Project description:A novel approach to single cell analysis to reveal intrinsic differences in immune marker expression in unstimulated macrophages from BALB/c and C57BL/6 mouse strains.
Project description:The bacterial HflK-HflC membrane complex is a member of the highly conserved family of SPFH proteins, which are present in all domains of life and include eukaryotic stomatins, flotillins, and prohibitins. These proteins organize cell membranes and are involved in various processes. However, the exact physiological functions of most bacterial SPFH proteins remain unclear. Here, we report that the HflK-HflC complex in Escherichia coli is required for growth under high aeration. The absence of this complex causes an aerobic growth defect due to a reduced abundance of IspG, a crucial enzyme in the isoprenoid biosynthetic pathway. This reduction leads to lower levels of ubiquinone, reduced respiration, lower ATP levels, and misregulated expression of respiratory genes. The regulation of aerobic respiration by the HflK-HflC complex resembles the mitochondrial respiratory defects caused by prohibitin mutations in mammalian and yeast cells, suggesting a functional commonality between these bacterial and eukaryotic SPFH proteins.
Project description:Chemoreceptors enable bacteria to modulate their swimming behavior in accordance with the perceived environmental cues. Bacteria exhibit large diversity of stimuli sensed by their chemoreceptors, whereas the output of receptors to the chemotaxis signaling pathway that controls flagellar motor is typically highly conserved. Here, we characterize a unique chemoreceptor-like protein, Tls, which is found in the B2 phylogroup of Escherichia coli that includes many extraintestinal pathogenic (ExPEC) strains. Instead of mediating chemotactic signaling, Tls controls motility by repressing the expression of flagellar genes, and thus cell motility, apparently by sequestering the transcriptional master activator of flagellar genes, FlhDC. Furthermore, we observe that the subcellular localization of Tls, the sequestration of FlhDC, and the repression of flagellar genes and motility are all abolished during growth on porous medium, indicating that this regulation may be mechanosensitive. Deletion of tls in a uropathogenic E. coli leads to reduced attachment to the urinary tract cells, but an increased migration to and/or proliferation in the murine gut, a pathogen reservoir niche, thus implicating Tls in the regulation of motility during infection.
Project description:The coordination of cell cycle progression and flagellar synthesis is a complex process in motile bacteria. In γ-proteobacteria, the localization of the flagellum to the cell pole is mediated by the SRP-type GTPase FlhF. However, the mechanism of action of FlhF, and its relationship with the cell pole landmark protein HubP remain unclear. In this study, we discovered a novel protein called FipA that is required for normal FlhF activity and function in polar flagellar synthesis. We demonstrated that membrane-localized FipA interacts with FlhF and is required for normal flagellar synthesis in Vibrio parahaemolyticus, Pseudomonas putida, and Shewanella putrefaciens, and it does so independently of the polar localization mediated by HubP. FipA exhibits a dynamic localization pattern and is present at the designated pole before flagellar synthesis begins, suggesting its role in licensing flagellar formation. This discovery provides insight into a new pathway for regulating flagellum synthesis and coordinating cellular organization in bacteria that rely on polar flagellation and FlhF-dependent localization.
Project description:E.coli Dilution were performed and mixed with standard HeLa in 1:1, 1:2, 1:4, 1:5 and 1:10 HeLa. These samples were measured both in DIA and DDA mode using timsTOF-pro-2 instrument. .
Project description:Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER encounter structure (ERMES) and Tom70 are part of two cooperative and partially redundant ER-to-mitochondria transfer routes. If the ER-to-mitochondria transfer is prevented, many mitochondrial precursor proteins accumulate non-productively on the ER surface, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.
