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Abstract 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.

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.

Abstract 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

Abstract 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‐1β 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.

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.

Abstract 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.

Abstract 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 (

Summary: 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.

Abstract 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

The authors report that endothelial protocadherin FAT1 inhibits endothelial proliferation and angiogenesis by promoting degradation of the transcriptional cofactors YAP and TAZ by direct interaction with the E3 ubiquitin ligase Mind Bomb-2 (MIB2).

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.

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.

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163highCD206high tumor‐associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine‐triggered signaling, mirrored by an impaired transcriptional response to interferons and IL‐6 in monocyte‐derived macrophages by AA. This inhibition of pro‐inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune‐regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA‐mediated interference with STAT1 phosphorylation. Inhibition of interferon‐triggered STAT1 phosphorylation by AA was reversed by water‐soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.

BackgroundAcute 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.MethodsThis 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.ResultsLinear 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.ConclusionOur 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.

Abstract Background Basal cell adhesion molecule (BCAM) is a laminin α5 (LAMA5) binding membrane‐bound protein with a putative role in cancer. Besides full‐length BCAM1, an isoform lacking most of the cytoplasmic domain (BCAM2), and a soluble form (sBCAM) of unknown function are known. In ovarian carcinoma (OC), all BCAM forms are abundant and associated with poor survival, yet BCAM's contribution to peritoneal metastatic spread remains enigmatic. Methods Biochemical, omics‐based and real‐time cell assays were employed to identify the source of sBCAM and metastasis‐related functions of different BCAM forms. OC cells, explanted omentum and a mouse model of peritoneal colonisation were used in loss‐ and gain‐of‐function experiments. Results We identified ADAM10 as a major BCAM sheddase produced by OC cells and identified proteolytic cleavage sites proximal to the transmembrane domain. Recombinant soluble BCAM inhibited single‐cell adhesion and migration identically to membrane‐bound isoforms, confirming its biological activity in OC. Intriguingly, this seemingly anti‐tumorigenic potential of BCAM contrasts with a novel pro‐metastatic function discovered in the present study. Thus, all queried BCAM forms decreased the compactness of tumour cell spheroids by inhibiting LAMA5 – integrin β1 interactions, promoted spheroid dispersion in a three‐dimensional collagen matrix, induced clearance of mesothelial cells at spheroid attachment sites in vitro and enhanced invasion of spheroids into omental tissue both ex vivo and in vivo. Conclusions Membrane‐bound BCAM as well as sBCAM shed by ADAM10 act as decoys rather than signalling receptors to modulate metastasis‐related functions. While BCAM appears to have tumour‐suppressive effects on single cells, it promotes the dispersion of OC cell spheroids by regulating LAMA5‐integrin‐β1‐dependent compaction and thereby facilitating invasion of metastatic target sites. As peritoneal dissemination is majorly mediated by spheroids, these findings offer an explanation for the association of BCAM with a poor clinical outcome of OC, suggesting novel therapeutic options.

Abstract Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free‐flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix‐free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state‐of‐the‐art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin‐positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.

