RESUMO
The study of the cellular secretome using proteomic techniques continues to capture the attention of the research community across a broad range of topics in biomedical research. Due to their untargeted nature, independence from the model system employed, historically superior depth of analysis, as well as comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based proteomic assays have massively gained in analytical depth, which together with their high sensitivity, dynamic range coverage as well as high throughput capabilities render them exquisitely suited to secretome analysis. In this review, we revisit the analytical challenges implied by secretomics and provide an overview of affinity-based proteomic platforms currently available for such analyses, using the study of the tumor secretome as an example for basic and translational research.
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Polygenic scores (PGS) can identify individuals at risk of adverse health events and guide genetics-based personalized medicine. However, it is not clear how well PGS translate between different populations, limiting their application to well-studied ethnicities. Proteins are intermediate traits linking genetic predisposition and environmental factors to disease, with numerous blood circulating protein levels representing functional readouts of disease-related processes. We hypothesized that studying the genetic architecture of a comprehensive set of blood-circulating proteins between a European and an Arab population could shed fresh light on the translatability of PGS to understudied populations. We therefore conducted a genome-wide association study with whole-genome sequencing data using 1301 proteins measured on the SOMAscan aptamer-based affinity proteomics platform in 2935 samples of Qatar Biobank and evaluated the replication of protein quantitative traits (pQTLs) from European studies in an Arab population. Then, we investigated the colocalization of shared pQTL signals between the two populations. Finally, we compared the performance of protein PGS derived from a Caucasian population in a European and an Arab cohort. We found that the majority of shared pQTL signals (81.8%) colocalized between both populations. About one-third of the genetic protein heritability was explained by protein PGS derived from a European cohort, with protein PGS performing ~20% better in Europeans when compared to Arabs. Our results are relevant for the translation of PGS to non-Caucasian populations, as well as for future efforts to extend genetic research to understudied populations.
Assuntos
Árabes , Locos de Características Quantitativas , População Branca , Humanos , Árabes/genética , Estudo de Associação Genômica Ampla , População Branca/genética , Genética PopulacionalRESUMO
Reprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.
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Reprogramação Celular , Montagem e Desmontagem da Cromatina , Animais , Linhagem Celular , Cromatina/metabolismo , DNA Helicases/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Fator 4 Semelhante a Kruppel , Camundongos , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.
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The HSP70 co-chaperone BAG3 targets unfolded proteins to degradation via chaperone assisted selective autophagy (CASA), thereby playing pivotal roles in the proteostasis of adult cardiomyocytes (CMs). However, the complex functions of BAG3 for regulating autophagy in cardiac disease are not completely understood. Here, we demonstrate that conditional inactivation of Bag3 in murine CMs leads to age-dependent dysregulation of autophagy, associated with progressive cardiomyopathy. Surprisingly, Bag3-deficient CMs show increased canonical and non-canonical autophagic flux in the juvenile period when first signs of cardiac dysfunction appear, but reduced autophagy during later stages of the disease. Juvenile Bag3-deficient CMs are characterized by decreased levels of soluble proteins involved in synchronous contraction of the heart, including the gap junction protein Connexin 43 (CX43). Reiterative administration of chloroquine (CQ), an inhibitor of canonical and non-canonical autophagy, but not inactivation of Atg5, restores normal concentrations of soluble cardiac proteins in juvenile Bag3-deficient CMs without an increase of detergent-insoluble proteins, leading to complete recovery of early-stage cardiac dysfunction in Bag3-deficient mice. We conclude that loss of Bag3 in CMs leads to age-dependent differences in autophagy and cardiac dysfunction. Increased non-canonical autophagic flux in the juvenile period removes soluble proteins involved in cardiac contraction, leading to early-stage cardiomyopathy, which is prevented by reiterative CQ treatment.
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Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Autofagia , Cardiomiopatias , Miócitos Cardíacos , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Cloroquina/farmacologia , Camundongos KnockoutRESUMO
Chromatin remodeling complexes have functions in transcriptional regulation and chromosome maintenance, but it is mostly unknown how the function of these normally ubiquitous complexes is specified in the cellular context. Here, we describe that the evolutionary conserved long non-coding RNA linc-MYH regulates the composition of the INO80 chromatin remodeler complex in muscle stem cells and prevents interaction with WDR5 and the transcription factor YY1. Linc-MYH acts as a selective molecular switch in trans that governs the pro-proliferative function of the ubiquitous INO80 complex but does not affect its role in maintaining genomic stability. The molecular switch is essential for restricting generation of quiescent MuSCs and proliferation of myoblasts in homeostasis and regeneration. Since linc-MYH is expressed in proliferating myoblasts but not in quiescent MuSCs, we reason that the extent of myoblast proliferation has decisive effects on the size of the quiescent MuSC pool.
