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1.
Cell ; 178(2): 316-329.e18, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31257023

RESUMO

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular Tumoral , Movimento Celular , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Feminino , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Estimativa de Kaplan-Meier , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional
2.
Mol Cell ; 84(7): 1224-1242.e13, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38458201

RESUMO

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.


Assuntos
Ciclinas , Reparo de Erro de Pareamento de DNA , Animais , Ciclinas/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Interfase , Mamíferos/metabolismo
3.
J Virol ; 97(10): e0050723, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37768083

RESUMO

IMPORTANCE: Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana , Proteínas de Membrana , Proteínas de Membrana Transportadoras , SARS-CoV-2 , Replicação Viral , Humanos , COVID-19/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/metabolismo
4.
Acta Neuropathol ; 147(1): 27, 2024 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-38289539

RESUMO

The prevalence of epilepsy is increased among Alzheimer's Disease (AD) patients and cognitive impairment is common among people with epilepsy. Epilepsy and AD are linked but the shared pathophysiological changes remain poorly defined. We aim to identify protein differences associated with epilepsy and AD using published proteomics datasets. We observed a highly significant overlap in protein differences in epilepsy and AD: 89% (689/777) of proteins altered in the hippocampus of epilepsy patients were significantly altered in advanced AD. Of the proteins altered in both epilepsy and AD, 340 were altered in the same direction, while 216 proteins were altered in the opposite direction. Synapse and mitochondrial proteins were markedly decreased in epilepsy and AD, suggesting common disease mechanisms. In contrast, ribosome proteins were increased in epilepsy but decreased in AD. Notably, many of the proteins altered in epilepsy interact with tau or are regulated by tau expression. This suggests that tau likely mediates common protein changes in epilepsy and AD. Immunohistochemistry for Aß and multiple phosphorylated tau species (pTau396/404, pTau217, pTau231) showed a trend for increased intraneuronal pTau217 and pTau231 but no phosphorylated tau aggregates or amyloid plaques in epilepsy hippocampal sections. Our results provide insights into common mechanisms in epilepsy and AD and highlights the potential role of tau in mediating common pathological protein changes in epilepsy and AD.


Assuntos
Doença de Alzheimer , Epilepsia , Humanos , Proteômica , Encéfalo , Proteínas Ribossômicas
5.
Acta Neuropathol ; 147(1): 91, 2024 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-38772917

RESUMO

APOEε4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). Although APOEε4 is known to promote Aß pathology, recent data also support an effect of APOE polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across APOE genotypes in the frontal cortex of 10 advanced AD cases (n = 5 APOEε3/ε3 and n = 5 APOEε4/ε4), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-Aß 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases (n = 11 APOEε3/ε3 and n = 10 APOEε4/ε4). Our dataset includes 1130 and 1330 proteins enriched in IPPHF1 samples from APOEε3/ε3 and APOEε4/ε4 groups (fold change ≥ 1.50, IPPHF1 vs IPIgG ctrl). We identified 80 and 68 proteins as probable pTau interactors in APOEε3/ε3 and APOEε4/ε4 groups, respectively (SAINT score ≥ 0.80; false discovery rate (FDR) ≤ 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOEε3/ε3 vs APOEε4/ε4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOEε4/ε4 vs APOEε3/ε3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOEε4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOEε4/ε4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to Aß-affected brain regions in APOEε4 carriers, paving the way to the identification of new therapeutic targets.


Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Proteínas tau , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Genótipo , Fosforilação , Proteômica , Proteínas tau/metabolismo , Proteínas tau/genética
6.
Neuropathol Appl Neurobiol ; 48(1): e12746, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34164845

