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Multilevel changes in the Mevalonate Pathway in Reduced Autophagy Aging Skeletal Muscle Cell Models

ABSTRACT: Autophagy is an essential cellular pathway that maintains homeostasis in the cell and healthy function overtime. Decline in autophagy function with age has been observed in skeletal muscle which leads to deterioration of muscle function. Defective autophagy has been linked with a reduction in the post-translational protein of prenylation bringing into question the association defective autophagy has with the mevalonate pathway. In this work, cell models of reduced autophagy function in L6 rat myoblast cells were used to study the response of cells to reduced autophagy with a focus on the mevalonate pathway and protein prenylation. The first cell model was an ATG7 knockout produced using CRISPR/Cas9 to remove the expression of functional ATG7 protein. The second cell model was an ATG7 siRNA treatment to reduce the expression of ATG7 protein. Mass spectrometry was applied to study the expression changes of prenylated proteins with enrichment for prenylated proteins by an isoprenoid analog. Gene expression of prenylated proteins and enzymes in the mevalonate pathway was assessed using quantitative polymerase chain reaction. Overall prenylation function was evaluated using mass cytometry with an isoprenoid analog and reporter attached using copper catalyzed cycloaddition. Both cell models indicated a direct impact on the mevalonate pathway related to autophagy decline, but there were differences between the two models. These cell models provide key findings that autophagy and the mevalonate pathway are interconnected at the gene, protein and functional level, in skeletal muscle indicating an area to target for further study in the understanding of the aging phenotype.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Yuanzhe Chen

LAB HEAD: Mark D Distefano

PROVIDER: PXD037896 | Pride | 2026-02-12

REPOSITORIES: Pride

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			Action	DRS
	ATG7-KnockOut_vs_WT_Comparison_.sps	Other
	ATG7_KD_profile_proteinGroups.txt	Txt
	ATG7_KO_Profile__proteinGroups.txt	Txt
	ATG7_KnockOut_Profile.sps	Other
	ATG7_Knockdown_Profile.sps	Other

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Project description:Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. Given the widespread involvement of prenylation in protein localization, membrane trafficking, and cellular signaling, dysregulation of prenylation and prenylated proteins have been implicated in the pathogenesis of numerous diseases. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. First docking experiments were used to examine how lipid length might impact prenyltransferase selectivity. Next, chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to four used in this study. Enzyme kinetics were used to evaluate these molecules as substrates for prenyltransferases in vitro. In cellulo metabolic labeling, enrichment and proteomic analysis were performed, resulting in the labeling of 8 proteins for C10AlkOPP (lipid length = 12 atoms), 70 proteins for C15AlkOPP (lipid length = 16 atoms), 41 proteins for C15hAlkOPP (lipid length = 17 atoms) and 7 proteins for C20AlkOPP (lipid length = 20 atoms). While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

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Multilevel changes in the Mevalonate Pathway in Reduced Autophagy Aging Skeletal Muscle Cell Models

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