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ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hipertrofia/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , RNA Longo não Codificante/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proliferação de Células , Cromatina , DNA Glicosilases/genética , Proteínas de Ligação a DNA/genética , Epigenômica , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Mioblastos/citologia , RNA Longo não Codificante/genética , RNA não Traduzido , Regeneração/fisiologia , Transcriptoma , Fator de Transcrição YY1/genéticaRESUMO
Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.
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Propiolactona , Proteômica , Humanos , Propiolactona/farmacologia , Propiolactona/metabolismo , Propiolactona/química , Feminino , Biomarcadores/sangue , Biomarcadores/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Inativação de Vírus/efeitos dos fármacos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologiaRESUMO
BACKGROUND AND AIMS: Atherosclerosis is the main cause of stroke and coronary heart disease (CHD), both leading mortality causes worldwide. Proteomics, as a high-throughput method, could provide helpful insights into the pathological mechanisms underlying atherosclerosis. In this study, we characterized the associations of plasma protein levels with CHD and with carotid intima-media thickness (CIMT), as a surrogate measure of atherosclerosis. METHODS: The discovery phase included 1000 participants from the KORA F4 study, whose plasma protein levels were quantified using the aptamer-based SOMAscan proteomics platform. We evaluated the associations of plasma protein levels with CHD using logistic regression, and with CIMT using linear regression. For both outcomes we applied two models: an age-sex adjusted model, and a model additionally adjusted for body mass index, smoking status, physical activity, diabetes status, hypertension status, low density lipoprotein, high density lipoprotein, and triglyceride levels (fully-adjusted model). The replication phase included a matched case-control sample from the independent KORA F3 study, using ELISA-based measurements of galectin-4. Pathway analysis was performed with nominally associated proteins (p-value < 0.05) from the fully-adjusted model. RESULTS: In the KORA F4 sample, after Bonferroni correction, we found CHD to be associated with five proteins using the age-sex adjusted model: galectin-4 (LGALS4), renin (REN), cathepsin H (CTSH), and coagulation factors X and Xa (F10). The fully-adjusted model yielded only the positive association of galectin-4 (OR = 1.58, 95% CI = 1.30-1.93), which was successfully replicated in the KORA F3 sample (OR = 1.40, 95% CI = 1.09-1.88). For CIMT, we found four proteins to be associated using the age-sex adjusted model namely: cytoplasmic protein NCK1 (NCK1), insulin-like growth factor-binding protein 2 (IGFBP2), growth hormone receptor (GHR), and GDNF family receptor alpha-1 (GFRA1). After assessing the fully-adjusted model, only NCK1 remained significant (ß = 0.017, p-value = 1.39e-06). Upstream regulators of galectin-4 and NCK1 identified from pathway analysis were predicted to be involved in inflammation pathways. CONCLUSIONS: Our proteome-wide association study identified galectin-4 to be associated with CHD and NCK1 to be associated with CIMT. Inflammatory pathways underlying the identified associations highlight the importance of inflammation in the development and progression of CHD.
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Biomarcadores , Proteínas Sanguíneas , Espessura Intima-Media Carotídea , Doença das Coronárias , Valor Preditivo dos Testes , Proteômica , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Estudos de Casos e Controles , Doença das Coronárias/sangue , Doença das Coronárias/diagnóstico , Doença das Coronárias/epidemiologia , Doença das Coronárias/diagnóstico por imagem , Doenças das Artérias Carótidas/sangue , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/epidemiologia , Proteoma , Alemanha/epidemiologia , Fatores de Risco , Medição de Risco , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , AdultoRESUMO
BACKGROUND: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1. METHODS: Different Adar1-mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis. RESULTS: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7. Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1-mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1-deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes. CONCLUSIONS: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.
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Adenosina Desaminase , Insuficiência Cardíaca , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Inosina/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/metabolismo , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , RNARESUMO
RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Hipertensão Pulmonar , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Hipertensão Arterial Pulmonar , Animais , Proliferação de Células , Células Cultivadas , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , Remodelação Vascular/fisiologiaRESUMO
Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.
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COVID-19 , Inibidores de Hidroximetilglutaril-CoA Redutases , Lipoproteínas HDL , Proteoma , SARS-CoV-2 , Humanos , Proteoma/metabolismo , Masculino , COVID-19/sangue , COVID-19/virologia , COVID-19/complicações , Feminino , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Pessoa de Meia-Idade , SARS-CoV-2/efeitos dos fármacos , Idoso , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Síndrome de COVID-19 Pós-Aguda , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Tratamento Farmacológico da COVID-19 , AdultoRESUMO
The immunoreceptor NKG2D, which is expressed on NK cells and T cell subsets is critically involved in tumor immune surveillance. This applies in particular to acute myeloid leukemia (AML), which evades immune detection by downregulation of NKG2D ligands (NKG2D-L), including MICA. The absence of NKG2D-L on AML cells is moreover associated with leukemia stem cell characteristics. The NKG2D/NKG2D-L system thus qualifies as an interesting and promising therapeutic target.Here we aimed to identify transcription factors susceptible to pharmacological stimulation resulting in the expression of the NKG2D-L MICA in AML cells to restore anti-tumor activity. Using a CRISPR-based engineered ChIP (enChIP) assay for the MICA promoter region and readout by mass spectrometry-based proteomics, we identified the transcription factor krüppel-like factor 4 (KLF4) as associated with the promoter. We demonstrated that the MICA promoter comprises functional binding sites for KLF4 and genetic as well as pharmacological gain- and loss-of-function experiments revealed inducible MICA expression to be mediated by KLF4.Furthermore, induction in AML cells was achieved with the small compound APTO253, a KLF4 activator, which also inhibits MYC expression and causes DNA damage. This induction in turn yielded increased expression and cell surface presentation of MICA, thus rendering AML cells more susceptible to NK cell-mediated killing. These data unravel a novel link between APTO253 and the innate anti-tumor immune response providing a rationale for targeting AML cells via APTO253-dependent KFL4/MICA induction to allow elimination by endogenous or transplanted NK and T cells in vivo. Video Abstract.
