Project description:Global food production is reliant on the application of finite phosphorus (P) fertilisers. Numerous negative consequences associated with intensive P fertilisation have resulted in a high demand to find alternative sustainable methods that will enhance crop P uptake. Bacteroidetes, primarily from the genus Flavobacterium, have recently been shown to be abundant members of the plant microbiome, but their general ecological role and potential to mobilise P in the rhizosphere remains very poorly characterised. Here, we sought to determine the P mobilisation potential of Flavobacterium strains isolated from the rhizosphere of oilseed rape (Brassica napus L.). In contrast to other abundant rhizosphere bacteria, such as Pseudomonas, all Flavobacterium strains exhibited constitutive phosphatase activity independent of external phosphate (Pi) concentrations. Interestingly, a combination of exoproteomic analysis and molecular microbiology techniques revealed that Flavobacterium have a complex and largely unique repertoire of proteins to mobilise and acquire Pi. This includes the expression of novel, as yet unidentified, phosphatases, and numerous proteins of unknown function. We also discovered that Flavobacterium expresses certain SusCD-like transporters, whose role is typically associated with specialised carbon acquisition, in response to Pi-starvation. Furthermore, the genes encoding these unusual Pi-responsive proteins were enriched in plant-associated Flavobacterium strains suggesting that this machinery represents niche-adaptive strategies for overcoming P scarcity in this genus. We propose that abundant rhizosphere-dwelling Flavobacterium spp. have evolved unique mechanisms for coping with Pi-stress which may provide novel solutions for future sustainable agricultural practices.
Project description:We present here evidence that histones H3.1 and H4 can be imported into the nucleus as monomers in human cells. Using a tether-and-release system to study the cytosolic phase and import dynamics of newly synthesised histones, we find that H3.1 and H4 can be maintained as stable monomers in the cytosol in a tethered state. Cytosolically tethered histones are bound tightly to Importin- proteins (predominantly IPO4), but not to the histone specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, dissociation with IPO4 and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones under steady-state conditions reveals an excess of H3 specifically associated with sNASP, suggesting that NASP can maintain a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol, with sNASP acting as a potential nuclear receptor for monomeric histone H3.
Project description:Many bacterial species are known to recover peptidoglycan (PG) fragments released from remodelling of their cell walls during growth and cell division. These PG fragments not only provide an essential energy resource, especially in nutrient restricted environments, but also play a critical role in influencing infection. Yet whether mycobacteria have the capacity to recycle their PG, or not, has yet to be resolved. In this study we show that NagA, an N-acetylglucosamine-6-phosphate (GlcNAc-6-P) deacetylase, is essential for coordinating the recycling of an amino sugar component released from the mycobacterial cell wall. We show that NagA is exclusively responsible for GlcNAc-6-P deacetylation and is pivotal for the de novo synthesis of core cell wall building blocks. Indeed, a nagA mutant exhibited impaired cell wall, defective biofilm formation, and enhanced susceptibility to PG targeting agents. Moreover, uptake analysis and profiling of the amino-sugar pool revealed that NagA inactivation blocks N-acetylglucosamine (GlcNAc) import and has a pronounced effect on the fate and levels of the intracellular amino sugar pool. Loss of NagA led to the up- and down-regulation of proteins involved in cell wall biosynthesis, thereby altering cell wall homeostasis. Overall, our data highlights the importance of an overlooked yet conserved component in an important PG salvage pathway in mycobacteria, in which NagA provides a unique GlcNAc sensing mechanism, thus acting as a checkpoint for regulating the recovery and reuse of PG fragments.
Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved Bromo-Adjacent Homology (BAH) module of BAHCC1 (BAHCC1BAH) ‘recognizes’ H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and ChIP-seq-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-lysine-binding ‘cage’ formed by BAHCC1BAH, mediating co-localization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is overexpressed in human acute leukemias and interacts with transcriptional co-repressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH:H3K27me3 interaction, causes de-repression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germ-line mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study unveils a novel H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH ‘reader’.
Project description:This project investigated the effect of Cln3-deficiency on protein secretion in the social amoeba Dictyostelium discoideum by performing LC-MS/MS on conditioned media harvested from starved WT and cln3- cells.
Project description:Molecular chaperones are critical to maintaining intracellular proteostasis and have been shown to have a protective role against alpha-synuclein mediated toxicity. Co-chaperone proteins regulate the activity of molecular chaperones and connect the chaperone network to protein degradation and cell death pathways. Bcl-2 associated athanogene 5 (BAG5) is a co-chaperone that modulates proteostasis by inhibiting the activity of Hsp70 and several E3 ubiquitin ligases, resulting in enhanced neurodegeneration in models of Parkinson’s disease (PD). Here we identify a novel interaction between BAG5 and p62/sequestosome-1 (SQSTM1), suggesting that BAG5 may bridge the chaperone network to autophagy-mediated protein degradation. The interaction was discovered using affinity purifaction followed by mass spectrometry to identify BAG5 interacting proteins and was validated by subsequent in vitro immunoprecipitation studies.
Project description:The recent identification of catalytically active peptidylglycine -amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). Homologs of prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) were identified in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectroscopy to identify proteins recovered from the soluble secretome of mating gametes and searched for evidence that the putative peptidergic processing enzymes were functional.After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact or were subjected to cleavage were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the soluble mating secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites and had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.
Project description:Putative binding partners of IQGAP-related protein IqgC were identified by LC-MS/MS. Recombinant GST-tagged IqgC was purified from E. coli and immobilised to glutathione agarose. Interactors from cell lysate of D discoideum AX2 cells were applied to prepared affinity resin and bound proteins were identified by LC-MS/MS.
Project description:We characterized the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.
Project description:Plasmepsin X (PM X) is an aspartate protease expressed in the malaria parasie Plasmodium falciparum. Following synthesis PM X undergoes proteolytic processing. This project aims to understand the significance of PM X processing. To identify the cleavage sites, the processed PM X bands were excised from gels and subjected to LC-MSMS.