Project description:Members of the Protein Kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified over three-hundred putative targets of PKD3. Further, biochemical analysis revealed that PKD3 regulates cAMP-dependent protein kinase A (PKA) activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes
Project description:The tRNA linked repeat sequence rrB was tagged in either 5'- or 3'- end with the MS2 aptamer and expressed from a plasmid in E. coli. Using the MS2 aptamer, the RNAs were affinity purified after cell lysis. The co-purifying proteins were identified by NanoLC-MS/MS and compared to the proteins co-purifying with the MS2 sequence alone.
Project description:Bacteria spatially confine cellular processes like protease secretion and signal transduction in membrane platforms termed functional membrane microdomains, which in certain organisational and functional features resemble the lipid rafts of eukaryotic cells. However, a rigorous understanding of their composition, assembly and biological significance is unknown. Here we use the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) to show that the organization of these platforms requires a preferential interaction between unphosphorylated membrane saccharolipids and the scaffold protein flotillin. This interaction leads to their accumulation in specific membrane microdomains concomitantly to the attraction of membrane-associated multimeric complexes, for which flotillin promotes efficient oligomerization. One of these harbored proteins is the penicillin-binding protein PBP2a, responsible for penicillin resistance in MRSA. We took PBP2a as showcase to demonstrate that flotillin mutants are also defective in PBP2a oligomerization and activity. Thus, perturbation of microdomains assembly, using commercially available drugs, interferes with PBP2a oligomerization and causes a relapse of MRSA penicillin resistance in vitro and in vivo, resulting in MRSA infections susceptible to conventional penicillin treatments. Our study shows that bacterial cells organize sophisticated programs for cellular compartmentalization and unravels a novel strategy to develop antimicrobial therapies for multi-drug resistance pathogens.
Project description:The oncogenic transcription factor Myc is a pleiotropic regulator of RNA Polymerase II (RNAPII)-dependent transcription, DNA replication and DNA damage response pathways. Myc is stringently regulated by the ubiquitin system - for example, ubiquitination controls recruitment of the elongation factor Paf1c, which is critical for several Myc functions. Curiously, a key Myc-targeting deubiquitinase Usp28 also controls cellular response to DNA damage via the mediator protein 53bp1. Usp28 forms stable dimers, but the biological role of Usp28 dimerization is unknown. We show that dimerization limits Usp28 activity and restricts recruitment of Paf1c by Myc. Expression of monomeric Usp28 leads to ectopic Paf1c recruitment and resolution of transcription-replication conflicts, accelerating DNA synthesis. Strikingly, 53bp1 selectively interacts with and stabilizes dimeric Usp28 - depletion of 53bp1 favors formation of Usp28 monomers deregulating DNA replication. Genotoxic stress disrupts 53bp1-Usp28 complexes, promotes formation of Usp28 monomers and recruitment of Paf1 by Myc. This triggers ectopic DNA synthesis during early response to genotoxins, amplifying DNA damage. We propose that dimerization of Usp28 limits aberrant replication at transcriptionally active chromatin to maintain genome stability.
Project description:Forkhead Box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promote nuclear localization and activation of FoxO proteins, which - depending on the cellular context - leads to cell cycle arrest or apoptosis. Moreover, FoxOs can control oxidative stress resistance and cell metabolism. In endothelial cells (ECs), they additionally regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxO transcription factors exert a tumor-suppressive function, due to their critical role in regulating proliferation and survival. Others and we have previously shown that FoxOs can regulate these processes via two different mechanisms: either by direct binding to FoxO-responsive elements (FRE) at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners, which might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed Transformation/Transcription Domain-Associated Protein (TRRAP), a member of multiple histone acetyltransferase (HAT) complexes, as novel binding partner of FoxO family proteins. TRRAP is required to support FoxO3 transactivation and FoxO3-dependent apoptosis in ECs via transcriptional activation of the proapoptotic Bcl-2 family member BIM. Moreover, FoxO-TRRAP interaction might explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking FRE sequences.
Project description:Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. We screened a large cohort of unselected WTs and revealed frequencies of 3 % for MYCN P44L and 0.8 % for MAX R60Q, associated with a higher risk of relapse in the case of MYCN. Biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. Nevertheless, we could identify a number of novel N-MYC partner proteins, and several of these are known for their oncogenic potential. Correlated expression in WT samples suggests a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.
Project description:SPT6 is a histone chaperone that tightly binds RNA polymerase II (POL2) during transcription elongation. However, its primary role in POL2 transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in POL2 behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for POL2 processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in POL2 termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the absence of SPT6. In conclusion, targeted protein degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and POL2 elongation factor.
Project description:DYT-TOR1A dystonia is a movement disorder characterized by involuntary muscle contractions. Despite being the most common monogenetic form of dystonia, its pathophysiolofy remains unclear. With a reduced penetrance of about 30%, there is a suggestion that extragenetic factors are needed to develeop a dystonic phenotype. In the present study, we induced a sciatic nerve crush injury in a genetically predisposed DYT-TOR1A mouse model (DYT1KI) to evoke a dystonic phenotype. Subsequently, we employed a multi-omic approach to uncover novel pathophysiological pathways associated with DYT-TOR1A dystonia. Utilizing a deep-learning-based characterization of the dystonic phenotype, we observed that nerve-injured DYT1KI animals exhibited significantly more dystonia-like movement (DLM) compared to naive DYT1KI animals, with noticeable effects as early as two weeks post-surgery. Moreover, nerve-injured DT1KI mice displayed significantly more DLM than their wildtype (wt) counterparts starting 6 weeks post-injury. In the cerebellum of nerve-injured wt mice, multi-omice analysis indicated regulatory changes in translation-related processes, a phenomenon not observed in the cerebellum of nerve-injured DYT1KI mice; instead, these changes were localized to the cortex and striatum. Our findings suggest a failure of translational compensatory mechanisms in the cerebellum of phenotypic DY1KI mice displaying DLM, while dysregulations in translation in the cortex and striatum likely contribute to the promotion of the dystonic phenotype.
Project description:The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14 and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.
Project description:In contrast to the general ubiquitin activation and transfer cascade, we show that Uba1 binds to ubiquitin conjugating enzymes (Ubc) even in the absence of ubiquitin under mild oxidizing conditions. We show that this binding is ATP-dependent and leads to a disulfide-linkage between the catalytic cysteine residues as demonstrated with gel shift assays and visualized in a high-resolution structure of a covalent Uba1Ubc13 complex. The structure provides additional insights into the Uba1-Ubc binding mode and reveals unexpected structural changes in the N-terminus of Uba1, leading to a more open ATP-binding pocket. Immunoprecipitation experiments with yeast cells in combination with mass spectrometry revealed that several Uba~Ubc complexes are generated in vivo under oxidative stress conditions.