Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals.

Chronic infection with the neurotropic parasite Toxoplasma gondii has been implicated in the risk for several neuropsychiatric disorders. The mechanisms, by which the parasite may alter neural function and behavior of the host, are not yet understood completely. Here, a novel proteomic approach using mass spectrometry was employed to investigate the alterations in synaptic protein composition in a murine model of chronic toxoplasmosis. In a candidate-based strategy, immunoblot analysis and immunohistochemistry were applied to investigate the expression levels of key synaptic proteins in glutamatergic signaling. A comparison of the synaptosomal protein composition revealed distinct changes upon infection, with multiple proteins such as EAAT2, Shank3, AMPA receptor, and NMDA receptor subunits being downregulated, whereas inflammation-related proteins showed an upregulation. Treatment with the antiparasitic agent sulfadiazine strongly reduced tachyzoite levels and diminished neuroinflammatory mediators. However, in both conditions, a significant number of latent cysts persisted in the brain. Conversely, infection-related alterations of key synaptic protein levels could be partly reversed by the treatment. These results provide evidence for profound changes especially in synaptic protein composition in T. gondii-infected mice with a downregulation of pivotal components of glutamatergic neurotransmission. Our results suggest that the detected synaptic alterations are a consequence of the distinct neuroinflammatory milieu caused by the neurotropic parasite.

Inflammation, aberrant proteostasis, and energy depletion are hallmarks of neurodegenerative diseases such as multiple sclerosis (MS). However, the interplay between inflammation, proteasomal dysfunction in neurons, and its consequences for neuronal integrity remains unclear. Using transcriptional, proteomic, and functional analyses of proteasomal subunits in inflamed neurons, we found that interferon-γ-mediated induction of the immunoproteasome subunit, proteasome 20S beta 8 (PSMB8) impairs the proteasomal balance, resulting in reduced proteasome activity. This reduction causes the accumulation of phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key metabolic regulator, leading to enhanced neuronal glycolysis, reduced pentose phosphate pathway activity, oxidative injury, and ferroptosis. Neuron-specific genetic and systemic pharmacological targeting of PSMB8 or PFKFB3 protected neurons in vitro and in a mouse model of MS. Our findings provide a unifying explanation for proteasomal dysfunction in MS and possibly other neurodegenerative diseases, linking inflammation to metabolic disruption, and presenting an opportunity for targeted neuroprotective therapies.

Astrocytes exhibit regional heterogeneity in morphology, function and molecular composition to support and modulate neuronal function and signaling in a region‐specific manner. To characterize regional heterogeneity of astrocytic proteomes of different brain regions we established an inducible Aldh1l1‐methionyl‐tRNA‐synthetase^L274G (MetRS^L274G) mouse line that allows astrocyte‐specific metabolic labeling of newly synthesized proteins by azidonorleucine (ANL) in vivo and subsequent isolation of tagged proteins by click chemistry. We analyzed astrocytic proteins from four different brain regions by mass spectrometry. The induced expression of MetRS^L274G is restricted to astrocytes and identified proteins show a high overlap with proteins compiled in “AstroProt,” a newly established database for astrocytic proteins. Gene enrichment analysis reveals a high similarity among brain regions with subtle differences in enriched biological processes and in abundances of key astrocytic proteins for hippocampus, cortex and striatum. However, the cerebellar proteome stands out with proteins being highly associated with the calcium signaling pathway or with bipolar disorder. Subregional analysis of single astrocyte TAMRA intensities in hippocampal layers indicates distinct subregional heterogeneity of astrocytes and highlights the applicability of our toolbox to study differences of astrocytic proteomes in vivo.

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4⁺ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

The primary mRNA sequence determines its secondary structure and the repertoire of interacting RNA-binding proteins (RBPs). The resulting mRNA ribonucleoprotein complex (mRNP) then influences all stages of the life of an mRNA. Here, we determined the mRNP composition of individual Kaposi sarcoma herpesviral (KSHV) mRNAs. Like all herpesviruses, KSHV switches between a latent and lytic stage of the viral life cycle. During reactivation from latency, the viral RNA regulator ORF57 ensures the translation of viral mRNAs by increasing their mRNA stability and nuclear export. We optimized an LNA/DNA mixmer RNA capture protocol for both transfection and viral infection settings. In combination with eCLIP, we confirmed that ORF57 directly binds to an AU-rich RNA motif, which may enable ORF57 to discriminate viral from cellular RNAs based on the nucleotide bias of KSHV lytic RNAs. In addition, we captured the RBPome of two ORF57-dependent viral transcripts and identified the host RNA processing factor SRSF3 as a key regulator of viral replication.