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NADPH oxidase organizer 1 (NoxO1) is a scaffold cytoplasmic subunit of the reactive oxygen species (ROS) forming Nox1 complex and involved in angiogenesis, differentiation, and atherosclerosis. We found that overexpression of NoxO1 without simultaneous overexpression of any other component of the active Nox1 complex inhibited EGF-induced wound closure and signaling, while NoxO1 KO yielded the opposite effect. Accordingly, we hypothesize NoxO1 to exert Nox1 independent functions. Using the BioID technique, we identified ErbB2 interacting protein (Erbin) as novel interaction partner of NoxO1. Colocalization of NoxO1 with EGFR, as well as with Erbin validated this finding. EGF treatment interrupted colocalization of NoxO1 and EGFR. EGF mediated kinase activation was delayed in NoxO1 overexpressing cells, while knockout of NoxO1 had the opposite effect. In conclusion, Erbin was identified as a novel NoxO1 interacting protein. Through the subsequent interaction of NoxO1 and EGFR, NoxO1 interferes with EGF signaling. The results of this study suggest a potential role of NoxO1 as an adaptor protein with functions beyond the well-established enabling of Nox1 mediated ROS formation.
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Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation.
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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 used, 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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Neoplasias , Proteómica , Humanos , Proteómica/métodos , Neoplasias/metabolismo , Inmunoensayo/métodos , Secretoma/metabolismo , Animales , Espectrometría de Masas/métodosRESUMEN
In-depth multiomic phenotyping provides molecular insights into complex physiological processes and their pathologies. Here, we report on integrating 18 diverse deep molecular phenotyping (omics-) technologies applied to urine, blood, and saliva samples from 391 participants of the multiethnic diabetes Qatar Metabolomics Study of Diabetes (QMDiab). Using 6,304 quantitative molecular traits with 1,221,345 genetic variants, methylation at 470,837 DNA CpG sites, and gene expression of 57,000 transcripts, we determine (1) within-platform partial correlations, (2) between-platform mutual best correlations, and (3) genome-, epigenome-, transcriptome-, and phenome-wide associations. Combined into a molecular network of > 34,000 statistically significant trait-trait links in biofluids, our study portrays "The Molecular Human". We describe the variances explained by each omics in the phenotypes (age, sex, BMI, and diabetes state), platform complementarity, and the inherent correlation structures of multiomics data. Further, we construct multi-molecular network of diabetes subtypes. Finally, we generated an open-access web interface to "The Molecular Human" ( http://comics.metabolomix.com ), providing interactive data exploration and hypotheses generation possibilities.
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Fenotipo , Humanos , Masculino , Femenino , Metabolómica/métodos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Metilación de ADN , Transcriptoma , Persona de Mediana Edad , Estudio de Asociación del Genoma Completo , Qatar/epidemiología , Epigenoma , Adulto , Islas de CpG/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , MultiómicaRESUMEN
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 Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Autofagia , Cardiomiopatías , Miocitos Cardíacos , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/deficiencia , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratones , Miocardio/metabolismo , Miocardio/patología , Cloroquina/farmacología , Ratones NoqueadosRESUMEN
Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.
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Carcinoma Ductal Pancreático , Progresión de la Enfermedad , Vesículas Extracelulares , Interleucina-33 , Mastocitos , Neoplasias Pancreáticas , Microambiente Tumoral , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Proliferación Celular , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Interleucina-33/genética , Mastocitos/metabolismo , Mastocitos/inmunología , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/inmunologíaRESUMEN
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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Carcinoma Ductal Pancreático , Quinasas DyrK , Macrófagos , Neoplasias Pancreáticas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Microambiente Tumoral , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fagocitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Altered protein levels in the aqueous humor (AH) may be a valuable source of novel biomarkers in neurodegenerative retinal disease. The proximity of this body fluid to the disease focus, and its corresponding enrichment for tissue specific proteins, renders it an excellent matrix to study underlying molecular mechanisms. Novel proteomic methods accordingly hold large potential for insight into pathologies based on the composition of the AH proteome, including primary open angle glaucoma (POAG). Recent mass spectrometry-based studies use novel approaches to tackle the challenges arising from the combination of low available sample volume and protein concentration, thereby increasing proteome coverage. But despite significant improvements in mass spectrometry (MS), a different class of proteomic technologies is poised to majorly impact the analysis of ocular biofluids. Affinity proteomic workflows, having become available commercially recently, have started to complement data obtained by MS and likely will grow into a crucial tool for ophthalmological biomarker research. This review highlights corresponding approaches in proteome analysis of aqueous humor and discusses recent findings on alterations of the AH proteome in POAG.
