LC-MS/MS quantification of proteome derived from wild-type and agp2Δ mutant strains of Yeast.
Ontology highlight
ABSTRACT: Characterization of wild-type and agp2Δ mutant strains of Saccharomyces cerevisiae proteome derived from their respective crude plasma membrane fractions.
Project description:Description: These date form the basis for supplementary table 2 and supplementary figure 6 of the manuscript "Modelling glycan processing reveals Golgi-enzyme homeostasis upon trafficking defects and cellular differentiation" by P Fisher, H Spencer, J Thomas-Oates, AJ Wood, D Ungar in Cell Reports
Project description:Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phosphorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and transient nature of these “docking” interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38α. We subsequently screened a library consisting of ~12,000 sequences from the human proteome, revealing a large number of MAPK-selective interactors, including many not conforming to previously defined docking motifs. Analysis of p38α/JNK1 exchange mutants identified specific docking groove residues mediating selective binding. Finally, we verified that docking sequences identified in the screen could function in substrate recruitment in vitro and in cultured cells. Collectively, these studies establish an approach for characterization of MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK MAP kinases.
Project description:Plant stress caused by pathogens or though abiotic means (e.g. drought or temperature) reduces agricultural yields, causing substantial economic losses while reducing food security at the global level. It is critical to recognize how plants perceive stress signals to elicit responses for survival. Endogenous plant peptidases and their peptide products play an important role in the signaling of plant immune processes. Thimet oligopeptidases (TOPs) are zinc-dependent peptide hydrolases with a conserved HEXXH active site motif. These metallopeptidases are critical components in plant response to oxidative stress triggered by pathogens or abiotic factors and are required for a fully functioning immune response to certain pathogens. Further characterization of plant TOPs and their peptide substrates would provide insights into their contribution to defense signaling, stress perception, and plant adaptation pathways. Herein, a quantitative mass spectrometry-based peptidomics approach was implemented to characterize the Arabidopsis thaliana plant peptidome and in the context TOPs (Fig. 1). A comparison between wild type (Col-0) and top1top2 null mutant revealed putative direct and indirect TOPs substrates in vivo.
Project description:The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis we have identified Collapsin Response Mediator Protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)- mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wildtype CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wildtype CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.
Project description:Analysis of sectreted proteins from pure media, LX and Heps breast cancer cells grown in conditioned media. All samples were prepared in biological triplicates.
Project description:Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment.
Project description:Maintaining skeletal muscle mass is of high importance as muscle atrophy like during sarcopenia or cachexia lead to a decrease in independence and a higher risk of morbidity and mortality. A leading compound in the treatment against ageing and cancer is rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). Whether the treatment with mTORC1 inhibitors would work at a cost of losing muscle mass is unclear, as most studies have been focusing on the role of mTORC1 specifically during hypertrophy. In order to answer this question we developed an inducible muscle specific knockout mouse model in which raptor can be ablated during adulthood to eliminate mTORC1 activity. We analysed the muscles after different time points and found that after 3 months the mice showed a fiber shift towards slower fiber types, a loss in oxidative capacity but only very few myopathic features. After 5 months the myopathic features became more apparent, however it did not largely affect the ex vivo muscle force. Surprisingly despite the myopathy we did not see a significant loss of muscle mass even after 5 months, that we hypothesised based on mTORC1s central role in protein synthesis. We assume that the myopathy after long-term mTORC1 inactivation is mostly a result of secondary effects through the loss of mitochondria, alterations in metabolism and in cytoskeletal components. In conclusion, during skeletal muscle maintenance mTORC1 is more essential for metabolic processes than it is for maintaining basal muscle mass.Maintaining skeletal muscle mass is of high importance as muscle atrophy like during sarcopenia or cachexia lead to a decrease in independence and a higher risk of morbidity and mortality. A leading compound in the treatment against ageing and cancer is rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). Whether the treatment with mTORC1 inhibitors would work at a cost of losing muscle mass is unclear, as most studies have been focusing on the role of mTORC1 specifically during hypertrophy. In order to answer this question we developed an inducible muscle specific knockout mouse model in which raptor can be ablated during adulthood to eliminate mTORC1 activity. We analysed the muscles after different time points and found that after 3 months the mice showed a fiber shift towards slower fiber types, a loss in oxidative capacity but only very few myopathic features. After 5 months the myopathic features became more apparent, however it did not largely affect the ex vivo muscle force. Surprisingly despite the myopathy we did not see a significant loss of muscle mass even after 5 months, that we hypothesised based on mTORC1s central role in protein synthesis. We assume that the myopathy after long-term mTORC1 inactivation is mostly a result of secondary effects through the loss of mitochondria, alterations in metabolism and in cytoskeletal components. In conclusion, during skeletal muscle maintenance mTORC1 is more essential for metabolic processes than it is for maintaining basal muscle mass.
Project description:Metabolic heterogeneity modulates productivity, antibiotic resistance and cancer aggressiveness. Since metabolic fluxes represent the functional output of metabolism, with glycolytic flux correlating with highly-productive phenotypes and cancer, such flux map will be indicative of the cellular metabolic state. Therefore, the quantification of metabolic fluxes is vital to identify the existence of metabolic subpopulations and to understand the process of their emergence at the single-cell level. However, so far inference of metabolic fluxes in individual cells is not possible as no method is available. Here, we developed a biosensor for glycolytic flux measurements in single yeast cells drawing on the robust correlation between fructose-1,6-bisphosphate (FBP) and flux levels in yeast, and using the B. subtilis FBP-binding transcription factor CggR. We followed a systematic engineering approach starting from promoter design, computational protein design and protein engineering, accompanied by strict characterization of the biosensor using different biochemical methods, proteomics, metabolomics and physiological analyses. As proof of principle, we applied the biosensor in vivo in the search for metabolic subpopulations in yeast cultures and, using fluorescence microscopy, we demonstrated that quiescent yeast cells have low glycolytic fluxes in comparison to coexisting dividing cells. We anticipate that our biosensor will contribute with unprecedented resolution for the study of metabolic subpopulations, to understand how and why metabolic subpopulations emerge and, very importantly, give clues on how to counteract the undesirable effects of such.
Project description:Photoageing in skin is commonly recognised by architectural remodelling of dermal extracellular matrix components. Mass spectrometry was previously used to identify tissue-specific patterns of fibrillin-1 and collagen VI peptide spectrum matches (PXD008450). This study aimed to determine if the same mass spectrometry-based approach could detect peptide spectrum match patterns and significantly differences in relative abundance of peptide sequences characteristic of damage following exposure to UVR of co-purified suspensions of fibrillin and collagen VI microfibrils. Human dermal fibroblast-derived suspensions of microfibrils were irradiated with either broadband UVB or solar simulated radiation (SSR). UVR-induced molecular damage was characterised by proteolytic peptide generation with elastase followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). This allowed the molecular scale identification of UV-induced structural changes within two skin matrix assemblies. The proteomic approaches used have the potential to facilitate the rapid, protein-specific identification of differential molecular fingerprints of damage in key extracellular matrix proteins.