Acetyl-CoA flux regulates the proteome and acetyl-proteome to maintain intracellular metabolic crosstalk - Acetylation Stoichiometry Dataset
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
John Denu Luigi Puglielli
PROVIDER: MSV000083855 | MassIVE | Fri May 24 13:18:00 BST 2019
SECONDARY ACCESSION(S): PXD014013
REPOSITORIES: MassIVE
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Project description:AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and degenerative diseases. Here, we investigate two models of AT-1 dysregulation and altered acetyl-CoA flux: AT-1S113R/+ mice, a model of AT-1 haploinsufficiency, and AT-1 sTg mice, a model of AT-1 overexpression. The animals display distinct metabolic adaptation across intracellular compartments, including reprogramming of lipid metabolism and mitochondria bioenergetics. Mechanistically, the perturbations to AT-1-dependent acetyl-CoA flux result in global and specific changes in both the proteome and the acetyl-proteome (protein acetylation). Collectively, our results suggest that AT-1 acts as an important metabolic regulator that maintains acetyl-CoA homeostasis by promoting functional crosstalk between different intracellular organelles.
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Project description:The acetyl-CoA transporter, AT-1 (also referred to as SLC33A1), is a key member of the endoplasmic reticulum (ER) acetylation machinery; it transports acetyl-CoA from the cytosol into the ER lumen where it serves as donor of the acetyl group for Nε-lysine acetylation 1,2. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and age-associated human diseases 3-7. Mice with reduced or increased AT-1 expression mimic associated human diseases 8-10. In this study, we investigated the pervasive effects that dysregulated AT-1 has on intracellular acetyl-CoA homeostasis. Specifically, we used AT-1S113R/+ mice 8, a model of AT-1 haploinsufficiency, and AT-1 sTg mice 10, a model of AT-1 overexpression. We found that reduced AT-1 activity in AT-1S113R/+ mice led to increased availability of acetyl-CoA in the cytosol and spontaneous steatosis. Conversely, increased AT-1 activity decreased the availability of acetyl-CoA in the cytosol and made the animals resistant to diet-induced steatosis. Both models displayed significant metabolic adaptation involving different cellular organelles and compartments. Mechanistically, the metabolic adaptation was driven by changes in both protein levels (proteome) and stoichiometry of acetylation (acetylome) within fundamental pathways. Collectively, our results suggest that AT-1 acts as an important metabolic regulator that maintains acetyl-CoA homeostasis by promoting functional “cross-talk” between different intracellular organelles and compartments.
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Project description:Nε-lysine acetylation has emerged as a central mechanism to maintain quality control and protein homeostasis within the Endoplasmic Reticulum (ER) and secretory pathway. The ER acetylation machinery includes AT-1/SLC33A1, a membrane transporter that translocates acetyl-CoA from the cytosol to the ER lumen, and ATase1 and ATase2, two ER membrane-bound acetyltransferases that acetylate ER cargo proteins. Dysfunctional AT-1, as caused by loss-of-function mutations or gene duplication events, results in neurodevelopmental or neurodegenerative disorders. Experiments in our lab have demonstrated that these human diseases can be effectively recapitulated in mouse models. In this thesis, we used two models of dysregulated acetyl-CoA flux: AT-1S113R/+, a model of AT-1 haploinsufficiency, and AT-1 sTg, a model of systemic overexpression of AT-1. First, we examined upstream processes of cytosol-to-ER acetyl-CoA flux by evaluating the cytosolic pool of acetyl-CoA. The aberrant AT-1 models demonstrated distinct metabolic reprogramming of lipid metabolism and mitochondria bioenergetics. Dysregulated acetyl-CoA flux resulted in global changes at the level of the proteome and the acetyl-proteome. Second, we examined the downstream consequences of cytosol-to-ER acetyl-CoA flux. Specifically, we investigated the engagement and functional organization of the secretory pathway and used N-glycoproteomics to determine the quality of secreted proteins. Aberrant AT-1 models demonstrated reorganization of the ER, Golgi, and ERGIC, as well as a delay in glycoproteins clearing the Golgi apparatus. Additionally, AT-1 sTg mice showed a marked delay in protein trafficking to the cell surface. The N-glycoproteome revealed significant alterations, highlighting changes in the quality of the secretome.
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Project description:Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.
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