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The prediction of protein localization, such as in the extracellular space, from high-throughput data is essential for functional downstream inference. It is well accepted that some secreted proteins go through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide. However, a large number of proteins have been found to reach the extracellular space by following unconventional secretory pathways. There remains a demand for reliable prediction of unconventional protein secretion (UPS). Here, we present OutCyte, a fast and accurate tool for the prediction of UPS, which for the first time has been built upon experimentally determined UPS proteins. OutCyte mediates the prediction of protein secretion in two steps: first, proteins with N-terminal signals are accurately filtered out; second, proteins without N-terminal signals are classified as UPS or intracellular proteins based on physicochemical features directly generated from their amino acid sequences. We are convinced that OutCyte will play a relevant role in the annotation of experimental data and will therefore contribute to further characterization of the extracellular nature of proteins by considering the commonly neglected UPS proteins. OutCyte has been implemented as a web server at www.outcyte.com .

Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium Synechocystis sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.

Class I glutaredoxins (GRXs) are nearly ubiquitous proteins that catalyse the glutathione (GSH)-dependent reduction of mainly glutathionylated substrates. In land plants, a third class of GRXs has evolved (class III). Class III GRXs regulate the activity of TGA transcription factors through yet unexplored mechanisms. Here we show that Arabidopsis thaliana class III GRX ROXY9 is inactive as an oxidoreductase on widely used model substrates. Glutathionylation of the active site cysteine, a prerequisite for enzymatic activity, occurs only under highly oxidizing conditions established by the GSH/glutathione disulfide (GSSG) redox couple, while class I GRXs are readily glutathionylated even at very negative GSH/GSSG redox potentials. Thus, structural alterations in the GSH binding site leading to an altered GSH binding mode likely explain the enzymatic inactivity of ROXY9. This might have evolved to avoid overlapping functions with class I GRXs and raises questions of whether ROXY9 regulates TGA substrates through redox regulation. Plant-specific class III glutaredoxins regulate the activity of TGA transcription factors. Here, the authors show that that ROXY9, a member of the class III of glutaredoxins, lacks oxidoreductase activity due to unfavourable positioning of glutathione. Consequently, class III glutaredoxins may not regulate gene expression through redox modifications of target proteins.

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii , an invasive intracellular apicomplexan protozoan parasite of global importance. T . gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host’s intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T . gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T . gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T . gondii . Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T . gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T . gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2-coordinated cell-autonomous host defense mechanism against T . gondii .

Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of ‘DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)’. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted ‘skin aging–associated secreted proteins (SAASP)’. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of ‘metabolism’ and ‘adherens junction interactions’ was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

The non-photochemical quenching of light energy as heat (NPQ) is an important photoprotective mechanism that is activated in plants when light absorption exceeds the capacity of light utilization in photosynthesis. The PsbS protein plays a central role in this process and is supposed to activate NPQ through specific, light-regulated interactions with photosystem (PS) II antenna proteins. However, NPQ-specific interaction partners of PsbS in the thylakoid membrane are still unknown. Here, we have determined the localization and protein interactions of PsbS in thylakoid membranes in the NPQ-inactive (dark) and NPQ-active (light) states. Our results corroborate a localization of PsbS in PSII supercomplexes and support the model that the light activation of NPQ is based on the monomerization of dimeric PsbS and a light-induced enhanced interaction of PsbS with Lhcb1, the major component of trimeric light-harvesting complexes in PSII. In photosynthesis, excess light energy is dissipated as heat by non-photochemical quenching (NPQ). The localization of proteins and their interactions in thylakoid membranes under high and low light conditions suggests that NPQ involves monomerization of the dimeric protein PsbS and interaction of the resulting monomers with components of trimeric light-harvesting complexes of photosystem II.

