Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1, as well as glutamate dehydrogenase and succinate dehydrogenase, all of whose activities are reduced in Sirt5 KO BAT. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamic acid significantly decreases its activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.

Brown adipose tissue (BAT) from Sirt5 floxed mice and brown fat-specific Sirt5 KO mice (5-BKO) housed at RT and cold acclimated to 5 ⁰C were subjected to succinylation affinity enrichment and subsequent mass spectrometric analysis. For each of the 4 mouse groups we analyzed BAT from 5-6 mice. We analyzed the samples using an adapted form of the automated PTM identification and quantification using exclusively DIA (PIQED) workflow as described previously. Data independent acquisition was used for label free quantification.

Brown adipose tissue (BAT) from 1) Sirt5 floxed mice housed at RT 2) Sirt5 floxed mice housed cold acclimated to 5 ⁰C 3) brown fat-specific Sirt5 KO mice (5-BKO) housed at RT 4) brown fat-specific Sirt5 KO mice (5-BKO) housed cold acclimated to 5 ⁰C succinylation and acetylation immuno affinity enrichment protein expression changes