Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CHIP controls the expression of the neuropeptide precursor protein VGF

ABSTRACT: CHIP is a neuroprotective E3-ubiquitin ligase that supports longevity and healthy ageing. Loss of CHIP function has a major impact on life expectancy in animal models, whilst in humans’ mutations that compromise the E3-ligase activity of CHIP are causative for forms of Spinocerebellar Ataxia (SCA) that are accompanied by cognitive decline and/or dementia. The pathways regulated by CHIP to maintain neuronal health remain to be discovered. Gene-edited neuroblastoma cells were produced and used as a model to study the effects of CHIP loss on the steady state proteome in the absence of proteotoxic stress. Label free quantitative proteomic analysis (SWATH-MS) highlighted VGF, a member of the neuropeptide precursor family of proteins, as being a dominant protein responding to loss of CHIP function. By studying the dependence of VGF expression on CHIP using SILAC and RNA-Seq we have defined a role for the ligase in regulated neuropeptide expression.

INSTRUMENT(S): TripleTOF 5600, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Neuroblast

DISEASE(S): Spinocerebellar Ataxia Type 31

SUBMITTER: Lenka Hernychova

LAB HEAD: Kathryn Ball

PROVIDER: PXD016299 | Pride | 2021-07-29

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Pathway Analysis of Dominant CHIP E3 Ligase-Dependent Protein Steady State Changes Highlight a Role in Neuronal Cell Membrane Integrity Linked to the Actin Cytoskeleton

Project description:Functional integrity of the E3-ubiquitin ligase CHIP is essential for healthy aging in the brain. By virtue of its association with the core molecular chaperone machinery, studies into the physiological role(s) of CHIP have been dominated by its involvement in the quality control and degradation of unfolded, aggregated and/or mutated proteins. To elucidate the dominant CHIP-dependent changes in protein steady state levels in human neurons we conducted an unbiased proteomic analysis in genetically engineered wild-type and CHIP knockout iPSC-derived cortical neurons. Rather than a broad effect on protein homeostasis, pathway analysis highlighted a focused cohort of CHIP-responsive proteins involved in actin cytoskeleton signalling and membrane integrity. In support of the pathway analysis CHIP KO cell-lines had enhanced sensitivity to mechanical and chemical membrane damage. Consistent with recent studies on the identification of a non-canonical HSP-independent E3-activity for CHIP, the lack of general quality control defects in knock-out cells suggests that the major readout of CHIP function in cortical neurons is the regulation of native folded substrates involved in maintaining cellular ‘health’. Thus, CHIP-mediated regulation of the of cytoskeletal- and membrane-related proteins is likely to make a vital contribute to the neuroprotective activity of CHIP.

2021-07-29 | PXD021404 | Pride

LC-MS/MS proteomics analysis of neuronal cells and secretomes with timsTOF Pro

Project description:The neuropeptide VGF was recently proposed as a neurodegeneration biomarker. The Parkinson's disease-related protein leucine-rich repeat kinase 2 (LRRK2) regulates endolysosomal dynamics, a process that involves SNARE-mediated membrane fusion and could regulate secretion. Here we investigate potential biochemical and functional links between LRRK2 and v-SNAREs. We find that LRRK2 directly interacts with the v-SNAREs VAMP4 and VAMP7. Secretomics reveals VGF secretory defects in VAMP4 and VAMP7 knockout (KO) neuronal cells. In contrast, VAMP2 KO "regulated secretion-null" and ATG5 KO "autophagy-null" cells release more VGF. VGF is partially associated with extracellular vesicles and LAMP1+ endolysosomes. LRRK2 expression increases VGF perinuclear localization and impairs its secretion. Retention using selective hooks (RUSH) assays show that a pool of VGF traffics through VAMP4+ and VAMP7+ compartments, and LRRK2 expression delays its transport to the cell periphery. Overexpression of LRRK2 or VAMP7-longin domain impairs VGF peripheral localization in primary cultured neurons. Altogether, our results suggest that LRRK2 might regulate VGF secretion via interaction with VAMP4 and VAMP7.

2023-01-31 | PXD028321 | Pride

CHIP phosphorylation regulates proteostasis

Project description:Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we demonstrate that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein halflife, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKGmediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

2021-09-09 | PXD021531 | Pride

ChIP-MS reveals the local chromatin composition by label-free quantitative proteomics

Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.