Somatic mutations in hematopoietic stem/progenitor cells (HSPCs) can lead to clonal hematopoiesis of indeterminate potential (CHIP) and progression to myelodysplastic syndromes (MDS). Using single-cell and anatomical profiling of a large cohort of human bone marrow (BM), we show that the HSPC BM niche in CHIP and MDS is undergoing inflammatory remodeling. This includes loss of CXCL12⁺ adipogenic stromal cells and the emergence of a distinct population of inflammatory mesenchymal stromal cells (iMSCs), which arise in CHIP and become more prevalent in MDS. Functional studies in primary BM HSPC-MSC co-cultures reveals that healthy aged and CHIP HSPCs activate stromal support, while MDS HSPCs fail to do so. In contrast, MDS blasts further suppress HSPC support and trigger inflammation, indicating disease-stage-specific stromal disruption. In parallel, we show that iMSCs retain partial support and angiogenic potential in MDS, coinciding with expanded BM vasculature. Additionally, we identify IFN-responsive T cells that preferentially interact with iMSCs, potentially reinforcing local inflammation. These findings position iMSCs as central mediators of early BM niche dysfunction and potential therapeutic targets for intercepting pre-malignant hematopoiesis. Clonal haematopoiesis of indeterminate potential is driven by somatic mutations in hematopoietic stem/progenitor cells and may progress to myelodysplastic syndromes (MDS). Here authors show that the two conditions share a similar pattern of bone marrow remodeling, characterized by the emergence of inflammatory mesenchymal stromal cells and IFN-responsive T cells, reinforcing their shared etio-pathology.

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (M pro ), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that M pro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit M pro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites. Here the authors demonstrate that the SARS-CoV-2 main protease (Mpro) is subject to redox regulation in vitro, reversibly switching between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues.

Genome-wide association studies in combination with functional analyses identify a genetic variant that explains why anti-tumour necrosis factor therapy, used in several autoimmune diseases, exacerbates multiple sclerosis. Genetic variation in multiple sclerosis Recent genome-wide association studies (GWAS) have indicated an association between multiple sclerosis and a single-nucleotide polymorphism in the TNFRSF1A gene that encodes tumour necrosis factor (TNF) receptor 1 (TNFR1). TNF has previously been implicated in autoimmunity and TNF antagonists are effective treatments in several autoimmune diseases, but not in multiple sclerosis. Interestingly, GWAS evidence shows no link between TNFRSF1A and multiple sclerosis. This study compares GWAS results across different autoimmune conditions, as well as findings from functional and biophysical investigations, to show that multiple sclerosis-associated genetic risk at the TNFR1 locus results in the generation of a novel, endogenous TNF antagonist. This genetic-risk effect parallels the effects of anti-TNF therapy, which has been reported — in rare cases — to induce clinical onset of multiple sclerosis. Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS) 1 , it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these variants make to disease risk has raised questions regarding their medical relevance 2 . Here we have investigated a single nucleotide polymorphism (SNP) in the TNFRSF1A gene, that encodes tumour necrosis factor receptor 1 (TNFR1), which was discovered through GWAS to be associated with multiple sclerosis (MS) 3 , 4 , but not with other autoimmune conditions such as rheumatoid arthritis 5 , psoriasis 6 and Crohn’s disease 7 . By analysing MS GWAS 3 , 4 data in conjunction with the 1000 Genomes Project data 8 we provide genetic evidence that strongly implicates this SNP, rs1800693, as the causal variant in the TNFRSF1A region. We further substantiate this through functional studies showing that the MS risk allele directs expression of a novel, soluble form of TNFR1 that can block TNF. Importantly, TNF-blocking drugs can promote onset or exacerbation of MS 9 , 10 , 11 , but they have proven highly efficacious in the treatment of autoimmune diseases for which there is no association with rs1800693. This indicates that the clinical experience with these drugs parallels the disease association of rs1800693, and that the MS-associated TNFR1 variant mimics the effect of TNF-blocking drugs. Hence, our study demonstrates that clinical practice can be informed by comparing GWAS across common autoimmune diseases and by investigating the functional consequences of the disease-associated genetic variation.

Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione–disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S -glutathionylation of the NAD ⁺-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S -glutathionylation to vertebrate development and successful embryonic angiogenesis.