Abstract Background The metabolic syndrome (MetS), defined by the simultaneous clustering of cardio-metabolic risk factors, is a significant worldwide public health burden with an estimated 25% prevalence worldwide. The pathogenesis of MetS is not entirely clear and the use of molecular level data could help uncover common pathogenic pathways behind the observed clustering. Methods Using a highly multiplexed aptamer-based affinity proteomics platform, we examined associations between plasma proteins and prevalent and incident MetS in the KORA cohort (n = 998) and replicated our results for prevalent MetS in the HUNT3 study (n = 923). We applied logistic regression models adjusted for age, sex, smoking status, and physical activity. We used the bootstrap ranking algorithm of least absolute shrinkage and selection operator (LASSO) to select a predictive model from the incident MetS associated proteins and used area under the curve (AUC) to assess its performance. Finally, we investigated the causal effect of the replicated proteins on MetS using two-sample Mendelian randomization. Results Prevalent MetS was associated with 116 proteins, of which 53 replicated in HUNT. These included previously reported proteins like leptin, and new proteins like NTR domain-containing protein 2 and endoplasmic reticulum protein 29. Incident MetS was associated with 14 proteins in KORA, of which 13 overlap the prevalent MetS associated proteins with soluble advanced glycosylation end product-specific receptor (sRAGE) being unique to incident MetS. The LASSO selected an eight-protein predictive model with an (AUC = 0.75; 95% CI = 0.71–0.79) in KORA. Mendelian randomization suggested causal effects of three proteins on MetS, namely apolipoprotein E2 (APOE2) (Wald-Ratio = − 0.12, Wald-p = 3.63e−13), apolipoprotein B (APOB) (Wald-Ratio = − 0.09, Wald-p = 2.54e−04) and proto-oncogene tyrosine-protein kinase receptor (RET) (Wald-Ratio = 0.10, Wald-p = 5.40e−04). Conclusions Our findings offer new insights into the plasma proteome underlying MetS and identify new protein associations. We reveal possible casual effects of APOE2, APOB and RET on MetS. Our results highlight protein candidates that could potentially serve as targets for prevention and therapy.

Abstract The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

Blood circulating proteins reflect biological processes, thus providing insight into complex traits. Here the authors study the relationship between 1000 plasma proteins and body mass index (BMI), highlighting widespread proteome changes and causal relationships between BMI and specific proteins.

The order of DNA methylation and histone modifications during transcription remained unclear. Here the authors show that HMGA2 induces DNA nicks at TGFB1-responsive genes, promoting nucleosome incorporation containing γ-H2AX, which is required for repair-mediated DNA demethylation and transcription.

Abstract The oligosaccharide required for asparagine (N)-linked glycosylation of proteins in the endoplasmic reticulum (ER) is donated by the glycolipid Glc3Man9GlcNAc2-PP-dolichol. Remarkably, whereas glycosylation occurs in the ER lumen, the initial steps of Glc3Man9GlcNAc2-PP-dolichol synthesis generate the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic side of the ER. Glycolipid assembly is completed only after M5-DLO is translocated to the luminal side. The membrane protein (M5-DLO scramblase) that mediates M5-DLO translocation across the ER membrane has not been identified, despite its importance for N-glycosylation. Building on our ability to recapitulate scramblase activity in proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based 'activity correlation profiling' approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Data curation prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins is necessary for M5-DLO scramblase activity. Our results instead strongly suggest that M5-DLO scramblase activity is due to a protein, or protein complex, whose activity is regulated at the level of quaternary structure.

While biallelic mutations of the SLC26A4 gene cause non-syndromic hearing loss with enlarged vestibular aqueducts or Pendred syndrome, a considerable number of patients carry mono-allelic mutations. Here the authors identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4.

DNA methylation is associated with complex traits and the expression of genes and proteins. Here, Zaghlool et al. perform epigenome-wide association studies for 1,123 plasma proteins, replicate obtained protein (p)QTMs in an independent cohort and find overlap of pQTMs with expression QTMs and previously reported disease associations.

Protein biomarkers have been identified across many age-related morbidities. However, characterising epigenetic influences could further inform disease predictions. Here, we leverage epigenome-wide data to study links between the DNA methylation (DNAm) signatures of the circulating proteome and incident diseases. Using data from four cohorts, we trained and tested epigenetic scores (EpiScores) for 953 plasma proteins, identifying 109 scores that explained between 1% and 58% of the variance in protein levels after adjusting for known protein quantitative trait loci (pQTL) genetic effects. By projecting these EpiScores into an independent sample (Generation Scotland; n = 9537) and relating them to incident morbidities over a follow-up of 14 years, we uncovered 130 EpiScore-disease associations. These associations were largely independent of immune cell proportions, common lifestyle and health factors, and biological aging. Notably, we found that our diabetes-associated EpiScores highlighted previous top biomarker associations from proteome-wide assessments of diabetes. These EpiScores for protein levels can therefore be a valuable resource for disease prediction and risk stratification.

