RESUMO
X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.
Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X , Animais , DNA Helicases/isolamento & purificação , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Proteínas Nucleares/isolamento & purificação , Proteína Nuclear Ligada ao XRESUMO
BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.
Assuntos
Sistemas CRISPR-Cas , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Humanos , Técnicas de Inativação de Genes/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Anticorpos Monoclonais/biossíntese , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismoRESUMO
OBJECTIVE: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. CONCLUSIONS: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.
Assuntos
Processamento Alternativo , Artérias Carótidas/metabolismo , Estenose das Carótidas/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Remodelação Vascular , Animais , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Estenose das Carótidas/patologia , Estenose das Carótidas/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Matriz Extracelular/patologia , Fibronectinas/deficiência , Fibronectinas/genética , Mecanotransdução Celular , Camundongos Knockout , Isoformas de Proteínas , Fluxo Sanguíneo Regional , Estresse MecânicoRESUMO
The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Guanosina Pentafosfato/biossíntese , Guanosina Pentafosfato/fisiologia , Amidofosforribosiltransferase/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Nucleotídeos de Guanina/biossíntese , Nucleotídeos de Guanina/fisiologia , Guanosina Tetrafosfato , Purinas/antagonistas & inibidores , Purinas/biossínteseRESUMO
In the version of the article originally published, two sets of labels on the x axis of the graph in Fig. 5b were in reverse order. In the 'PurF' row, the locations of 'N48A' and 'R45A' should be switched, and in the row below those of '4.1' and the minus sign should be switched. Shown below are the original and corrected versions of Fig. 5b. The error has been corrected in the HTML and PDF versions of the article.
RESUMO
The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Proteômica/métodos , Tenascina/fisiologia , Adenocarcinoma/metabolismo , Animais , Anexina A2/metabolismo , Sistemas CRISPR-Cas , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Metástase Neoplásica , Prognóstico , Proteínas S100/metabolismo , Fator Nuclear 1 de Tireoide/metabolismo , Resultado do Tratamento , Microambiente TumoralRESUMO
DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Cromatina/química , Cromatina/efeitos da radiação , Montagem e Desmontagem da Cromatina/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação/efeitos da radiação , Fator B de Elongação Transcricional Positiva/metabolismo , Isoformas de Proteínas/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/química , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.
Assuntos
Matriz Extracelular/química , Neoplasias Ovarianas/química , Proteômica/métodos , Neoplasias de Mama Triplo Negativas/química , Mama/citologia , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/análise , Feminino , Humanos , Espectrometria de Massas , Anotação de Sequência Molecular , Omento/citologia , Neoplasias Ovarianas/secundário , Neoplasias Ovarianas/ultraestrutura , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/ultraestruturaRESUMO
Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.
Assuntos
Diferenciação Celular/genética , Fertilidade/genética , Células Germinativas/fisiologia , Sirtuína 1/metabolismo , Espermatogênese/genética , Acetilação , Animais , Diferenciação Celular/fisiologia , Montagem e Desmontagem da Cromatina/genética , Cromatografia Líquida , Feminino , Fertilidade/fisiologia , Imunofluorescência , Histonas/metabolismo , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional/genética , Sirtuína 1/deficiência , Espectrometria de Massas em Tandem , Testículo/metabolismoRESUMO
MS-based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti-acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine-containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot-to-lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.
Assuntos
Proteínas de Ligação a DNA/genética , Histonas/química , Lisina/análise , Peptídeos/química , Engenharia de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Acetilação , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteômica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ≈ 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.
Assuntos
Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/mortalidade , Animais , Anexina A2/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Anidrase Carbônica III/genética , Transformação Celular Neoplásica/genética , Receptores ErbB/análise , Receptores ErbB/metabolismo , Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Prognóstico , Proteínas S100/genética , Transdução de Sinais , Sobrevida , Transplante Heterólogo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Constipation negatively affects quality of life (QOL), however, the specific mechanisms through which this relationship occurs are unclear. The present study examined anxiety and depression as potential mediators of the relationship between constipation severity and QOL in a sample of 142 constipated patients. Results indicated that depression symptom severity mediated the relationship between constipation severity and mental health-related QOL. For patients meeting diagnostic criteria for Major Depressive Disorder, indirect effects were observed in the relationship between constipation severity and both physical and mental health-related QOL. Anxiety did not contribute to this model. Treating depression may be useful in improving QOL in severely constipated patients, which highlights the importance of psychological screening and treatment referrals in primary care settings.
Assuntos
Transtornos de Ansiedade/psicologia , Constipação Intestinal/psicologia , Transtorno Depressivo Maior/psicologia , Qualidade de Vida/psicologia , Adulto , Idoso , Transtornos de Ansiedade/diagnóstico , Transtornos de Ansiedade/epidemiologia , Comorbidade , Constipação Intestinal/epidemiologia , Constipação Intestinal/terapia , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/epidemiologia , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Atenção Primária à Saúde , Encaminhamento e Consulta , Inquéritos e QuestionáriosRESUMO
Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.
