ABSTRACT: HDXMS raw data for various states of EFSAM including various mutants, and the apo and calcium-bound forms. The ID runs that were used to identify peptides in PLGS are also included.
Project description:Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, such calcium transients trigger pre-synaptic vesicle release and mediate post-synaptic plasticity. It is challenging to capture the molecular events associated with these localized and ephemeral calcium signals, however. Here we report the development of an engineered biotin ligase that combines the power of genetically encoded calcium indicators with protein proximity labeling. The enzyme, Cal-ID, biotinylates nearby proteins in response to elevated local calcium levels. The biotinylated proteins can be visualized by microscopy and identified via mass spectrometry. Cal-ID mass spectrometry applied to HEK293T cells identified cell cycle-dependent calcium signaling microdomains at centrosomes. Our results from mouse primary neurons indicated active calcium signaling near calcium extrusion sites on the plasma membrane. Therefore, we propose Cal-ID as a biochemical recorder of calcium signaling in living cells.
Project description:A microarray analysis to investigate transcriptional responses in the ectomycorrhizal fungus Paxillus involutus, after providing various sources of nitrogen. Five different sources of nitrogen representing various degrees of complexity were provided as patches in peat microcosms for fungal ingrowth: ammonium phosphate (APO), ammonium sulphate (ASU), glutamine (GLN), bovine serum albumin (BSA), and chitin (CHI). After fungal establishment of patches total RNA was isolated and used for global transcriptional analyses using cDNA microarrays. The entire design involved 9 slides (in the range DW2_01--DW2_13), 14 labelled extracts, and 3 biological replicates (R1-R3). The experiment was designed as a loop combining the 5 diffrent samples and including dye-swaps and biological replication. COMMENT: In our analysis of the raw dataset, 3 channels were removed due to poor signal quality: array 12707648b, Cy5 (635nm) channel (GLN R1 sample); array 12707650c, Cy5 (635nm) channel (APO R1 sample); array 12707651b, Cy5 (635nm) channel (APO R1 sample).
Project description:Nonsense-mediated mRNA decay (NMD) functions to degrade transcripts bearing premature stop codon (PTC) and is a crucial regulator of gene expression. NMD and the UPF3B gene have been implicated as the cause of various forms of intellectual disability (ID) and other neurological symptoms. Here, we reports three patients with global developmental delay carrying hemizygous deletions of the UPF2 gene, another important member of the NMD pathway and direct interacting partner of UPF3B.
Project description:Continuous labelling HDX on dN-YME1L, hexYME1L, hexYME1L_E600Q in apo, ATP-bound, ADP-bound and ATP and Ni2+-bound states. Folders are organized per state. Peptide mapping and undeuterated controls are included.
Project description:Nonsense-mediated mRNA decay (NMD) functions to degrade transcripts bearing premature stop codon (PTC) and is a crucial regulator of gene expression. NMD and the UPF3B gene have been implicated as the cause of various forms of intellectual disability (ID) and other neurological symptoms. Here, we reports three patients with global developmental delay carrying hemizygous deletions of the UPF2 gene, another important member of the NMD pathway and direct interacting partner of UPF3B. Using RNA-SEQ on lymphoblastoid cells from UPF2 deletion patients, we identified 1009 differently expressed genes (DEGs). 38% of these DEGs overlapped with DEGs identified in UPF3B patients. More importantly, 95% of all DEGs in either UPF2 or UPF3B patients share the same trend of de-regulation. This demonstrates that the transcriptome deregulation in these two patient groups is similar and that UPF2 should be considered as a new candidate gene for ID in man. We expanded our inq`uiries and performed a comprehensive search for copy number variations (CNVs) encompassing all NMD genes in cohorts of ID patients and controls. We found that UPF2, UPF3A, Y14, SMG6 and EIF4A3 are frequently deleted and/or duplicated in ID patients. These CNVs are likely to be the root of the problems or to act as predisposing factors. Our results suggest that dosage imbalance of NMD factors is associated with ID and further emphasize the importance of NMD in normal learning and memory processes.
Project description:The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.