2024-03-05 | PXD039627 | Pride

Mass spectrometric evidence for neuropeptide-amidating enzymes in C. elegans

Project description:Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules, and are widely involved in the regulation of various physiological processes. The nematode C. elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans. However, the enzymes required for the last step in the production of many bioactive peptides – the carboxyterminal amidation reaction – have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in wild-type animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase (PHM) and a peptidylglycine α-amidating monooxygenase (PAM) had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans. In summary, the key steps in neuropeptide-processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

2018-03-12 | PXD008942 | Pride

Comparative RNA-Seq in mouse prelimbic cortex (PL)

Project description:Major depressive disorder (MDD) is a prevalent and life-threatening illness in modern society. The susceptibility to MDD is profoundly influenced by environmental factors, such as stressful lifestyle or traumatic events, which could impose maladaptive transcriptional program through epigenetic regulation. However, the underlying molecular mechanisms remain elusive. Here we report that stress-susceptible rodents exhibit lower levels of histone crotonylation in the medial prefrontal cortex (mPFC), concurrent with regional upregulation of chromodomain Y-like (CDYL), a crotonyl-CoA hydratase and histone methyllysine reader. Overexpression of CDYL in the prelimbic cortex (PL), a sub-region of mPFC, increases microdefeat-induced social avoidance behaviors and anhedonia in mice. Conversely, knockdown of CDYL in PL prevents chronic social defeat stress-induced depression-like behaviors. Mechanistically, we show that CDYL inhibits structural synaptic plasticity mainly by transcriptional repression of neuropeptide VGF, and this activity is dependent on its dual effect on regional histone crotonylation and H3K27 tri-methylation on the VGF promoter. Together, our data indicate CDYL plays a critical role in regulating stress-induced depression-like behaviors, providing a potential therapeutic target for MDD.

2018-11-15 | GSE120293 | GEO

VGF-derived peptide attenuates development of type 2 diabetes via enhancement of islet beta cell survival and function survival and function

Project description:Deterioration of functional islet β-cell mass is the final step in progression to Type 2 diabetes. We previously reported that overexpression of Nkx6.1 in rat islets has the dual effects of enhancing glucose-stimulated insulin secretion (GSIS) and increasing β-cell replication. Here we show that Nkx6.1 strongly upregulates the prohormone VGF in rat islets and that VGF is both necessary and sufficient for Nkx6.1-mediated enhancement of GSIS. Moreover, the VGF-derived peptide TLQP-21 potentiates GSIS in rat and human islets and improves glucose tolerance in vivo. Chronic injection of TLQP-21 in pre-diabetic ZDF rats preserves islet mass and slows diabetes onset. TLQP-21 prevents islet cell apoptosis by a pathway similar to that used by GLP-1, but independent of the GLP-1, GIP, or VIP receptors. Unlike GLP-1, TLQP-21 does not inhibit gastric emptying or increase heart rate. We conclude that a TLQP-21 is a novel agent for enhancing islet β-cell survival and function. We utilized a “sample x reference” experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1, the mouse form of Pdx1, or the beta-galactosidase enzyme. 3-5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

2012-05-09 | E-GEOD-37852 | biostudies-arrayexpress

The tumor antigen PRAME is a substrate recognition subunit of a Cul2-based ubiquitin ligase and is associated with active NFY promoters

Project description:The human tumor antigen PRAME (Preferentially expressed antigen of melanoma) is frequently overexpressed in tumors. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumorigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. Genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at NF-Y promoters that are transcriptionally active, suggesting a role in gene activation. Our results are consistent with the involvement of the PRAME ubiquitin ligase complex in NF-Y-mediated transcriptional regulation. ChIP-seq binding profiles of PRAME (ChIP-seq using the preimmune serum was used as negative control), NFYA, and NFYB, and expression analysis by RNA-seq in K562 human leukemia cell line

2011-07-29 | E-GEOD-26439 | biostudies-arrayexpress

CHN-1 and CHN-1 L141R interacting proteins

Project description:Proteostasis is achieved by quality control pathways that support the generation of correctly folded proteins, prevent protein misfolding and remove toxic proteins. The quality control E3 ligase CHIP ubiquitylates damaged proteins consigned by chaperone partners for disposal through the endo-lysosomal pathway, proteasomal degradation, or autophagy. Additionally, CHIP has been reported to modulate essential signaling pathways by precisely delivering a myriad of native proteins to destined fates. We aimed at understanding the substrate specificity and processivity through a “structure to function” approach, by examining the modeled 3D structure of the C. elegans ortholog of CHIP, CHN-1, based on the reported structure of murine CHIP. Using different model organisms and various genetic and biochemical analyses, we demonstrate that monomeric CHN-1/CHIP has preserved ubiquitylation activity and promotes longevity via the IIS pathway. The lack of monomer results in premature aging and neurodegenerative disorder. Our data reveal that autoubiquitylation plays an important role in the alteration between monomer and dimer. Together, the conserved dimer-monomer transition provides a molecular switch regulating CHIP activity in response to proteotoxic stress and aging.

2022-08-25 | PXD025375 | Pride

Cross-linking Analysis (HSP70-HSP90-CHIP)

Project description:Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case of Hsp70 and Hsp90, which in concert with the cochaperone CHIP –an E3 ligase–, direct their bound substrate to degradation through ubiquitination. We have generated complexes between the chaperone (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). The two ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinate the substrate, and this is done with a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, show the substrate located between the chaperone and the cochaperone, which suggests an ubiquitination mechanism. Both complexes are extremely flexible, which is crucial for the ubiquitination process.

2019-03-06 | PXD009777 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data