Autoantibodies Targeting Nephrin in Podocytopathies.

BACKGROUND: Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. METHODS: We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. RESULTS: The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. CONCLUSIONS: In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

HUMOS: How to Understand My Orbitrap Spectrum?-An Interactive Web-Based Tool to Teach the Basics of Mass-Spectrometry-Based Proteomics.

The Orbitrap mass analyzer can provide high mass accuracy and throughput, which has significantly improved proteome research and made this type of instrumentation one of the most frequently applied in proteomics. The efficient use of Orbitrap mass spectrometers requires training. Students in the field of proteomics can benefit from a deeper understanding of the Orbitrap technology to comprehend mass spectral interpretation, troubleshooting, and judgment of experimental settings. Unfortunately, the cost of high-end mass spectrometers limits the implementation of this type of equipment in educational laboratories. Guided by these concerns, we developed an eLearning web application called HUMOS aimed to help teach Orbitrap mass spectrometry. Although a typical proteomics experiment includes the use of several different technologies, such as liquid chromatography, mass spectrometry, and bioinformatics, the learning objectives of HUMOS are focused on mass spectrometry. HUMOS models a mass spectrum of a peptide mixture, allowing us to investigate the influence of mass spectral acquisition parameters. By changing the parameters and observing the differences, students can learn more about the mass spectral resolution, duty cycle, throughput of the analysis, ion accumulation, and spectral dynamic range and get familiar with advanced spectral acquisition methods, such as BoxCar. HUMOS is an open-source software published under the Apache license; the live installation is available at http://humos.bmb.sdu.dk.

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

Remodeling of ER-plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis.

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.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.

Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Genome-wide identification and functional analysis of Apobec-1-mediated C-to-U RNA editing in mouse small intestine and liver.

BACKGROUND: RNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored. RESULTS: Deep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required. CONCLUSIONS: These studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific.

Membrane glucocorticoid receptor activation induces proteomic changes aligning with classical glucocorticoid effects.

Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function.

Identification of differentially expressed proteins in curcumin-treated prostate cancer cell lines.

Due to high prevalence and slow progression of prostate cancer, primary prevention appears to be attractive strategy for its eradication. During the last decade, curcumin (diferuloylmethane), a natural compound from the root of turmeric (Curcuma longa), was described as a potent chemopreventive agent. Curcumin exhibits anti-inflammatory, anticarcinogenic, antiproliferative, antiangiogenic, and antioxidant properties in various cancer cell models. This study was designed to identify proteins involved in the anticancer activity of curcumin in androgen-dependent (22Rv1) and -independent (PC-3) human prostate cancer cell lines using two-dimensional difference in gel electrophoresis (2D-DIGE). Out of 425 differentially expressed spots, we describe here the MALDI-TOF-MS analysis of 192 spots of interest, selected by their expression profile. This approach allowed the identification of 60 differentially expressed proteins (32 in 22Rv1 cells and 47 in PC-3 cells). Nineteen proteins are regulated in both cell lines. Further bioinformatic analysis shows that proteins modulated by curcumin are implicated in protein folding (such as heat-shock protein PPP2R1A; RNA splicing proteins RBM17, DDX39; cell death proteins HMGB1 and NPM1; proteins involved in androgen receptor signaling, NPM1 and FKBP4/FKBP52), and that this compound could have an impact on miR-141, miR-152, and miR-183 expression. Taken together, these data support the hypothesis that curcumin is an interesting chemopreventive agent as it modulates the expression of proteins that potentially contribute to prostate carcinogenesis.

Proteomic profiling of rapid non-genomic and concomitant genomic effects of acute restraint stress on rat thymocytes.

In order to investigate rapid non-genomic effects of acute stress, rats were restrained for 15 min which was sufficient to activate the hypothalamus-pituitary-adrenal (HPA) axis but too short to induce massive genomic effects of cortisol. Subcellular fractions of thymocytes (cytosol, nucleus, membrane) were investigated using quantitative 2D DIGE with MALDI-TOF/TOF mass spectrometry. In total, 108 proteins with differential subcellular localizations were identified. The specificity of the changes induced by psychological stress was reflected by the prominent modulation of proteins involved in the HPA and sympathoadrenal medullar (SAM) axis such as HMGB1 and NHERF1. Intracellular trafficking was characterized by a dominant protein exodus from the cytosol. Real translocation was observed for 9 proteins with 6 that shuttled from the cytosol to the nucleus (HYOU1, HNRPF, HNRPC, STRAP, PSA1, PPA1) and 3 from the nucleus to the cytosol (HMGB1, NHERF1, PSMA1). Proteins showing subcellular reshuffling were largely involved in transcription and translation processes (39 of 108) with a significant enrichment of RNA splicing factors. Bioinformatics analysis revealed significant enrichment for protein kinase A and 14-3-3 signaling, probably reflecting real non-genomic effects. This is the first study investigating rapid effects of stress-induced HPA activation in vivo at the proteome level.

Cortisol is a potent modulator of lipopolysaccharide-induced interferon signaling in macrophages.

The effects of cortisol (CORT) on resting and lipopolysaccharide (LPS)-activated monocyte-derived THP-1 macrophages were investigated by proteomics. Forty-seven proteins were found to be modulated, 20 by CORT, 11 by LPS, and 16 by CORT and LPS. Cortisol-sensitive chaperones and cytoskeletal proteins were mostly repressed. HCLS1, MGN, and MX1 were new proteins identified to be under the transcriptional control of this steroid and new CORT-sensitive variants of MX1, SYWC and IFIT3 were found. FKBP51, a known CORT target gene, showed the strongest response to CORT and synergism with LPS. In resting THP-1 macrophages, 18 proteins were modulated by CORT, with 15 being down-regulated. Activation of macrophages by LPS was associated with enhanced expression of immune response and metabolic proteins. In activated macrophages, CORT had a more equilibrated effect and almost all metabolism-related proteins were up-regulated, whereas immune response proteins were mostly down-regulated. The majority of the LPS up-regulated immune response-related proteins are known interferon (IFN) target genes (IFIT3, MX1, SYWC, PSME2) suggesting activation of the IRF3 signaling pathway. They were all suppressed by CORT. This is the first proteomics study to investigate the effects of CORT on activated immune cells.