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International audience; The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.

Hyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described. In the current study we investigated a possible role of neutralizing autoantibodies against anti-inflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-α2, IFN-γ, IFN-ω and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations. In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN- nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF-α signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1ß signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality.To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB., Mitochondria in skeletal muscle have distinct localization and properties through unclear mechanisms. Here, the authors use complexome profiling and immunoprecipitations to identify PERM1 as a MICOS-MIB complex interactor that also binds ankyrin B, suggesting PERM1 directs the mitochondria to the membrane.

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly interconnected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3 (also known as Fermt3). In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5–10% of the kindlin-3 level of wild-type mice. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro-generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts, but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1(-/-) mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Background Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. Methods Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. Results Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. Conclusion Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups.

AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of Serum Response Factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise.CONCLUSION: Our results identify a new exercise signaling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.

Conference Report

Introduction: Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma. Methods: BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities. Results: The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications. Conclusions: A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. Disclosures Stilgenbauer: Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.