Assuntos
Células Endoteliais , Mecanotransdução Celular , Camundongos , Animais , Mecanotransdução Celular/fisiologia , Engenharia Tecidual/métodos , Morfogênese , Diferenciação Celular , Matriz ExtracelularRESUMO
PURPOSE: The purposes of this study were: (1) to examine the efficacy of anorectal biofeedback (AB) for constipation compared to a biofeedback control (BC) treatment and (2) to examine the extent to which self-reported childhood sexual/physical abuse predicted biofeedback outcome. METHODS: Twenty-one patients with pelvic floor dyssynergia were randomized to either (1) an AB arm, where patients learned to isolate the anal sphincter using an electromyography probe, or (2) a BC arm that controlled for the nonspecific effects of biofeedback, where patients learned to relax trapezius or temporalis muscles with EMG feedback. Both treatments were delivered by registered nurses for six sessions. Prior to randomization and post-treatment, patients completed the validated Constipation Severity Instrument and two measures of quality of life (QOL), the Irritable Bowel Syndrome-QOL, and the SF-36. Generalized estimating equations examined the within-group and between-group differences over time. RESULTS: Pre- and post-treatment data were obtained for six AB and nine BC patients. AB patients' overall constipation severity scores decreased by 35.5% (vs. 15.3%), and their obstructive defecation symptom scores decreased by 37.9% (vs. 19.7%) compared to BC. A similar pattern was shown on the IBS-QOL. On the SF-36 Mental Health Composite (MCS), AB scores improved 28.0% compared to BC scores, which worsened 12.7%. Those without (vs. with) a childhood sexual/physical abuse history showed improvement on the MCS post-biofeedback. CONCLUSIONS: While our sample was statistically underpowered, AB produced clinical improvements in constipation severity and QOL.
Assuntos
Canal Anal/fisiopatologia , Biorretroalimentação Psicológica/métodos , Constipação Intestinal/fisiopatologia , Constipação Intestinal/terapia , Reto/fisiopatologia , Criança , Abuso Sexual na Infância , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Qualidade de Vida , Resultado do TratamentoRESUMO
Background: Industry-standard guidance on method development and validation of hybrid LC-MS/MS assays for protein biomarkers, particularly on evaluation of parallelism, is lacking. Methods: Using a protein endogenous to humans and mice as a model analyte, a quantitative hybrid LC-MS/MS workflow was developed using a surrogate matrix approach with a recombinant form of the protein as the calibrant. Results: The developed workflow identified a surrogate matrix, established parallelism between the surrogate and authentic matrices and assessed parallelism between the recombinant and authentic forms of the protein. The final method was qualified using precision and accuracy with recovery assessments. Conclusion: The established workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for endogenous protein biomarkers.
Assuntos
Proteínas , Espectrometria de Massas em Tandem , Animais , Biomarcadores/metabolismo , Cromatografia Líquida/métodos , Humanos , Camundongos , Espectrometria de Massas em Tandem/métodos , Fluxo de TrabalhoRESUMO
BACKGROUND: Significant concern exists regarding the effect of proctectomy on sexual function in patients with IBD. Little is known about gender-specific differences. OBJECTIVE: This study aimed to examine sexual function and quality of life in men and women with IBD before and after proctectomy. DESIGN: This is a prospective cohort study. SETTING: The study was conducted at a colorectal surgery center. PATIENTS: The patients included in this study have IBD and underwent proctectomy or proctocolectomy. INTERVENTION: The treatment provided was proctectomy or proctocolectomy. MAIN OUTCOME MEASURES: Validated questionnaires were used to assess sexual function, quality of life, bowel habits, and urinary symptoms, and were completed before and 6 months after surgery. RESULTS: Sixty-six participants (41 men and 25 women) were evaluated at baseline and 6 months after proctocolectomy or completion proctectomy. A total of 48 IPAAs (31 men and 17 women) and 18 end ileostomies (10 men and 8 women) were created. Men reported improved scores on the International Index of Erectile Function (P = .003), a modified Sexual Function Questionnaire (P = .001), Inflammatory Bowel Disease Quality of Life (P < .001), and SF-36 (Mental Component Summary, P = .003; Physical Component Summary, P = .001) after surgery. Women had improvement in the desire subscale of the Female Sexual Function Index (P = .03), Inflammatory Bowel Disease Quality of Life scores (P = .04), and SF-36 (Mental Component Summary, P = .02; Physical Component Summary, P = .02). There was no gender difference in the magnitude of change in scores before and after surgery for any of the measures. LIMITATIONS: Small sample size and sexually inactivity in 50% of cohort may have had an impact on our findings. CONCLUSIONS: Both men and women reported improvements in general and IBD-specific quality of life after surgery, but only men demonstrated several areas of improved sexual function. Women reported improved sexual desire but no other sexual function improvement. The postsurgical gender difference in sexual function, despite similar improvements in quality of life, may be accounted for by unexamined aspects of female sexual function.
Assuntos
Procedimentos Cirúrgicos do Sistema Digestório/métodos , Doenças Inflamatórias Intestinais/fisiopatologia , Doenças Inflamatórias Intestinais/cirurgia , Disfunções Sexuais Fisiológicas/epidemiologia , Adulto , Distribuição de Qui-Quadrado , Feminino , Humanos , Ileostomia , Masculino , Estudos Prospectivos , Qualidade de Vida , Fatores de Risco , Fatores Sexuais , Estatísticas não Paramétricas , Inquéritos e Questionários , Resultado do TratamentoRESUMO
ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
Assuntos
Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1RESUMO
The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , HumanosRESUMO
We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.