: Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings.
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BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. METHODS: Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for in vitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth in vivo. Mouse PDAC cells derived from the KrasLSL-G12D/+;Tp53L/+;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis in vivo. RESULTS: High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration in vitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin ß4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin ß4 protein, which may have accounted for the suppression of metastasis. CONCLUSIONS: Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Tirosina , Integrina beta4/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Transportadores de Sulfato , Proteínas de Membrana/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.
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Aldeído Oxirredutases/antagonistas & inibidores , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Tretinoína/metabolismo , HumanosRESUMO
Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.
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Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.
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Proteínas do Capsídeo/metabolismo , Chaperonina com TCP-1/metabolismo , Chaperonas Moleculares/metabolismo , Reoviridae/metabolismo , Proteínas do Capsídeo/química , Chaperonina com TCP-1/química , Microscopia Crioeletrônica , Espectrometria de Massas , Chaperonas Moleculares/química , Conformação Proteica , Dobramento de Proteína , ProteostaseRESUMO
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV+, 11 MCV-) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV large T antigen, CDKN2AIP, SERPINB5, and TRIM29) that discriminate MCV+ and MCV- MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be upregulated in virus-infected samples. Remarkably, 4 (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify potentially novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
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Antígenos Virais de Tumores/metabolismo , Biomarcadores/metabolismo , Carcinoma de Célula de Merkel/diagnóstico , Infecções por Polyomavirus/complicações , Proteoma/metabolismo , Neoplasias Cutâneas/diagnóstico , Infecções Tumorais por Vírus/complicações , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/virologia , Formaldeído/química , Humanos , Poliomavírus das Células de Merkel/fisiologia , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Proteoma/análise , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologiaRESUMO
Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.
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Degradação Associada com o Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cromatografia Líquida , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Ergosterol/biossíntese , Ergosterol/metabolismo , Leupeptinas/farmacologia , Oxigenases de Função Mista/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Proteômica , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Massas em Tandem , UbiquitinaçãoRESUMO
Since many anticancer therapies target DNA and DNA damage response pathways, biomarkers of DNA damage endpoints may prove valuable in basic and clinical cancer research. Ataxia telangiectasia-mutated (ATM) kinase is the principal regulator of cellular responses to DNA double-strand breaks (DSBs). In humans, ATM autophosphorylation at serine 1981 (p-S1981) is an immediate molecular response to nascent DSBs and ionizing radiation (IR). Here we describe the analytical characteristics and fit-for-purpose validation of a quantitative dual-labeled immunoblot that simultaneously measures p-S1981-ATM and pan-ATM in human peripheral blood mononuclear cells (PBMCs) following ex vivo exposure to 2â¯Gy IR, facilitating the calculation of %p-ATM. To validate our assay, we isolated PBMCs from 41 volunteers. We report that the median basal level of p-S1981-ATM and pan-ATM was 2.4 and 49.5â¯ng/107 PBMCs, respectively, resulting in %p-ATM of 4%. Following exposure of PBMCs to 2â¯Gy IR, p-S1981-ATM levels increased 12-fold to 29.8â¯ng/107 PBMCs resulting in %p-ATM of 63%. Interestingly, we show that PBMCs from women have a 2.6-fold greater median p-S1981-ATM level following IR exposure than men (44.4 versus 16.9â¯ng/107 cells; p < 0.01). This results in a significantly greater %p-ATM for women (68% versus 49%; pâ¯<⯠0.01). Our rigorous description of the analytical characteristics and reproducibility of phosphoprotein immunoblotting, along with our finding that the ATM DNA damage response is greater in women, has far reaching implications for biomedical researchers.
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Proteínas Mutadas de Ataxia Telangiectasia/análise , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Immunoblotting/métodos , Linfócitos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Mutadas de Ataxia Telangiectasia/química , DNA/metabolismo , DNA/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Linfócitos/efeitos da radiação , Masculino , Fosforilação , Radiação Ionizante , Reprodutibilidade dos TestesRESUMO
Aging is an ill-defined process that increases the risk of morbidity and mortality. Aging is also heterogeneous meaning that biological and chronological age can differ. Here, we used unbiased differential mass spectrometry to quantify thousands of proteins in mouse liver and select those that that consistently change in expression as mice age. A panel of 14 proteins from inbred C57BL/6 mice was used to equate chronological and biological age in this reference population, against which other mice could be compared. This "biological age calculator" identified two strains of f1 hybrid mice as biologically younger than inbred mice and progeroid mice as being biologically older. In an independent validation experiment, the calculator identified mice treated with rapamycin, known to extend lifespan of mice, as 18% younger than mice fed a placebo diet. This demonstrates that it is possible to measure subtle changes in biologic age in mammals using a proteomics approach.
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Envelhecimento/metabolismo , Fígado/metabolismo , Proteínas/metabolismo , Animais , Feminino , Masculino , Espectrometria de Massas , Camundongos , Valores de ReferênciaRESUMO
The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.