Abstract Background Transcoelomic spread is the major route of metastasis of ovarian high‐grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumour microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. Methods Based on transcriptomic analysis of tumour cells, tumour‐associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analysing clinical correlations and potentially pro‐metastatic functions, including tumour cell migration, pro‐inflammatory signal transduction and TAM expansion. Results The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumour cells, are the major source of most cytokines and growth factors in the omental microenvironment (n = 176 vs. n = 13). Many of these factors target tumour cells, are linked to metastasis and are associated with a short survival. Likewise, tumour stroma cells play a major role in extracellular‐matrix‐triggered signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumour cell migration and adhesion via WNT4 which is highly expressed by CAF; (ii) induces pro‐tumourigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro‐inflammatory signaling, at least in part via a HSP70–NF‐κB pathway. Conclusions The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.

Abstract Background Chronic heart failure (CHF) leads to diaphragm myopathy that significantly impairs quality of life and worsens prognosis. In this study, we aimed to assess the efficacy of a recently discovered small‐molecule inhibitor of MuRF1 in treating CHF‐induced diaphragm myopathy and loss of contractile function. Methods Myocardial infarction was induced in mice by ligation of the left anterior descending coronary artery. Sham‐operated animals (sham) served as controls. One week post‐left anterior descending coronary artery ligation animals were randomized into two groups—one group was fed control rodent chow, whereas the other group was fed a diet containing 0.1% of the compound ID#704946—a recently described MuRF1‐interfering small molecule. Echocardiography confirmed development of CHF after 10 weeks. Functional and molecular analysis of the diaphragm was subsequently performed. Results Chronic heart failure induced diaphragm fibre atrophy and contractile dysfunction by ~20%, as well as decreased activity of enzymes involved in mitochondrial energy production (P

The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl‐LPA species are strongly elevated in ascites versus plasma and are associated with short relapse‐free survival. Data derived from transcriptome and secretome analyses of primary ascite‐derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor‐associated macrophages play an essential role as main producers of PLA2G7 and autotaxin. The latter conclusion is consistent with mass spectrometry‐based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor‐associated macrophages, but not tumor cells, are able to produce 20:4 acyl‐LPA in lipid‐free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type‐selective manner: While tumor cells express predominantly LPAR1‐3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type‐selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA‐induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.

Abnormal alveolar development and homeostasis are common features of pulmonary disease. Here the authors show that Myh10 expression is reduced in emphysema patients, and that Myh10 loss of function impairs alveolar formation and lung morphogenesis via upregulation of matrix metalloproteinase activity and altered matrix remodeling.

Genetic variation can influence levels of disease-related plasma proteins and, thus, contribute to the pathogenesis of complex diseases. Here, the authors perform genome-wide QTL analysis for 71 plasma proteins to identify causal proteins for coronary heart disease and provide a molecular QTL browser.

In scholarly publications, multiple graphical elements such as plots or raster images are traditionally combined into a single compound figure using panels arranged in a grid. Package multipanelfigure is a GPL-3 licensed R package that eases assembly of such compound figures, allowing for straightforward integration of R specific plots using base graphics, lattice and ggplot2 plots, as well as grid grobs in general. Also provided are facilities to incorporate R external graphical output stored as SVG, PNG, JPEG, or TIFF formatted files.

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles, but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here, we focused on CDKAL1, linked by GWASs to diabetes. Through transcript profiling, we find that expression of the metallothionein (MT) gene family, also linked by GWASs to diabetes, is significantly downregulated in CDKAL1−/− cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression.

Individual genetic variation can affect the levels of protein in blood, but detailed data sets linking these two types of data are rare. Here, the authors carry out a genome-wide association study of levels of over a thousand different proteins, and describe many new SNP-protein interactions.

