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1.
J Neurochem ; 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38814273

RESUMO

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.

2.
bioRxiv ; 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38645048

RESUMO

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.

3.
medRxiv ; 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38496591

RESUMO

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.

4.
Cell Mol Gastroenterol Hepatol ; 16(3): 473-495, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37192689

RESUMO

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/metabolismo
5.
Biomolecules ; 13(1)2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36671485

RESUMO

The "block and lock" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The "block" refers to a compound's ability to inhibit latent HIV-1 proviral transcription, while the "lock" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.


Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/metabolismo , Quinases Ativadas por p21/metabolismo , Latência Viral , Leucócitos Mononucleares/metabolismo
6.
Physiol Rep ; 10(15): e15415, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35924321

RESUMO

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.


Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Dieta Hiperlipídica , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Oxirredução , Complexo Piruvato Desidrogenase/metabolismo , Piruvatos , Volume Sistólico
8.
Commun Biol ; 4(1): 1420, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34934174

RESUMO

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.


Assuntos
Aldeído Oxirredutases/antagonistas & inibidores , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Tretinoína/metabolismo , Humanos
9.
NAR Cancer ; 3(4): zcab044, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34806016

RESUMO

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.

10.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836586

RESUMO

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.


Assuntos
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 , Proteostase
11.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723040

RESUMO

Host mitochondrial association (HMA) is a well-known phenomenon during Toxoplasma gondii infection of the host cell. The T. gondii locus mitochondrial association factor 1 (MAF1) is required for HMA and MAF1 encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the T. gondii vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.


Assuntos
Interações Hospedeiro-Parasita , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Proteínas de Transporte , Expressão Ectópica do Gene , Imunofluorescência , Interações Hospedeiro-Parasita/genética , Espectrometria de Massas , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Vacúolos/metabolismo , Virulência
12.
JCI Insight ; 5(22)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33055416

RESUMO

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.


Assuntos
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/virologia
13.
Mol Cell Proteomics ; 19(11): 1896-1909, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32868373

RESUMO

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.


Assuntos
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 , Ubiquitinação
14.
JAMA Psychiatry ; 77(1): 86-95, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31642882

RESUMO

Importance: Findings from unbiased genetic studies have consistently implicated synaptic protein networks in schizophrenia, but the molecular pathologic features within these networks and their contribution to the synaptic and circuit deficits thought to underlie disease symptoms remain unknown. Objective: To determine whether protein levels are altered within synapses from the primary auditory cortex (A1) of individuals with schizophrenia and, if so, whether these differences are restricted to the synapse or occur throughout the gray matter. Design, Setting, and Participants: This paired case-control study included tissue samples from individuals with schizophrenia obtained from the Allegheny County Office of the Medical Examiner. An independent panel of health care professionals made consensus DSM-IV diagnoses. Each tissue sample from an individual with schizophrenia was matched by sex, age, and postmortem interval with 1 sample from an unaffected control individual. Targeted mass spectrometry was used to measure protein levels in A1 gray matter homogenate and synaptosome preparations. All experimenters were blinded to diagnosis. Mass spectrometry data were collected from September 26 through November 4, 2016, and analyzed from November 3, 2016, to July 15, 2019. Main Outcomes and Measures: Primary measures were homogenate and synaptosome protein levels and their coregulation network features. Hypotheses generated before data collection were (1) that levels of canonical postsynaptic proteins in A1 synaptosome preparations would differ between individuals with schizophrenia and controls and (2) that these differences would not be explained by changes in total A1 homogenate protein levels. Results: Synaptosome and homogenate protein levels were investigated in 48 individuals with a schizophrenia diagnosis and 48 controls (mean age in both groups, 48 years [range, 17-83 years]); each group included 35 males (73%) and 13 females (27%). Robust alterations (statistical cutoff set at an adjusted Limma P < .05) were observed in synaptosome levels of canonical mitochondrial and postsynaptic proteins that were highly coregulated and not readily explained by postmortem interval, antipsychotic drug treatment, synaptosome yield, or underlying alterations in homogenate protein levels. Conclusions and Relevance: These findings suggest a robust and highly coordinated rearrangement of the synaptic proteome. In line with unbiased genetic findings, alterations in synaptic levels of postsynaptic proteins were identified, providing a road map to identify the specific cells and circuits that are impaired in individuals with schizophrenia A1.


Assuntos
Córtex Auditivo/metabolismo , Proteoma/metabolismo , Esquizofrenia/metabolismo , Sinapses/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Feminino , Substância Cinzenta/metabolismo , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Mitocondriais/metabolismo , Mapas de Interação de Proteínas , Sinaptossomos/metabolismo , Adulto Jovem
15.
DNA Repair (Amst) ; 80: 1-7, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176958

RESUMO

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 2 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 49.5 ng/107 PBMCs, respectively, resulting in %p-ATM of 4%. Following exposure of PBMCs to 2 Gy IR, p-S1981-ATM levels increased 12-fold to 29.8 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 16.9 ng/107 cells; p < 0.01). This results in a significantly greater %p-ATM for women (68% versus 49%; p <  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.


Assuntos
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 Testes
16.
Aging (Albany NY) ; 11(3): 1045-1061, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30745468

RESUMO

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.


Assuntos
Envelhecimento/metabolismo , Fígado/metabolismo , Proteínas/metabolismo , Animais , Feminino , Masculino , Espectrometria de Massas , Camundongos , Valores de Referência
17.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30630894

RESUMO

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.


Assuntos
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/métodos
18.
Neuropsychopharmacology ; 44(4): 743-748, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30390066

RESUMO

The mammalian neocortex is organized into layers distinguished by the size, packing density, and connectivity of their constituent neurons. Many neuropsychiatric illnesses are complex trait disorders with etiologic factors converging on neuronal protein networks. Cortical pathology of neuropsychiatric diseases, such as schizophrenia, is often restricted to, or more pronounced in, certain cortical layers, suggesting that genetic vulnerabilities manifest with laminar specificity. Thus, the ability to investigate cortical layer-specific protein levels in human postmortem brain is highly desirable. Here, we developed and validated a laser capture microdissection-mass spectrometry (LCM-MS) approach to quantify over 200 proteins in cortical layers 3 and 5 of two cohorts of human subjects as well as a monkey model of postmortem interval. LCM-MS was readily implementable and reliably identified protein patterns that differed between cortical layers 3 and 5. Our findings suggest that LCM-MS facilitates the precise quantification of proteins within individual cortical layers from human postmortem brain tissue, providing a powerful tool in the study of neuropsychiatric disease.


Assuntos
Microdissecção e Captura a Laser/normas , Espectrometria de Massas/normas , Córtex Pré-Frontal/química , Córtex Pré-Frontal/patologia , Adulto , Idoso , Animais , Autopsia , Estudos de Coortes , Humanos , Microdissecção e Captura a Laser/métodos , Macaca fascicularis , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
19.
Am J Psychiatry ; 175(10): 999-1009, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021459

RESUMO

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.


Assuntos
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/metabolismo
20.
Redox Biol ; 17: 259-273, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29747066

RESUMO

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.


Assuntos
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/metabolismo
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