Project description:Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and how it affects antibiotic resistance. Our study demonstrated the ubiquitous presence of crotonylation in E.coli and its connection to bacterial resistance to polymyxin. We first identified the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies suggested that YjgM upregulates the crotonylation of the substrate protein PmrA, which is crucial for controlling E. coli's resistance to polymycin, thereby boosting PmrA's affinity for binding to the promoter of eptA, which in turn promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discovered that PmrA's crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective for creating antibacterial medicines.
Project description:Many animal species employ a chromosome-based mechanism of sex determination, which has led to coordinate evolution of dosage compensation systems. Dosage compensation not only corrects the imbalance in the number of X-chromosomes between the sexes, but is also hypothesized to correct dosage imbalance within cells due to mono-allelic X expression and bi-allelic autosomal expression, by upregulating X-linked genes (termed M-CM-"M-BM-^@M-BM-^XOhnoM-CM-"M-BM-^@M-BM-^Ys hypothesisM-CM-"M-BM-^@M-BM-^Y). Although this hypothesis is well supported by expression analyses of individual X-linked genes and by array-based transcriptome analyses, a recent study claimed that no such X upregulation exists in mammals and C. elegans based on RNA-sequencing and proteomics analyses. We provide RNA-seq RNA-seq analysis of mouse female PGK12.1 ES cells with two active X chromosomes and confirmed that the X chromosome is upregulated, consistent with the previous microarray study. Examination of expression of X-linked and autosomal genes in mouse female ES cells with two active X chromosomes.
Project description:Many proteins undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in the cytoplasmic protein GDP-mannose pyrophosphorylase A (GMPPA) cause a syndrome characterized by alacrima, achalasia, mental retardation and myopathic alterations. GMPPA acts as feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA enhances incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we show that loss of GMPPA affects the functionality of the Golgi apparatus using different approaches. First, we show a fragmentation of the Golgi apparatus in skeletal muscle fibers and in neurons of GMPPA KO mice. A major reorganization is also evident by mass spectrometry of KO tissues with a regulation of several ER- and Golgi-resident proteins. We further show that loss of GMPPA increases the retention of α-DG in the ER. Notably, mannose supplementation can mimic changes in ER and Golgi structure and function in WT cells. In summary, our data underline the importance of a balanced mannose homeostasis for structure and function of the secretory pathway.
Project description:we first expressed AAa and AAg in a eukaryotic system to investigate whether the proteins could be sulfated in vivo. For this purpose we used insect cells as a model of the Anopheles mosquito. Specifically, codon-optimized sequences encoding AAa and AAg were designed as N-terminal fusions with the honeybee mellitin signal sequence in order to direct the recombinant proteins to the secretion pathway and were expressed in Trichoplusia ni insect cells. Following expression, the cell medium containing the secreted proteins was analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS).
Project description:We further expanded on previous optimizations to isolate the plasma peptidome and compared various methods to maximize identifications with speed and ease. Previous studies have reported loss of polypeptides binding to high abundant proteins during depletion strategies. We hypothesized that rapid chaotropic denaturation of plasma with urea under reducing conditions would liberate non-covalently bound peptides to improve recovery during protein depletion. We also compared depletion strategies to isolate the peptidome including protein precipitation and removal with either TCA, acetone or acetonitrile (AcN). Following centrifugation of precipitated proteins, the supernatant containing peptides was collected. For acetone and AcN precipitations, an additional vacuum centrifugation step was required followed by resuspension of peptides in aqueous buffer. Our comparison of peptidome isolation also included removal of proteins with size-exclusion 10 kDa MWCO filters. All peptide isolations were acidified to 0.1% TFA, adjusted to 5% acetonitrile and desalted with HLB-SPE. Peptides were analysed by single-shot nanoUHPLC-MS/MS employing both HCD and EThcD and quantified by LFQ
Project description:The endoplasmic reticulum (ER) functions in protein and lipid synthesis, calcium ion flux, and inter-organelle communication, all of which are driven by the ER proteome landscape. ER is remodeled in part through autophagy-dependent protein turnover involving membrane-embedded ER-phagy receptors1,2. A refined tubular ER network is formed in neurons within highly polarized dendrites and axons3,4. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, associated with hyper-excitability,5 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy6,7. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we employ genetic, proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy during conversion of stem cells to induced neurons in vitro. Through analysis of single and combinatorial ER-phagy receptor mutants coupled with an allelic series computational framework, we delineate the extent to which each of five receptors contributes to both the magnitude of ER turnover by autophagy and the selectivity of clearance for individual ER proteins. We define specific subsets of reticulon-domain containing ER-tubule shaping proteins or luminal proteins as preferred clients for autophagic turnover via distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant accumulation of ER in axonal structures in ER-phagy receptor or autophagy-deficient cells. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping this critical organelle during transitions in cell states.