RESUMO

AIMS: Hippocampal findings are implicated in the pathogenesis of sudden unexplained death in childhood (SUDC), although some studies have identified similar findings in sudden explained death in childhood (SEDC) cases. We blindly reviewed hippocampal histology in SUDC and SEDC controls. METHODS: Hippocampal haematoxylin and eosin (H&E) slides (n = 67; 36 SUDC, 31 controls) from clinical and forensic collaborators were evaluated by nine blinded reviewers: three board-certified forensic pathologists, three neuropathologists and three dual-certified neuropathologists/forensic pathologists. RESULTS: Among nine reviewers, about 50% of hippocampal sections were rated as abnormal (52.5% SUDC, 53.0% controls), with no difference by cause of death (COD) (p = 0.16) or febrile seizure history (p = 0.90). There was little agreement among nine reviewers on whether a slide was within normal range (Fleiss' κ = 0.014, p = 0.47). Within reviewer groups, there were no findings more frequent in SUDC compared with controls, with variability in pyramidal neuron and dentate gyrus findings. Across reviewer groups, there was concordance for bilamination and granule cell loss. Neither SUDC (51.2%) nor control (55.9%) slides were considered contributory to determining COD (p = 0.41). CONCLUSIONS: The lack of an association of hippocampal findings in SUDC and controls, as well as inconsistency of observations by multiple blinded reviewers, indicates discrepancy with previous studies and an inability to reliably identify hippocampal maldevelopment associated with sudden death (HMASD). These findings underscore a need for larger studies to standardise evaluation of hippocampal findings, identifying the range of normal variation and changes unrelated to SUDC or febrile seizures. Molecular studies may help identify novel immunohistological markers that inform on COD.


Assuntos
Neuropatologia , Convulsões Febris , Encéfalo/patologia , Criança , Morte Súbita/patologia , Hipocampo/patologia , Humanos , Convulsões Febris/complicações , Convulsões Febris/patologia
7.
Acta Neuropathol ; 143(5): 585-599, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35333953

RESUMO

Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10-15, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10-21, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10-6, z = 2.65) and CA1-3 (p = 4.7 × 10-6, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10-5) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10-3) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10-5) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10-3). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.


Assuntos
Proteômica , Convulsões Febris , Autopsia , Criança , Morte Súbita/etiologia , Morte Súbita/patologia , Hipocampo/patologia , Humanos , Convulsões Febris/complicações , Convulsões Febris/patologia
8.
Epilepsia ; 63(11): 2925-2936, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36053862

RESUMO

OBJECTIVE: Prolonged postictal generalized electroencephalographic suppression (PGES) is a potential biomarker for sudden unexpected death in epilepsy (SUDEP), which may be associated with dysfunctional autonomic responses and serotonin signaling. To better understand molecular mechanisms, PGES duration was correlated to 5HT1A and 5HT2A receptor protein expression and RNAseq from resected hippocampus and temporal cortex of temporal lobe epilepsy patients with seizures recorded in preoperative evaluation. METHODS: Analyses included 36 cases (age = 14-64 years, age at epilepsy onset = 0-51 years, epilepsy duration = 2-53 years, PGES duration = 0-93 s), with 13 cases in all hippocampal analyses. 5HT1A and 5HT2A protein was evaluated by Western blot and histologically in hippocampus (n = 16) and temporal cortex (n = 9). We correlated PGES duration to our previous RNAseq dataset for serotonin receptor expression and signaling pathways, as well as weighted gene correlation network analysis (WGCNA) to identify correlated gene clusters. RESULTS: In hippocampus, 5HT2A protein by Western blot positively correlated with PGES duration (p = .0024, R2  = .52), but 5HT1A did not (p = .87, R2  = .0020). In temporal cortex, 5HT1A and 5HT2A had lower expression and did not correlate with PGES duration. Histologically, PGES duration did not correlate with 5HT1A or 5HT2A expression in hippocampal CA4, dentate gyrus, or temporal cortex. RNAseq identified two serotonin receptors with expression that correlated with PGES duration in an exploratory analysis: HTR3B negatively correlated (p = .043, R2  = .26) and HTR4 positively correlated (p = .049, R2  = .25). WGCNA identified four modules correlated with PGES duration, including positive correlation with synaptic transcripts (p = .040, Pearson correlation r = .52), particularly potassium channels (KCNA4, KCNC4, KCNH1, KCNIP4, KCNJ3, KCNJ6, KCNK1). No modules were associated with serotonin receptor signaling. SIGNIFICANCE: Higher hippocampal 5HT2A receptor protein and potassium channel transcripts may reflect underlying mechanisms contributing to or resulting from prolonged PGES. Future studies with larger cohorts should assess functional analyses and additional brain regions to elucidate mechanisms underlying PGES and SUDEP risk.


Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Morte Súbita Inesperada na Epilepsia , Humanos , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Recém-Nascido , Lactente , Pré-Escolar , Criança , Serotonina , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/cirurgia , Eletroencefalografia/métodos , Epilepsia/patologia , Lobo Temporal/patologia , Hipocampo/patologia , Receptores de Serotonina/genética
9.
Proc Natl Acad Sci U S A ; 116(27): 13563-13572, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31217288

RESUMO

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Purinas/biossíntese , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Camundongos , Proteínas Repressoras/fisiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Fatores de Virulência/fisiologia
10.
Brain ; 143(9): 2803-2817, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32812023

RESUMO

Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer's disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer's disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer's disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteômica/métodos , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Cromatografia Líquida/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/fisiologia , Espectrometria de Massas em Tandem/métodos , Proteínas tau/análise , Proteínas tau/genética
11.
Brain ; 143(12): 3653-3671, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33206953

RESUMO

Epidemiological and experimental studies suggest that a disease-aggravating neuroinflammatory process is present at preclinical stages of Alzheimer's disease. Given that individuals with Down syndrome are at increased genetic risk of Alzheimer's disease and therefore develop the spectrum of Alzheimer's neuropathology in a uniform manner, they constitute an important population to study the evolution of neuroinflammation across the Alzheimer's continuum. Therefore, in this cross-sectional study, we characterized the brain inflammatory profile across the lifespan of individuals with Down syndrome. Microglial morphology and inflammatory cytokine expression were analysed by immunohistochemistry and electrochemiluminescent-based immunoassays in the frontal cortex from foetuses to adults with Down syndrome and control subjects (16 gestational weeks to 64 years), totalling 127 cases. Cytokine expression in mixed foetal primary cultures and hippocampus of adults with Down syndrome, as well as the effects of sex on cytokine expression were also analysed. A higher microglial soma size-to-process length ratio was observed in the frontal cortex of children and young adults with Down syndrome before the development of full-blown Alzheimer's pathology. Moreover, young adults with Down syndrome also displayed increased numbers of rod-like microglia. Increased levels of interleukin-8 and interleukin-10 were observed in children with Down syndrome (1-10 years; Down syndrome n = 5, controls n = 10) and higher levels of interleukin-1ß, interleukin-1α, interleukin-6, interleukin-8, interleukin-10, interleukin-15, eotaxin-3, interferon gamma-induced protein 10, macrophage-derived chemokine, and macrophage inflammatory protein-beta, were found in young adults with Down syndrome compared to euploid cases (13-25 years, Down syndrome n = 6, controls n = 24). Increased cytokine expression was also found in the conditioned media of mixed cortical primary cultures from second trimester foetuses with Down syndrome (Down syndrome n = 7, controls n = 7). Older adults with Down syndrome (39-68 years, Down syndrome n = 22, controls n = 16) displayed reduced levels of interleukin-10, interleukin-12p40, interferon-gamma and tumour necrosis factor-alpha. Microglia displayed larger somas and shorter processes. Moreover, an increase in dystrophic microglia and rod-like microglia aligning to neurons harbouring tau pathology were also observed. Sex stratification analyses revealed that females with Down syndrome had increased interleukin-6 and interleukin-8 levels compared to males with Down syndrome. Finally, multivariate projection methods identified specific cytokine patterns among individuals with Down syndrome. Our findings indicate the presence of an early and evolving neuroinflammatory phenotype across the lifespan in Down syndrome, a knowledge that is relevant for the discovery of stage-specific targets and for the design of possible anti-inflammatory trials against Alzheimer's disease in this population.