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Leucemia Mieloide Aguda , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Regulação para Cima , Ligantes , Fator 4 Semelhante a Kruppel , Antígenos de Histocompatibilidade Classe I/genética , Leucemia Mieloide Aguda/metabolismo , Linhagem Celular TumoralRESUMO
p97 is an ATP-dependent chaperone that plays an important role in endoplasmic reticulum-associated degradation but whose connections to turnover of soluble proteins remain sparse. Binding of p97 to substrates is mediated by cofactors that contain ubiquitin-binding domains. We employed "network proteomics" to show that p97 assembles with all of the 13 mammalian UBX-domain proteins. The UBX proteins that bind ubiquitin conjugates also interact with dozens of E3 ubiquitin ligases, only one of which had been previously linked to p97. In particular, UBXD7 links p97 to the ubiquitin ligase CUL2/VHL and its substrate hypoxia-inducible factor 1alpha (HIF1alpha). Depletion of p97 leads to accumulation of endogenous HIF1alpha and increased expression of a HIF1alpha target gene. The large number of ubiquitin ligases found associated with UBX proteins suggests that p97 plays a far broader role than previously anticipated in the global regulation of protein turnover.
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Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linhagem Celular , Humanos , Redes e Vias Metabólicas , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteoma , Ubiquitina-Proteína Ligases/metabolismo , Proteína com ValosinaRESUMO
Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis.
Assuntos
Miocárdio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proliferação de Células/genética , Endocárdio/metabolismo , Endotélio/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Genes erbB/genética , Coração/fisiologia , Ventrículos do Coração/metabolismo , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Organogênese/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.
Assuntos
Técnicas de Reprogramação Celular , Evolução Molecular Direcionada , Fatores do Domínio POU/genética , Animais , Fator 4 Semelhante a Kruppel , Camundongos , Engenharia de ProteínasRESUMO
The mitochondrial inner membrane glycerophospholipid cardiolipin (CL) associates with mitochondrial proteins to regulate their activities and facilitate protein complex and supercomplex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated. Trypanosoma brucei bloodstream forms use an unconventional ETC composed of glycerol-3-phosphate dehydrogenase and alternative oxidase (AOX), while the mitochondrial membrane potential (ΔΨm) is generated by the hydrolytic action of the Fo F1 -ATP synthase (aka Fo F1 -ATPase). We now report that the inducible depletion of cardiolipin synthase (TbCls) is essential for survival of T brucei bloodstream forms. Loss of CL caused a rapid drop in ATP levels and a decline in the ΔΨm. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of Fo F1 -ATPase subunits and AOX, resulting in a strong decline of glycerol-3-phosphate-stimulated oxygen consumption. The changes in cellular respiration preceded the observed decrease in Fo F1 -ATPase stability, suggesting that the AOX-mediated ETC is the first pathway responding to the decline in CL. Select proteins and pathways involved in glucose and amino acid metabolism were upregulated to counteract the CL depletion-induced drop in cellular ATP.
Assuntos
Cardiolipinas/genética , Metabolismo Energético/genética , Técnicas de Inativação de Genes , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/metabolismo , Cardiolipinas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Organismos Geneticamente Modificados , Oxirredutases/metabolismo , Consumo de Oxigênio/genética , Proteínas de Plantas/metabolismo , Proteoma , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Trypanosoma brucei brucei/classificaçãoRESUMO
RATIONALE: After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown. OBJECTIVE: To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis. METHODS AND RESULTS: Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo. CONCLUSIONS: We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Citocinese , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Técnicas de Cocultura , Proteínas da Matriz Extracelular/metabolismo , Feminino , Idade Gestacional , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitose , Proteínas do Tecido Nervoso/metabolismo , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.
Assuntos
Miócitos Cardíacos , Peixe-Zebra , Animais , Proliferação de Células , Glicólise , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Peixe-Zebra/metabolismoRESUMO
Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias/metabolismo , Fosforilação/fisiologia , Molécula 1 de Interação Estromal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteína ORAI1/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1007743.].