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Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain

ABSTRACT: Apolipoprotein E (ApoE) polymorphisms modify the risk of neurodegenerative disease. To elucidate how ApoE isoforms alter the proteome, we applied in vivo deuterium isotope labeling to transgenic mice modified to express the human ApoE gene (isoform 2, 3, or 4). We employed liquid-chromatography and mass-spectrometry which allowed us to simultaneously measure relative protein concentration and track the incorporation of heavy isotopes over a 32-day period. The conjunction of turnover data with concentration measurements provides insight as to how ApoE isoforms affect the synthesis and degradation of a wide variety of proteins. We tested the homeostatic regulation of >2700 ontologies and quantified significant isoform-dependent changes which link ApoE genotype to modulation of mitochondrial components, oxidative phosphorylation, and maintenance of protein quality. Here we demonstrate the application of turnover rate measurement to further characterize concentration changes and provide opportunities for further exploration into disease pathology. (Zuniga 2023)

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: John Price

LAB HEAD: John Calvin

PROVIDER: PXD044460 | Pride | 2024-11-25

REPOSITORIES: Pride

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Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).

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	A2D0F1_BC.raw	Raw
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	A2D0F1_BM_ID.raw	Raw
	A2D0F1_BM_Kin.raw	Raw
	A2D0F2_BC.raw	Raw

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Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain

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