A central reason behind the poor clinical outcome of patients with high-grade serous carcinoma (HGSC) of the ovary is the difficulty in reliably detecting early occurrence or recurrence of this malignancy. Biomarkers that provide reliable diagnosis of this disease are therefore urgently needed. Systematic proteomic methods that identify HGSC-associated molecules may provide such biomarkers. We applied the antibody-based proximity extension assay (PEA) platform (Olink) for the identification of proteins that are upregulated in the plasma of OC patients. Using binders targeting 368 different plasma proteins, we compared 20 plasma samples from HGSC patients (OC-plasma) with 20 plasma samples from individuals with non-malignant gynecologic disorders (N-plasma). We identified 176 proteins with significantly higher levels in OC-plasma compared to N-plasma by PEA (p < 0.05 by U-test; Benjamini-Hochberg corrected), which are mainly implicated in immune regulation and metastasis-associated processes, such as matrix remodeling, adhesion, migration and proliferation. A number of these proteins have not been reported in previous studies, such as BCAM, CDH6, DDR1, N2DL-2 (ULBP2), SPINT2, and WISP-1 (CCN4). Of these SPINT2, a protease inhibitor mainly derived from tumor cells within the HGSC microenvironment, showed the highest significance (p < 2 × 10−7) similar to the previously described IL-6 and PVRL4 (NECTIN4) proteins. Results were validated by means of the aptamer-based 1.3 k SOMAscan proteomic platform, which revealed a high inter-platform correlation with a median Spearman ρ of 0.62. Likewise, ELISA confirmed the PEA data for 10 out of 12 proteins analyzed, including SPINT2. These findings suggest that in contrast to other entities SPINT2 does not act as a tumor suppressor in HGSC. This is supported by data from the PRECOG and KM-Plotter meta-analysis databases, which point to a tumor-type-specific inverse association of SPINT2 gene expression with survival. Our data also demonstrate that both the PEA and SOMAscan affinity proteomics platforms bear considerable potential for the unbiased discovery of novel disease-associated biomarkers.

[This corrects the article DOI: 10.1371/journal.pgen.1007743.].

Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate ‘insulin-free’ model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.

Retinitis pigmentosa is often caused by mutations that affect the activity or transport of rhodopsin, but some mutations cause disease even though an apparently functional protein is produced. Here the authors show that three such enigmatic mutants retain scramblase activity but are unable to dimerize.

Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.

Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.

Most human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role in many complex disorders. One approach towards deciphering this human glycan code is to study natural variation in experimentally well characterized samples and cohorts. High-throughput capable large-scale methods that allow for the comprehensive determination of blood circulating proteins and their glycans have been recently developed, but so far, no study has investigated the link between both traits. Here we map for the first time the blood plasma proteome to its matching N-glycome by correlating the levels of 1116 blood circulating proteins with 113 N-glycan traits, determined in 344 samples from individuals of Arab, South-Asian, and Filipino descent, and then replicate our findings in 46 subjects of European ancestry. We report protein-specific N-glycosylation patterns, including a correlation of core fucosylated structures with immunoglobulin G (IgG) levels, and of trisialylated, trigalactosylated, and triantennary structures with heparin cofactor 2 (SERPIND2). Our study reveals a detailed picture of protein N-glycosylation and suggests new avenues for the investigation of its role and function in the associated complex disorders.

In the originally published version of this Article, financial support was not fully acknowledged. The sentence “KS was supported by the ‘Biomedical Research Program’ funds at Weill Cornell Medicine in Qatar, a program funded by the Qatar Foundation” has been added to the acknowledgement section in both the PDF and HTML versions of the Article.

Nature Communications 8: Article number: 14357 (2017); Published 27 February 2017; Updated 11 April 2017 The original version of the Supplementary Information attached to this Article did not include Supplementary Note 1 The HTML has now been updated to include a corrected version of the Supplementary Information.