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This proteomic study provides a nuanced mechanistic understanding of the signaling processes upon agonist binding to the melanocortin-3 receptor (MC3R). Utilizing thermal proteome profiling (TPP) combined with LC–MS, we uncovered the distinct influences of the endogenous agonists adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and γ-melanocyte-stimulating hormone (γ-MSH) on protein thermal stability and pathway activation. In our 2D-TPP study, transfected HEK293 cells for expression of MC3R were exposed to the three endogenous MC3R-ligands across several concentrations followed by incubation at several temperatures, centrifugation and LC–MS analysis of the resulting supernatants. This enabled us to assess the effects of type of ligand and concentration on the thermal stability of proteins in these cells. We employed a combination of multivariate analysis, differential expression, TPP and pathway analysis to deeply characterize the impact of MC3R activation on molecular mechanisms. All three ligands affected signaling pathways related to the immune system and energy homeostasis. While α-MSH significantly modulated the IL-6 pathway via STAT3, and γ-MSH prominently activated interferon signaling, ACTH uniquely affected NADPH-related proteins. All ligands shared involvement in the cAMP-PKA-CREB and varied impacts on PI3K and ERK pathways, crucial for energy metabolism. All proteomic data are available under DOI: https://doi.org/10.6019/PXD039945.

The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 x 10-6 with an enrichment in active promoter regions.

BACKGROUND: γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinate to regulate GABA-mediated T cell immunomodulation is still being examined.

METHODS: CD4⁺ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated in vitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca²⁺ imaging.

FINDINGS: We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4⁺ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABA_A receptor-subunit ρ2 expression, enhanced the GABA_A receptors-mediated currents and Ca²⁺ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner.

INTERPRETATION: These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions.

FUNDING: The Swedish Children's Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.

Given the rapid development of data-driven experimental procedures within the life sciences, it is important to equip students with proper skills and knowledge on how to obtain and interpret complex data. While laboratory exercises have for a long time been well established as a teaching method for life sciences, we consider there is room for improvement in how laboratory exercises are conducted. Hence, we designed a laboratory exercise course in which students at the graduate level in the European education system (M.Sc.) are challenged to pose their own biological question and write their own laboratory protocol for a proteomic study to investigate their hypothesis. Here, students are supported in their task with lectures and seminars that take them through the required details on experimental sample preparation, analysis with LC-MS, and proteomic data evaluation of biological function and relevance. According to student interviews, the inquiry-based learning concept we used here provided a deeper understanding of the laboratory protocols they wrote, according to which they eventually performed their own experiments.

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.

Filter-aided sample preparation (FASP) is widely used in bottom-upproteomics for tryptic digestion. However, the sample recovery yield of this methodis limited by the amount of the starting material. While similar to 100 ng of digested protein issufficient for thorough protein identification, proteomic information gets lost with aprotein content <10 mu g due to incomplete peptide recovery from thefilter. Wedeveloped and optimized aflexible well-plate mu FASP device and protocol that issuitable for an similar to 1 mu g protein sample. In 1 mu g of HeLa digest, we identified 1295 +/- 10proteins with mu FASP followed by analysis with liquid chromatography-massspectrometry. In contrast, only 524 +/- 5 proteins were identified with the standardFASP protocol, while 1395 +/- 4 proteins were identified in 20 mu g after standard FASPas a benchmark. Furthermore, we conducted a combined peptidomic and proteomicstudy of single pancreatic islets with well-plate mu FASP. Here, we separated neuropeptides and digested the remaining on-filterproteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved inglucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a usefultool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI:10.6019/PXD029039

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l-methadone and d-methadone and their major metabolites, l- and d-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated gamma-cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d-methadone concentrations were lower than those of l-methadone and the d-EDDP levels were lower than those of L-EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two-compartment pharmacokinetic model. l-methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady-state volume of distribution than d-methadone. d-methadone and d-EDDP were eliminated faster than their respective l-enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.

Alzheimer’s disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-β (Aβ) peptide. A potential therapeutic intervention in Alzheimer’s disease is to enhance Aβ degradation by increasing the activity of Aβ-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aβ42 degradation in the hippocampus of mice overexpressing the Aβ-precursor protein with the Swedish mutation (APPswe). Using LC–MS, we further evaluated the anti-Alzheimer’s disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC–MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer’s disease therapies based on SST peptides.