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For a long time, leaderless secreted proteins (LLSP) were neglected as artifacts derived from dying cells. It is now generally accepted that secretion of LLSP–as a part of the collective term unconventional protein secretion (UPS) - is an evolutionarily conserved process and that these LLSP are actively and selectively secreted from living cells bypassing the classical endoplasmic reticulum-Golgi pathway. However, the mechanism of UPS pathways, as well as the number of LLSP and which part of a protein is involved in the selection of LLSPs for secretion, are still enigmatic and await clarification. Secretomics-a proteomics-based approach to identify and quantify all proteins secreted by a cell-is inherently unbiased toward a particular secretion pathway and offers the opportunity to shed light on the UPS. Here, we will evaluate and present recent results of proteomic workflows allowing to obtain high-confident secretome data. Additionally, we address that cell culture conditions largely affect the composition of the secretome. This has to be kept in mind to control cell culture induced artifacts and adaptation stress in serum free conditions. Evaluation of click chemistry for secretome analysis of cells under serum-containing conditions showed a significant change in the cellular proteome with longer incubation time upon treatment with non-canonical amino acid azidohomoalanine. Finally, we showed that the number of LLSP far exceeds the number of secreted proteins annotated in Uniprot and ProteinAtlas. Thus, secretomics in combination with sophisticated microbioanalytical and sample preparation methods is well suited to provide a comprehensive picture of UPS.

T cell activation and differentiation is associated with metabolic reprogramming to cope with the increased bioenergetic demand and to provide metabolic intermediates for the biosynthesis of building blocks. Antigen receptor stimulation not only promotes the metabolic switch of lymphocytes but also triggers the uptake of calcium (Ca2+) from the cytosol into the mitochondrial matrix. Whether mitochondrial Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU) controls T cell metabolism and effector function remained, however, enigmatic. Using mice with T cell-specific deletion of MCU, we here show that genetic inactivation of mitochondrial Ca2+ uptake increased cytosolic Ca2+ levels following antigen receptor stimulation and store-operated Ca2+ entry (SOCE). However, ablation of MCU and the elevation of cytosolic Ca2+ did not affect mitochondrial respiration, differentiation and effector function of inflammatory and regulatory T cell subsets in vitro and in animal models of T cell-mediated autoimmunity and viral infection. These data suggest that MCU-mediated mitochondrial Ca2+ uptake is largely dispensable for murine T cell function. Our study has also important technical implications. Previous studies relied mostly on pharmacological inhibition or transient knockdown of mitochondrial Ca2+ uptake, but our results using mice with genetic deletion of MCU did not recapitulate these findings. The discrepancy of our study to previous reports hint at compensatory mechanisms in MCU-deficient mice and/or off-target effects of current MCU inhibitors.

Neurotransmitter release is mediated by an evolutionarily conserved machinery. The synaptic vesicle (SV) associated protein Mover/TPRGL/SVAP30 does not occur in all species and all synapses. Little is known about its molecular properties and how it may interact with the conserved components of the presynaptic machinery. Here, we show by deletion analysis that regions required for homomeric interaction of Mover are distributed across the entire molecule, including N-terminal, central and C-terminal regions. The same regions are also required for the accumulation of Mover in presynaptic terminals of cultured neurons. Mutating two phosphorylation sites in N-terminal regions did not affect these properties. In contrast, a point mutation in the predicted Calmodulin (CaM) binding sequence of Mover abolished both homomeric interaction and presynaptic targeting. We show that this sequence indeed binds Calmodulin, and that recombinant Mover increases Calmodulin signaling upon heterologous expression. Our data suggest that presynaptic accumulation of Mover requires homomeric interaction mediated by regions distributed across large areas of the protein, and corroborate the hypothesis that Mover functionally interacts with Calmodulin signaling.

Neutrophil Extracellular Traps (NETs) are produced by neutrophilic granulocytes and consist of decondensed chromatin decorated with antimicrobial peptides. They defend the organism against intruders and are released upon various stimuli including pathogens, mediators of inflammation, or chemical triggers. NET formation is also involved in inflammatory, cardiovascular, malignant diseases, and autoimmune disorders like rheumatoid arthritis, psoriasis, or systemic lupus erythematosus (SLE). In many autoimmune diseases like SLE or dermatomyositis, light of the ultraviolet-visible (UV-VIS) spectrum is well-known to trigger and aggravate disease severity. However, the underlying connection between NET formation, light exposure, and disease exacerbation remains elusive. We studied the effect of UVA (375 nm), blue (470 nm) and green (565 nm) light on NETosis in human neutrophils ex vivo. Our results show a dose- and wavelength-dependent induction of NETosis. Light-induced NETosis depended on the generation of extracellular reactive oxygen species (ROS) induced by riboflavin excitation and its subsequent reaction with tryptophan. The light-induced NETosis required both neutrophil elastase (NE) as well as myeloperoxidase (MPO) activation and induced histone citrullination. These findings suggest that NET formation as a response to light could be the hitherto missing link between elevated susceptibility to NET formation in autoimmune patients and photosensitivity for example in SLE and dermatomyositis patients. This novel connection could provide a clue for a deeper understanding of light-sensitive diseases in general and for the development of new pharmacological strategies to avoid disease exacerbation upon light exposure.

Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.