Assuntos
Síndrome de Down/patologia , Encefalite/patologia , Adolescente , Idoso , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Células Cultivadas , Criança , Pré-Escolar , Estudos Transversais , Citocinas/biossíntese , Progressão da Doença , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Lactente , Recém-Nascido , Longevidade , Masculino , Microglia/patologia , Pessoa de Meia-Idade , Gravidez , Tauopatias/patologia , Adulto Jovem
12.
Proc Natl Acad Sci U S A ; 115(3): E468-E477, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29282323

RESUMO

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Espectrometria de Massas/métodos , Receptor de Morte Celular Programada 1/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T/metabolismo , Animais , Biomarcadores Tumorais , Proliferação de Células/fisiologia , Citocinas/genética , Citocinas/metabolismo , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/genética
13.
Rapid Commun Mass Spectrom ; : e8962, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009686

RESUMO

RATIONALE: The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification. METHODS: We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification. RESULTS: Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously. CONCLUSIONS: Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics.

14.
Mol Cell Proteomics ; 16(4 suppl 1): S15-S28, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28196877

RESUMO

Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the Sa exoproteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains.


Assuntos
Espectrometria de Massas/métodos , Neutrófilos/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citotoxinas/genética , Citotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Genótipo , Humanos , Proteômica/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética
15.
Mass Spectrom Rev ; 36(5): 668-673, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-27741559

RESUMO

The evolution of data exchange in Mass Spectrometry spans decades and has ranged from human-readable text files representing individual scans or collections thereof (McDonald et al., 2004) through the official standard XML-based (Harold, Means, & Udemadu, 2005) data interchange standard (Deutsch, 2012), to increasingly compressed (Teleman et al., 2014) variants of this standard sometimes requiring purely binary adjunct files (Römpp et al., 2011). While the desire to maintain even partial human readability is understandable, the inherent mismatch between XML's textual and irregular format relative to the numeric and highly regular nature of actual spectral data, along with the explosive growth in dataset scales and the resulting need for efficient (binary and indexed) access has led to a phenomenon referred to as "technical drift" (Davis, 2013). While the drift is being continuously corrected using adjunct formats, compression schemes, and programs (Röst et al., 2015), we propose that the future of Mass Spectrometry Exchange Formats lies in the continued reliance and development of the PSI-MS (Mayer et al., 2014) controlled vocabulary, along with an expedited shift to an alternative, thriving and well-supported ecosystem for scientific data-exchange, storage, and access in binary form, namely that of HDF5 (Koranne, 2011). Indeed, pioneering efforts to leverage this universal, binary, and hierarchical data-format have already been published (Wilhelm et al., 2012; Rübel et al., 2013) though they have under-utilized self-description, a key property shared by HDF5 and XML. We demonstrate that a straightforward usage of plain ("vanilla") HDF5 yields immediate returns including, but not limited to, highly efficient data access, platform independent data viewers, a variety of libraries (Collette, 2014) for data retrieval and manipulation in many programming languages and remote data access through comprehensive RESTful data-servers. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:668-673, 2017.

16.
Nature ; 487(7408): 491-5, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22810586

RESUMO

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.


Assuntos
Genes Neoplásicos/genética , Genoma Humano/genética , Interações Hospedeiro-Patógeno , Neoplasias/genética , Neoplasias/metabolismo , Vírus Oncogênicos/patogenicidade , Proteínas Virais/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidade , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Neoplasias/patologia , Vírus Oncogênicos/genética , Vírus Oncogênicos/metabolismo , Fases de Leitura Aberta/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Papillomaviridae/patogenicidade , Polyomavirus/genética , Polyomavirus/metabolismo , Polyomavirus/patogenicidade , Receptores Notch/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética
17.
Mol Cell Proteomics ; 15(3): 1060-71, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26631509

RESUMO

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis.


