Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between beta-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.Peer reviewe

Skeletal muscle proteomes reveal downregulation of mitochondrial proteins in transition from prediabetes into type 2 diabetes

Skeletal muscle insulin resistance is a central defect in the pathogenesis of type 2 diabetes (T2D). Here, we analyzed skeletal muscle proteome in 148 vastus lateralis muscle biopsies obtained from men covering all glucose tolerance phenotypes: normal, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D. Skeletal muscle proteome was analyzed by a sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics technique. Our data indicate a downregulation in several proteins involved inmitochondrial electron transport or respiratory chain complex assembly already in IFG and IGT-muscles, with most profound decreases observed in T2D. Additional phosphoproteomic analysis reveals altered phosphorylation in several signaling pathways in IFG, IGT, and T2D muscles, including those regulating glucose metabolic processes, and the structure of muscle cells. These data reveal several alterations present in skeletalmuscle already in prediabetes and highlight impairedmitochondrial energy metabolism in the trajectory from prediabetes into T2D.Peer reviewe

Physical and functional interactome atlas of human receptor tyrosine kinases

Funding Information: We thank S. Miettinen for technical assistance and Professors Matthias Gstaiger, Aki Manninen, and Kaisa Lehti for critical reading and comments on the manuscript. This study was supported by grants from the Academy of Finland (nos. 288475 and 294173), the Sigrid Jusélius Foundation, the Finnish Cancer Foundation, the University of Helsinki Three‐year Research Grant, Biocentrum Helsinki, Biocentrum Finland, HiLIFE, Magnus Ehrnrooth Foundation, and the Instrumentarium Research Foundation. Open access funding enabled and organized by ProjektDEAL. Publisher Copyright: © 2022 The Authors. Published under the terms of the CC BY 4.0 license.Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK–protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.Peer reviewe

Askeleita kohti verkkokalvon irtauman jälkeisen arven lääkkeellistä estoa

Vertaisarvioitu.Verkkokalvon irtauma on näköä uhkaava sairaus, jossa solu- ja molekyylitason muutokset aiheuttavat akuutin tulehdusreaktion ja irtaantuneen verkkokalvon hapenpuutteen. Verkkokalvon irtauman hoidon kehitys perustuu edistyneen silmäkirurgian lisäksi lasiais- ja verkkokalvosairauksien solu- ja molekyylitason perusmekanismien ymmärtämiseen. Pitkittyneessä verkkokalvon irtaumassa syntyneen kollageenin ja muiden sidekudoskomponenttien hajotus häiriintyy ja kehittyy pysyvä kudosvaurio, proliferatiivinen vitreoretinopatia. Proteomiikkatutkimusten lisäksi transkriptomiikka, epigenomiikka ja metabolomiikka ovat lisänneet ymmärrystä verkkokalvon irtaumaan liittyvistä arpimuodostusmekanismeista. Tuoreet tutkimukset ovat osoittaneet, että kolesterolipitoisuutta pienentävät statiinit saattavat hillitä akuutin vaiheen tulehdusreaktioita, vähentää lasiaisen tulehduksellista sytokiinituotantoa ja parantaa verkkokalvon irtauman leikkausennustetta.Peer reviewe

Insights into Preservation of Blood Biomarkers in Biobank Samples

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FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2(+) breast cancer cells likewise led to increased MST1/2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2(+) MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2(+) breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2-induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.Peer reviewe

A multi-omics study to characterize the transdifferentiation of human dermal fibroblasts to osteoblast-like cells

Background: Various skeletal disorders display defects in osteoblast development and function. An in vitro model can help to understand underlying disease mechanisms. Currently, access to appropriate starting material for in vitro osteoblastic studies is limited. Native osteoblasts and their progenitors, the bone marrow mesenchymal stem cells, (MSCs) are problematic to isolate from affected patients and challenging to expand in vitro. Human dermal fibroblasts in vitro are a promising substitute source of cells. Method: We developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy and differentiated them into osteoblast-like cells with ss-glycerophosphate, ascorbic acid, and dexamethasone treatment. Osteoblastic phenotype was confirmed by staining for alkaline phosphatase (ALP), calcium and phosphate deposits (Alizarin Red, Von Kossa) and by a multi-omics approach (transcriptomic, proteomic, and phosphoproteomic analyses). Result: After 14 days of treatment, both fibroblasts and MSCs (reference cells) stained positive for ALP together with a significant increase in bone specific ALP (p = 0.04 and 0.004, respectively) compared to untreated cells. At a later time point, both cell types deposited minerals, indicating mineralization. In addition, fibroblasts and MSCs showed elevated expression of several osteogenic genes (e.g. ALPL, RUNX2, BMPs and SMADs), and decreased expression of SOX9. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell types (p_adj. = 0.003 and 0.004, respectively). Discussion: These data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system. This culturing system provides an alternative tool for bone biology research and skeletal tissue engineering.Peer reviewe

Predicting tubal factor infertility by using markers of humoral and cell-mediated immune response against Chlamydia trachomatis

Problem: The accuracy of Chlamydia trachomatis antibody test in predicting tubal factor infertility (TFI) is limited, and more accurate methods are needed. Cell-mediated immune response (CMI) is crucial in the resolution of pathogen, but it may play an important role in the pathogenesis of C trachomatis-associated tubal damage. We studied whether combining the markers of C trachomatis-induced CMI to humoral immune response improves the accuracy of serology in TFI prediction. Method of study: Our prospective study consists of 258 subfertile women, of whom 22 (8.5%) had TFI. Women with other causes for subfertility served as a reference group. Serum C trachomatis major outer membrane protein (MOMP) and chlamydial heat-shock protein 60 (cHSP60) IgG antibodies were measured by ELISA. CMI was studied by lymphocyte proliferation assay in vitro. Results: Serological markers were more prevalent in women with TFI than in other subfertile women (40.9% vs 12.3% for MOMP IgG and 27.3% vs 10.2% for cHSP60 IgG). The best test combination for TFI was C. trachomatis MOMP and cHSP60 antibody with an accuracy of 90.3%, sensitivity of 22.7% and specificity of 96.6%. Positive post-test probability of this combination was 54.2%, and negative post-test probability was 12.4%. Adding of the markers of CMI did not significantly improve the accuracy of serology in TFI prediction. Conclusion: The accuracy of TFI prediction increases when the combination of C trachomatis MOMP and cHSP60 antibody tests is used. C trachomatis-induced CMI was common in our study population, but the markers of CMI did not predict TFI.Peer reviewe

The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.Peer reviewe

A beta2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation

beta2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of beta2-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the b2-integrin (TTT/AAA-b2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-b2-integrin KI dendritic cells, which leads to a failure ofMRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of b2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of b2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.Peer reviewe