Project description:Treatment of Mycobacterium tuberculosis infections is a challenging task due to a growing number of resistant clinical isolates as well as an almost empty drug development pipeline. To identify new antibiotic hits, we screened a focused library of 400 synthetic compounds derived from a recently discovered molecule with promising anti-mycobacterial activity. A suite of more potent hit molecules was deciphered with sub-micromolar activity. Utilising tailored affinity-based probes for chemical proteomic investigations, we successfully pinpointed the mycolic acid transporter MmpL3 and two epoxide hydrolases, EphD and EphF, also linked to mycolic acid biosynthesis, as specific targets of the compounds. These targets were thoroughly and independently validated by activity assays, under- and overexpression, resistance generation, and proteomic studies. Structural refinement of the most potent hit molecules led to the development of a new lead compound that demonstrates enhanced biological activity in M. tuberculosis, low human cytotoxicity, and improved solubility and oral bioavailability − traits that are often challenging to achieve with anti-mycobacterial drugs. Overall, drug-likeness, as well as the dual mode of action, addressing the mycolic acid cell wall assembly at two distinct steps, holds significant potential for further in vivo applications.
Project description:Insulin resistance is a hallmark of type 2 diabetes, a highly heterogeneous condition with diverse pathological characteristics. Understanding the molecular adaptation to insulin resistance and its association with individual phenotypic traits is crucial for advancing precision medicine in diabetes. By utilizing cutting-edge proteomics technology, we mapped the proteome and phosphoproteome of basal and insulin-stimulated skeletal muscle from >120 individuals with varying degrees of insulin sensitivity, both with and without type 2 diabetes. Leveraging deep in vivo phenotyping data, we reveal that the basal proteome and phosphoproteome are strong predictors of insulin sensitivity. The insulin-stimulated phosphoproteome identified preserved and dysregulated signaling even in individuals with the most severe insulin resistance. Our study elucidates substantial differences in the male and female (phospho)proteome; however, the molecular signature associated with insulin resistance remains largely similar between sexes. These findings emphasize the importance of recognizing disease heterogeneity and advocate for the precision medicine approach for effective care in type 2 diabetes.
Project description:The field of graft preservation has made considerable strides in recent years improving outcomes related to solid organ restoration and regeneration. In lungs, the use of ex vivo lung perfusion (EVLP) in line with devices and treatments has shown promising results within preclinical and clinical studies with the potential to improve graft quality. The benefit of the therapy would be to render marginal and declined donor lungs suitable for transplantation, ultimately increasing the donor pool available for transplantation. Additionally, such therapies used in machine perfusion could also increase preservation time, facilitating logistical planning. Cytokine adsorption has been demonstrated as a potentially safe and effective therapy when applied to the EVLP circuit and post transplantation. The mechanism by which this treatment improves the donor lung on a molecular basis is not yet fully elucidated. We hypothesized that there were characteristic inflammatory and immunomodulatory differences between lungs treated with and without cytokine adsorption, reflecting in proteomic changes in gene ontology pathways and across inflammation-related proteins. In the current study we investigate the molecular mechanisms and signaling pathways of how cytokine adsorption impacts the lung function when used during EVLP and when used post transplantation as hemoperfusion in a porcine model. Lung tissue from EVLP and post lung transplantation were analyzed for their proteomic profile using mass spectrometry. The inflammatory and immune processes were compared between the treated and the non-treated groups to show the differences occurring between the forms of graft preservation.
Project description:Prediction of proteins and associated biological pathways from lipid analyses via MALDI MSI is a pressing chal-lenge. We introduced "dry proteomics," using MALDI MSI to validate spatial localization of identified optimal clusters in lipid or protein imaging. Consistent cluster appearance across omics images suggests association with specific lipid and protein pathways, forming the basis of dry proteomics. The methodology was refined using rat brain tissue as a model, then applied to human glioblastoma, a highly heterogeneous cancer. Sequen-tial tissue sections underwent omics MALDI MSI and unsupervised clustering. Differentiated lipid and protein clusters, with distinct spatial locations, were identified. Spatial omics analysis facilitated lipid and protein charac-terization, leading to a predictive model identifying clusters in any tissue based on unique lipid signatures and predicting associated protein pathways. Application to rat brain slices revealed diverse tissue subpopulations, including successfully predicted cerebellum areas. Similar analysis on 50 glioblastoma patients confirmed lipid-protein associations, correlating with patient prognosis.
Project description:The age and sex of studied animals profoundly impacts experimental outcomes in animal-based biomedical research. However, most preclinical studies in mice use a wide-spanning age range from 4 to 14 weeks and do not assess study parameters in male and female mice in parallel. This raises concerns regarding reproducibility and neglect of potentially relevant age and sex differences. Furthermore, the molecular setup of tissues in dependence of age and sex is unknown in naïve mice precluding efficient translational research. Here, we first compared two different mass spectrometric acquisition methods – DDA- and DIA-PASEF – in order to maximize the depth of proteome quantitation. We then employed an optimized workflow of quantitative proteomics based on DIA-PASEF followed by DIA-NN data analysis, and revealed significant differences in mouse paw skin and sciatic nerve (SCN) when comparing (i) male and female mice, and, in parallel, (ii) adolescent mice (4 weeks) with adult mice (14 weeks).
Project description:Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1) and other gluconeogenic enzymes. “GID” is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core and supramolecular module along with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex targeting Fbp1. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.
Project description:N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.
Project description:N6-methyladenosine (m6A) is the most prevalent mRNA modification with diverse regulatory roles in mammalian cells. While its functions are well-documented in mouse embryonic stem cells (mESCs), its role in human pluripotent stem cells (hPSCs) remains to be fully explored. METTL3 is the main enzyme responsible for m6A deposition. Here, using a METTL3 inducible knockout (iKO) system, we uncovered that, unlike in mESCs, METTL3 was indispensable for hPSC maintenance. Importantly, loss of METTL3 caused significant upregulation of pluripotency factors including naïve pluripotency genes and failure to exit pluripotency, thus impaired stem cell differentiation towards embryonic and extraembryonic cells including trophoblasts. Mechanistically, METTL3 iKO in hPSCs substantially increased expression and enhancer activities of two primate-specific transposable elements (TEs), SVA_D and HERVK/LTR5_Hs, which are normally modified by METTL3-dependent m6A. METTL3 loss activated SVA_D by lowering H3K9me3 deposition, and increased chromatin accessibility at LTR5_Hs through the naïve and other pluripotency factors. Conversely, we discovered that the activated SVA_D and LTR5_Hs loci positively regulated naïve gene expression by directly interacted with their promoters. These findings thus reveal that METTL3-dependent m6A RNA methylation has critical roles in suppressing TE expression and in the human pluripotency regulatory network.