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The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella , which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection. Flagellin proteins of Salmonella flagella are methylated. Here, the authors show that flagellin methylation facilitates adhesion of Salmonella to hydrophobic host-cell surfaces, and contributes to efficient gut colonization and host infection.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis. The ER chaperone BiP is critical for the unfolded protein response and tightly regulated through reversible AMPylation by FICD, but the structural basis is unknown. Here the authors use thiol-reactive nucleotide derivatives to stabilize the transient FICD:BiP complex and determine its crystal structure.

The Na,K-ATPase (NKA) is an essential ion transporter and signaling molecule in all animal tissues and believed to consist at least one α and one ß-subunit to form a functional enzyme. In the large milkweed bug, Oncopeltus fasciatus , adaptation to dietary cardiac glycosides (CGs), which can fatally block the NKA, has resulted in gene duplications leading to four α1-subunits. These differ in sensitivity to CGs, but resistance trades off against ion pumping activity, thus influencing the α1-subunits’ suitability for specific tissues. Besides, O. fasciatus possesses four different ß-subunits that can alter the NKA's kinetics and should play an essential role in the formation of cellular junctions. Proteomic analyses revealed the distribution and composition of α1/ß-complexes in the nervous tissue of O. fasciatus. The highly CG-resistant, but less active α1B and the highly active, but less resistant α1C predominated in the nervous tissue and co-occurred with ß2 and ß3, partly forming larger complexes than just heterodimers. Immunohistochemical analyses provided a fine scale resolution of the subunits’ distribution in different morphological structures of the nervous tissue. This may suggest that α1 as well as ß-subunits occur in isolation without the other subunit, which contradicts the present understanding that the two types of subunits have to associate to form functional complexes. An isolated occurrence was especially prominent for ß3 and βx, the enigmatic fourth and N-terminally largely truncated ß-subunit. We hypothesize that dimerization of these ß-subunits plays a role in cell–cell contacts.

Dataset integration is common practice to overcome limitations in statistically underpowered omics datasets. Proteome datasets display high technical variability and frequent missing values. Sophisticated strategies for batch effect reduction are lacking or rely on error-prone data imputation. Here we introduce HarmonizR, a data harmonization tool with appropriate missing value handling. The method exploits the structure of available data and matrix dissection for minimal data loss, without data imputation. This strategy implements two common batch effect reduction methods—ComBat and limma (removeBatchEffect() ). The HarmonizR strategy, evaluated on four exemplarily analyzed datasets with up to 23 batches, demonstrated successful data harmonization for different tissue preservation techniques, LC-MS/MS instrumentation setups, and quantification approaches. Compared to data imputation methods, HarmonizR was more efficient and performed superior regarding the detection of significant proteins. HarmonizR is an efficient tool for missing data tolerant experimental variance reduction and is easily adjustable for individual dataset properties and user preferences. Dataset integration is common practice to overcome limitations in statistically underpowered omics datasets. Here the authors present “HarmonizR”, a tool for missing data tolerant experimental variance reduction in large, integrated but independently generated datasets without data imputation, adjustable for individual dataset modalities, correction algorithm, and user preferences.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3' of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

Osteoporosis is caused by increased bone resorption and decreased bone formation. Intermittent administration of a fragment of Parathyroid hormone (PTH) activates osteoblast-mediated bone formation and is used in patients with severe osteoporosis. However, the mechanisms by which PTH elicits its anabolic effect are not fully elucidated. Here we show that the absence of the homeodomain protein TG-interacting factor 1 (Tgif1) impairs osteoblast differentiation and activity, leading to a reduced bone formation. Deletion of Tgif1 in osteoblasts and osteocytes decreases bone resorption due to an increased secretion of Semaphorin 3E (Sema3E), an osteoclast-inhibiting factor. Tgif1 is a PTH target gene and PTH treatment failed to increase bone formation and bone mass in Tgif1-deficient mice. Thus, our study identifies Tgif1 as a novel regulator of bone remodeling and an essential component of the PTH anabolic action. These insights contribute to a better understanding of bone metabolism and the anabolic function of PTH. Parathyroid hormone (PTH) is used to treat osteoporosis, but its therapeutic mechanism remains unclear. Here, the authors show that Tgif1 is a PTH target gene, and that its deletion impairs the function of osteoblasts and PTH-induced bone formation in mice.

Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. Here we show that tumors producing high levels of granulocyte colony-stimulating factor (G-CSF) promote the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma due to neutrophil reprogramming. Mechanistically, we identify tumor-driven activation of the G-CSF / nicotinamide phosphoribosyltransferase (NAMPT) signaling axis in neutrophil progenitors, resulting in impaired antibacterial functions, such as phagocytosis and neutrophil extracellular traps formation, and development of tissue-damaging neutrophil subsets. This disrupts lung tissue integrity and facilitates bacterial persistence. Importantly, targeting the G-CSF/NAMPT pathway prevents the generation of dysfunctional neutrophils and improves bacterial clearance in vivo. Our findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central driver of compromised antimicrobial defenses in cancer. Therapeutic strategies aimed at modulating G-CSF/NAMPT signaling could enhance infection control and survival for cancer patients. Cancer patients are at increased risk for severe bacterial infections due to immune dysfunction. Here, the authors show that chronic tumor-derived G-CSF drives NAMPT/NAD-dependent neutrophil dysfunction from the progenitor stage, and that targeting this pathway restores infection control.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids – a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease. Polyunsaturated Fatty Acids (PUFA), such as omega-3 fatty acids, are recognized for their lipid lowering and anti-inflammatory properties. Here, the authors show that endogenous lipid synthesis controls the use of PUFA and thus determine the therapeutic benefit of omega-3 fatty acid supplementation.

Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl- sn -glycero-3-phospho-(1’-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1 -defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling. Fatty acid unsaturation by stearoyl-CoA desaturase 1 (SCD1) protects against cellular stress through unclear mechanisms. Here the authors show 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) is an SCD1-derived signaling lipid that regulates stress-adaption, protects against cell death and promotes proliferation.

Background The survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy. Methods Screening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student’s T-test was performed. Results Among those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4 plus ) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4 plus cells) significantly increased polyglutamylation of β-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4 plus cells. EVs derived from TTLL4 plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer. Conclusions These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4 plus -derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.