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Lysophosphatidic acid (LPA) species, prevalent in the tumor microenvironment (TME), adversely impact various cancers. In ovarian cancer, the 18:0 and 20:4 LPA species are selectively associated with shorter relapse-free survival, indicating distinct effects on cellular signaling networks. Macrophages represent a cell type of high relevance in the TME, but the impact of LPA on these cells remains obscure. Here, we uncovered distinct LPA-species-specific responses in human monocyte-derived macrophages through unbiased phosphoproteomics, with 87 and 161 phosphosites upregulated by 20:4 and 18:0 LPA, respectively, and only 24 shared sites. Specificity was even more pronounced for downregulated phosphosites (163 versus 5 sites). Considering the high levels 20:4 LPA in the TME and its selective association with poor survival, this finding may hold significant implications. Pathway analysis pinpointed RHO/RAC1 GTPase signaling as the predominantly impacted target, including AHRGEF and DOCK guanine exchange factors, ARHGAP GTPase activating proteins, and regulatory protein kinases. Consistent with these findings, exposure to 20:4 resulted in strong alterations to the actin filament network and a consequent enhancement of macrophage migration. Moreover, 20:4 LPA induced p38 phosphorylation, a response not mirrored by 18:0 LPA, whereas the pattern for AKT was reversed. Furthermore, RNA profiling identified genes involved in cholesterol/lipid metabolism as selective targets of 20:4 LPA. These findings imply that the two LPA species cooperatively regulate different pathways to support functions essential for pro-tumorigenic macrophages within the TME. These include cellular survival via AKT activation and migration through RHO/RAC1 and p38 signaling.
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Lisofosfolípidos , Macrófagos , Proteómica , Transducción de Señal , Humanos , Lisofosfolípidos/metabolismo , Macrófagos/metabolismo , Proteómica/métodos , Fosforilación/efectos de los fármacos , Fosfoproteínas/metabolismoRESUMEN
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 , Grosor Intima-Media Carotídeo , Enfermedad Coronaria , Valor Predictivo de las Pruebas , Proteómica , Humanos , Masculino , Femenino , Persona de Mediana Edad , Anciano , Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Estudios de Casos y Controles , Enfermedad Coronaria/sangre , Enfermedad Coronaria/diagnóstico , Enfermedad Coronaria/epidemiología , Enfermedad Coronaria/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/epidemiología , Proteoma , Alemania/epidemiología , Factores de Riesgo , Medición de Riesgo , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , AdultoRESUMEN
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 , Proteómica , Humanos , Propiolactona/farmacología , Propiolactona/metabolismo , Propiolactona/química , Femenino , Biomarcadores/sangre , Biomarcadores/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Inactivación de Virus/efectos de los fármacos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/farmacologíaRESUMEN
BACKGROUND: IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation. METHODS: Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses. RESULTS: We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1ß as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC. CONCLUSIONS: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.
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Neoplasias Ováricas , Células Th17 , Humanos , Femenino , Interleucina-17/metabolismo , Citocinas/metabolismo , Neoplasias Ováricas/metabolismo , Inflamación/metabolismo , Microambiente TumoralRESUMEN
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 , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Lipoproteínas HDL , Proteoma , SARS-CoV-2 , Humanos , Proteoma/metabolismo , Masculino , COVID-19/sangre , COVID-19/virología , COVID-19/complicaciones , Femenino , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Persona de Mediana Edad , SARS-CoV-2/efectos de los fármacos , Anciano , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Síndrome Post Agudo de COVID-19 , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Tratamiento Farmacológico de COVID-19 , AdultoRESUMEN
A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated T cells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the T cell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.