Assuntos
Processamento Alternativo , Neoplasias Mamárias Experimentais/genética , Mutação , Proteômica/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Genoma , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas em Tandem , Transcriptoma
18.
Acta Neuropathol ; 133(6): 933-954, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28258398

RESUMO

Rapidly progressive Alzheimer's disease (rpAD) is a particularly aggressive form of Alzheimer's disease, with a median survival time of 7-10 months after diagnosis. Why these patients have such a rapid progression of Alzheimer's disease is currently unknown. To further understand pathological differences between rpAD and typical sporadic Alzheimer's disease (sAD) we used localized proteomics to analyze the protein differences in amyloid plaques in rpAD and sAD. Label-free quantitative LC-MS/MS was performed on amyloid plaques microdissected from rpAD and sAD patients (n = 22 for each patient group) and protein expression differences were quantified. On average, 913 ± 30 (mean ± SEM) proteins were quantified in plaques from each patient and 279 of these proteins were consistently found in plaques from every patient. We found significant differences in protein composition between rpAD and sAD plaques. We found that rpAD plaques contained significantly higher levels of neuronal proteins (p = 0.0017) and significantly lower levels of astrocytic proteins (p = 1.08 × 10-6). Unexpectedly, cumulative protein differences in rpAD plaques did not suggest accelerated typical sAD. Plaques from patients with rpAD were particularly abundant in synaptic proteins, especially those involved in synaptic vesicle release, highlighting the potential importance of synaptic dysfunction in the accelerated development of plaque pathology in rpAD. Combined, our data provide new direct evidence that amyloid plaques do not all have the same protein composition and that the proteomic differences in plaques could provide important insight into the factors that contribute to plaque development. The cumulative protein differences in rpAD plaques suggest rpAD may be a novel subtype of Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Placa Amiloide/metabolismo , Proteoma , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/patologia , Cromatografia Líquida , Estudos de Coortes , Progressão da Doença , Feminino , Imunofluorescência , Humanos , Masculino , Microdissecção , Microscopia Confocal , Pessoa de Meia-Idade , Neuritos/metabolismo , Neuritos/patologia , Placa Amiloide/patologia , Proteômica , Espectrometria de Massas em Tandem
19.
Proteomics ; 16(18): 2495-501, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27436706

RESUMO

Data sharing in the field of MS has advanced greatly thanks to innovations such as the standardized formats, data repositories, and publications guidelines. However, there is currently no data sharing mechanism that enables real-time data browsing and deep linking on a large scale: unrestricted data access (particularly at the quantitative level) ultimately requires the user to download a local copy of the relevant data files (e.g., in order to generate extracted ion chromatograms [XICs]). In this technical resource, we present a set of technologies (collectively termed OpenSlice) that enable the user to quantitatively query hundreds of hours of proteomics discovery data (i.e., nontargeted acquisition) in real time: the user is able to effectively generate XICs for arbitrary masses on the fly and across the entire dataset (so-called global ion chromatograms), interacting with the results through a very intuitive browser-based interface. A key design consideration underlying the OpenSlice approach is the notion that every aspect of the acquired data must be accessible through a RESTful uniform resource locator based application programming interface, up to and including individual chromatographic peaks (hence HyperPeaks). A publicly accessible demonstration of this technology based on the Clinical Proteomics Tumor Analysis Consortium CompRef dataset is made available at http://compref.fenyolab.org.


Assuntos
Cromatografia/métodos , Proteômica/métodos , Software , Humanos , Disseminação de Informação , Neoplasias/metabolismo , Interface Usuário-Computador
20.
J Proteome Res ; 15(3): 795-9, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26638927

RESUMO

Every molecular player in the cast of biology's central dogma is being sequenced and quantified with increasing ease and coverage. To bring the resulting genomic, transcriptomic, and proteomic data sets into coherence, tools must be developed that do not constrain data acquisition and analytics in any way but rather provide simple links across previously acquired data sets with minimal preprocessing and hassle. Here we present such a tool: PGx, which supports proteogenomic integration of mass spectrometry proteomics data with next-generation sequencing by mapping identified peptides onto their putative genomic coordinates.


Assuntos
Genômica/métodos , Proteômica/métodos , Análise de Sequência de Proteína/métodos , Bases de Dados de Proteínas , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Proteínas de Neoplasias/genética , Mapeamento de Peptídeos
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