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Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Junções Aderentes/metabolismo , Caderinas/genética , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actomiosina/metabolismo , Junções Aderentes/ultraestrutura , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Regulação da Expressão Gênica , Ontologia Genética , Camundongos , Anotação de Sequência Molecular , Miócitos Cardíacos/ultraestrutura , Cultura Primária de Células , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteômica/métodosRESUMO
OBJECTIVE: The presence of psychosis in Alzheimer's disease denotes a phenotype with more rapid cognitive deterioration than in Alzheimer's disease without psychosis. Discovery of novel pharmacotherapies that engage therapeutic targets for prevention or treatment of Alzheimer's disease with psychosis would benefit from identifying the neurobiology of resilience to psychosis in Alzheimer's disease. The primary objective of this study was to determine whether alterations in the synaptic proteome were associated with resilience to psychotic symptoms in Alzheimer's disease and, if present, were independent of neuropathologic burden. METHOD: Quantitative immunohistochemistry was used to measure multiple neuropathologies in dorsolateral prefrontal cortex from subjects with early and middle-stage Alzheimer's disease who differed in psychosis status. Synaptic proteins were quantified by liquid chromatography-mass spectrometry in gray matter homogenates from these subjects and from neuropathologically unaffected subjects. The synaptic proteome was similarly evaluated in cortical gray matter homogenate and in postsynaptic density fractions from an APPswe/PSEN1dE9 mouse model of amyloidosis with germline reduction in Kalrn, which has been shown to confer resilience to progression of psychosis-associated behaviors relative to APPswe/PSEN1dE9 alone. RESULTS: Subjects resilient to psychotic symptoms in Alzheimer's disease had higher levels of synaptic proteins compared with those with psychosis and unaffected control subjects. Neuropathologic burden predicted less than 20% of the variance in psychosis status and did not account for the synaptic protein level differences between groups. Reduction of Kalrn in APPswe/PSEN1dE9 mice resulted in higher levels of synaptic proteins in cortical homogenate and normalized protein levels in the postsynaptic density. CONCLUSIONS: Accumulation of synaptic proteins, particularly those that are enriched in the postsynaptic density, is associated with resilience to psychosis in Alzheimer's disease. One candidate mechanism for this synaptic proteome compensation is alteration in levels of proteins that facilitate the transport of synaptic proteins to and from the postsynaptic density.
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Doença de Alzheimer/psicologia , Proteínas do Tecido Nervoso/metabolismo , Proteoma , Transtornos Psicóticos/psicologia , Sinapses/metabolismo , Idoso de 80 Anos ou mais , Doença de Alzheimer/complicações , Doença de Alzheimer/metabolismo , Cromatografia Líquida , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Córtex Pré-Frontal/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transtornos Psicóticos/etiologia , Transtornos Psicóticos/metabolismoRESUMO
Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.
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Envelhecimento/genética , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mitocôndrias/genética , Animais , Antioxidantes/metabolismo , Senescência Celular/fisiologia , Óxidos N-Cíclicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.
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Apolipoproteína C-III/genética , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto , Triglicerídeos/metabolismo , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Apolipoproteína C-III/efeitos dos fármacos , Apolipoproteínas B/metabolismo , HDL-Colesterol/metabolismo , Cromatografia Líquida , Simulação por Computador , Doença das Coronárias/genética , Estudos Transversais , Feminino , Humanos , Immunoblotting , Metabolismo dos Lipídeos/genética , Lipoproteínas/efeitos dos fármacos , Lipoproteínas VLDL/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Fatores de Proteção , Espectrometria de Massas em TandemRESUMO
Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1.
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Dano ao DNA , Quinases Relacionadas a NIMA/metabolismo , Estresse Oxidativo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Quinases Relacionadas a NIMA/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Interferência de RNA , Complexo Shelterina , Telômero/genética , Telômero/efeitos da radiação , Proteínas de Ligação a Telômeros/genética , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with homologous to the E6-AP Cterminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758-1068) results in loss of UBE3B's ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29-58) results in loss of calmodulin binding and a significant increase in the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.
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Calmodulina/metabolismo , Mitocôndrias/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
It has been hypothesized that Alzheimer disease (AD) is primarily a disorder of the synapse. However, assessment of the synaptic proteome in AD subjects has been limited to a small number of proteins and often included subjects with end-stage pathology. Protein from prefrontal cortex gray matter of 59 AD subjects with mild to moderate dementia and 12 normal elderly subjects was assayed using targeted mass spectrometry to quantify 191 synaptically expressed proteins. The profile of synaptic protein expression clustered AD subjects into two groups. One of these was characterized by reduced expression of glutamate receptor proteins, significantly increased synaptic protein network coexpression, and associated withApolipoprotein E*4 (APOE*4) carrier status. The second group, by contrast, showed few differences from control subjects. A subset of AD subjects had altered prefrontal cortex synaptic proteostasis for glutamate receptors and their signaling partners. Efforts to therapeutically target glutamate receptors in AD may have outcomes dependent on APOE*4 genotype.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteína E4/genética , Ácido Glutâmico/metabolismo , Córtex Pré-Frontal/metabolismo , Sinapses/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Regulação para Baixo , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica/métodos , Receptores de Glutamato/metabolismo , Transdução de SinaisRESUMO
Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc-/- (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc-/- livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.
Assuntos
Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , Regeneração Hepática , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proliferação de Células , Tamanho Celular , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Hepatócitos/citologia , Hepatócitos/transplante , Fígado/citologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Triglicerídeos/metabolismoRESUMO
Recent studies have implicated the neuronal calcium-sensing protein visinin-like 1 protein (Vilip-1) as a peripheral biomarker in Alzheimer disease (AD), but little is known about expression of Vilip-1 in the brains of patients with AD. We used targeted and quantitative mass spectrometry to measure Vilip-1 peptide levels in the entorhinal cortex (ERC) and the superior frontal gyrus (SF) from cases with early to moderate stage AD, frontotemporal lobar degeneration (FTLD), and cognitively and neuropathologically normal elderly controls. We found that Vilip-1 levels were significantly lower in the ERC, but not in SF, of AD subjects compared to normal controls. In FTLD cases, Vilip-1 levels in the SF were significantly lower than in normal controls. These findings suggest a unique role for cerebrospinal fluid Vilip-1 as a biomarker of ERC neuron loss in AD.