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Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.
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Adipocitos , Placofilinas , Humanos , Moléculas de Adhesión Celular , Ciclo Celular/genética , División Celular , Obesidad/genética , Placofilinas/genéticaRESUMEN
BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , Humanos , Femenino , Proteómica , Proteoma , Hipoxia/terapiaRESUMEN
Background: Acute respiratory distress syndrome (ARDS) in corona virus disease 19 (COVID-19) is triggered by hyperinflammation, thus providing a rationale for immunosuppressive treatments. The Janus kinase inhibitor Ruxolitinib (Ruxo) has shown efficacy in severe and critical COVID-19. In this study, we hypothesized that Ruxo's mode of action in this condition is reflected by changes in the peripheral blood proteome. Methods: This study included 11 COVID-19 patients, who were treated at our center's Intensive Care Unit (ICU). All patients received standard-of-care treatment and n = 8 patients with ARDS received Ruxo in addition. Blood samples were collected before (day 0) and on days 1, 6, and 10 of Ruxo treatment or, respectively, ICU admission. Serum proteomes were analyzed by mass spectrometry (MS) and cytometric bead array. Results: Linear modeling of MS data yielded 27 significantly differentially regulated proteins on day 1, 69 on day 6 and 72 on day 10. Only five factors (IGLV10-54, PSMB1, PGLYRP1, APOA5, WARS1) were regulated both concordantly and significantly over time. Overrepresentation analysis revealed biological processes involving T-cells only on day 1, while a humoral immune response and complement activation were detected at day 6 and day 10. Pathway enrichment analysis identified the NRF2-pathway early under Ruxo treatment and Network map of SARS-CoV-2 signaling and Statin inhibition of cholesterol production at later time points. Conclusion: Our results indicate that the mechanism of action of Ruxo in COVID-19-ARDS can be related to both known effects of this drug as a modulator of T-cells and the SARS-CoV-2-infection.
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Rationale: Chronic obstructive pulmonary disease (COPD) is a complex disease characterized by airway obstruction and accelerated lung function decline. Our understanding of systemic protein biomarkers associated with COPD remains incomplete. Objectives: To determine what proteins and pathways are associated with impaired pulmonary function in a diverse population. Methods: We studied 6,722 participants across six cohort studies with both aptamer-based proteomic and spirometry data (4,566 predominantly White participants in a discovery analysis and 2,156 African American cohort participants in a validation). In linear regression models, we examined protein associations with baseline forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity (FVC). In linear mixed effects models, we investigated the associations of baseline protein levels with rate of FEV1 decline (ml/yr) in 2,777 participants with up to 7 years of follow-up spirometry. Results: We identified 254 proteins associated with FEV1 in our discovery analyses, with 80 proteins validated in the Jackson Heart Study. Novel validated protein associations include kallistatin serine protease inhibitor, growth differentiation factor 2, and tumor necrosis factor-like weak inducer of apoptosis (discovery ß = 0.0561, Q = 4.05 × 10-10; ß = 0.0421, Q = 1.12 × 10-3; and ß = 0.0358, Q = 1.67 × 10-3, respectively). In longitudinal analyses within cohorts with follow-up spirometry, we identified 15 proteins associated with FEV1 decline (Q < 0.05), including elafin leukocyte elastase inhibitor and mucin-associated TFF2 (trefoil factor 2; ß = -4.3 ml/yr, Q = 0.049; ß = -6.1 ml/yr, Q = 0.032, respectively). Pathways and processes highlighted by our study include aberrant extracellular matrix remodeling, enhanced innate immune response, dysregulation of angiogenesis, and coagulation. Conclusions: In this study, we identify and validate novel biomarkers and pathways associated with lung function traits in a racially diverse population. In addition, we identify novel protein markers associated with FEV1 decline. Several protein findings are supported by previously reported genetic signals, highlighting the plausibility of certain biologic pathways. These novel proteins might represent markers for risk stratification, as well as novel molecular targets for treatment of COPD.