Project description:The endoplasmic reticulum (ER) functions in protein and lipid synthesis, calcium ion flux, and inter-organelle communication, all of which are driven by the ER proteome landscape. ER is remodeled in part through autophagy-dependent protein turnover involving membrane-embedded ER-phagy receptors1,2. A refined tubular ER network is formed in neurons within highly polarized dendrites and axons3,4. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, associated with hyper-excitability,5 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy6,7. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we employ genetic, proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy during conversion of stem cells to induced neurons in vitro. Through analysis of single and combinatorial ER-phagy receptor mutants coupled with an allelic series computational framework, we delineate the extent to which each of five receptors contributes to both the magnitude of ER turnover by autophagy and the selectivity of clearance for individual ER proteins. We define specific subsets of reticulon-domain containing ER-tubule shaping proteins or luminal proteins as preferred clients for autophagic turnover via distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant accumulation of ER in axonal structures in ER-phagy receptor or autophagy-deficient cells. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping this critical organelle during transitions in cell states.
Project description:Synthetic peptides are commonly used in biomedical science for many applications in basic and translational research. Here, we assembled a large dataset of synthetic peptides whose identity was validated using mass spectrometry. We analyzed the mass spectra and used them for method validation as well as the creation of ground truth datasets and cognate databases. Contact: Michele Mishto, Head of the research group Molecular Immunology at King’s College London and the Francis Crick Institute, London (UK). Email: michele.mishto@kcl.ac.uk,
Project description:RNA splicing and the ubiquitin system allow the functional proteome to adapt in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here we show that the deregulation of the spliceosome B complex caused by USP39 deficiency leads to a novel splicing profile characterized by the use of cryptic 5′splice sites. Importantly, disruptive variants evade mRNA surveillance pathways and are translated into topologically incorrect proteins. These cryptic isoforms disrupt proteostasis and activate the unfolded protein response, causing ER stress-induced cell death. Human cells respond to this proteotoxicity by enhancing ubiquitin-mediated proteolysis and ER-phagy. Our findings show how cell death triggered by cryptic splicing can be mitigated, and provide insight into the molecular pathogenesis of diseases such as retinitis pigmentosa.
Project description:Cilia assembly, maintenance and the localization of signal-transduction proteins necessitate a functioning intraflagellar transport (IFT). The IFT machinery moves cargo along a microtubule scaffold from the proximal to the distal end of the cilium. This anterograde transport is facilitated by a large multiprotein complex IFT-B, which consists of 16 proteins in total and recruits motor protein Kinesin-2 for active movement. TTC30A and TTC30B are integral components of this complex and were shown before to have redundant function in context of IFT. One paralogue could compensate the loss of the other, preventing the disruption of IFT-B and thus a severe ciliogenesis defect with no formation of the cilium. Affinity-based protein complex analysis of endogenously tagged cells, generated via CRISPR/Cas9 system as described before, revealed paralogue specific protein interactors proposing the involvement of TTC30A or TTC30B in other ciliary functions, particularly Sonic hedgehog signaling (Shh). Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which intriguingly is also linked to a rare TTC30 variant. In this study we focus on the so far unknown interaction of TTC30A with protein kinase A catalytic subunit α PRKACA, which is a negative regulator of Shh pathway. For an in-depth analysis of this unique interaction and the influence on Shh, we used previously, via the CRISPR/Cas9 system, generated TTC30A or B single- and double-knockout hTERT-RPE1 cells as well as rescue cells harboring the TTC30 mutation. Our systematic approach revealed the paralogue specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA as well as the uptake of Smoothened into the cilium resulting in a downregulation of Sonic hedgehog signaling. Affinity-based protein complex analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in interaction pattern. These interactome alterations combined with Shh downregulation suggest a possible mechanism of how mutant TTC30A and respectively TTC30A KO are linked to synpolydactyly.