Project description:Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) MM cells were grown in RPMI-1640 medium supplemented with 10 % FBS and 0.68 mM L-glutamine, in a humidified incubator at 5% CO2 and 37 °C. The AMO1-CFZ medium was additionally added 90 nM CFZ, while the CFZ sensitive AMO1 cells were added vehicle (0.009 % DMSO) only. AMO1-CFZ was either harvested directly or grown for 1 week in CFZ free medium (vehicle only) prior to harvest. AMO1 and carfilzomib-resistant AMO1-CFZ cells were resuspended in 100 µl 1% sodium deoxycholate, 100 mM Tris-HCl pH 8.5, 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide (CAA), heated at 90 °C for 45 min and sonicated for 10 cycles (30 s ON/30 s OFF) using a Bioruptor pico sonicator. After centrifugation at 16000 × g for 10 min, 50 µg soluble protein from each sample was added 100 µl 0.1M ammonium bicarbonate, 0.5 µg trypsin and digested overnight at 37°C. Peptides were desalted using C18 spin columns, dried in a speedvac centrifuge and resuspended in 0.1% formic acid prior to MS analysis. Label-free quantitatative (LFQ) LC-MS/MS was performed on a timsTOF Pro (Bruker Daltonics) connected to a nanoElute (Bruker Daltonics) HPLC. Peptides were separated over a Bruker PepSep C18 (75 µm × 15 cm) column with running buffers A (0.1 % formic acid) and B (0.1 % formic acid in acetonitrile) using a 100 min gradient from 2 % B to 40 % B. The timsTof instrument was operated in the DDA PASEF mode with 10 PASEF scans per acquisition cycle and accumulation and ramp times of 100 ms each. The ‘target value’ was set to 20000 and dynamic exclusion was activated and set to 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 800.
Project description:Alternative to enzyme inhibition, small-molecule-induced protein degradation has emerged as a promising pharmacological modality for inactivating disease-relevant protein kinases. DYRK1A and DYRK1B are closely related protein kinases that are involved in pathological processes such as neurodegeneration, cancer development and adaptive immune homeostasis. Here we report the development of the first DYRK1 proteolysis targeting chimeras (PROTACs) that combine a new ATP- competitive DYRK1 inhibitor with ligands for the E3 ubiquitin ligase component Cereblon (CRBN) to induce ubiquitinylation and subsequent proteasomal degradation of DYRK1A and DYRK1B. The lead compound (DYR684) promoted fast, efficient, potent and selective degradation of endogenous DYRK1A in cell-based assays. Although DYR684 was also active against DYRK1B, we observed that an enzymatically inactive splicing variant of DYRK1B (p65) was resistant to degradation. Com- pared to competitive kinase inhibition, targeted degradation of DYRK1 by DYR684 provided improved suppression of down- stream signaling. Moreover, DYR684 sensitized SH-SY5Y neuroblastoma cells to the cytotoxic effects of the anticancer drug cisplatin. Collectively, our results identify DYRK1A and DYRK1B as viable targets for PROTAC-mediated degradation and qual- ify DYR684 as a suitable chemical probe for functional studies of the catalytically active DYRK1A and DYRK1B variants.
Project description:mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are spatially and temporally coordinated to ensure that only fully processed transcripts are released into the nucleoplasm for export from the nucleus. Here, we explore the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is involved in a RNP assembly checkpoint at the 3’-end of genes that is coupled to the release of the 3’-end processing complex after polyadenylation. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localizes to cleavage bodies, which are enriched for 3’-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3’-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, which is accompanied by a depletion of CPAC components from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites as well as delayed transcription termination, suggesting that the availability of CPAC components is insufficient to maintain normal levels of 3’-end processing. Our data is consistent with a model in which Dbp2 is involved in an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release.
Project description:In the methanogenic pathway from CO2 and H2, low potential electrons for CO2 reduction are generated by a flavin-based electron branching reaction catalysed by heterodisulphide reductase (Hdr) in complex with [NiFe]-hydrogenase (Mvh). The F420-reducing [NiFe]-hydrogenase (Frh) provides electrons for the methane formation pathway via the electron carrier F420. The production of both [NiFe]-hydrogenases in Methanothermobacter marburgensis is strongly down-regulated under strictly nickel-limited conditions. The Frh reaction is replaced by a coupled reaction with [Fe]-hydrogenase (Hmd), and the role of Mvh is taken over by F420-dependent electron donating proteins (Elp). Thus, Hmd provides all electrons for the reducing metabolism under these nickel-limited conditions.