Zhang Yuxun, Zhang Bob B, Bharathi Sivakama S, Bons Joanna, Rose Jacob P, Shah Samah, Dobrowolski Steven F, Sims-Lucas Sunder, Schilling Birgit, Goetzman Eric S
Department of Pediatrics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15224, USA.
The Buck Institute for Research on Aging, Novato, CA 94945, USA.
Biomolecules. 2024 Nov 26;14(12):1508. doi: 10.3390/biom14121508.
Lysine succinylation, and its reversal by sirtuin-5 (SIRT5), is known to modulate mitochondrial fatty acid β-oxidation (FAO). We recently showed that feeding mice dodecanedioic acid, a 12-carbon dicarboxylic acid (DC) that can be chain-shortened four rounds to succinyl-CoA, drives high-level protein hypersuccinylation in the peroxisome, particularly on peroxisomal FAO enzymes. However, the ability of SIRT5 to reverse DC-induced peroxisomal succinylation, or to regulate peroxisomal FAO in this context, remained unexplored. Here, we showed that feeding DC strongly recruits SIRT5 into hepatic peroxisomes. Knocking out SIRT5 impaired peroxisomal FAO as evidenced by reduced C-DC flux in liver homogenates and elevated levels of partially shortened DC catabolites in urine. Further, mass spectrometry revealed a trend toward less peroxisomal protein succinylation in SIRT5 knockout liver. This is consistent with a reduced flux of DC through the peroxisomal FAO pathway, thereby reducing the production of the succinyl-CoA that chemically reacts with lysine residues to produce protein succinylation. Mass spectrometry comparisons of site-level succinylation in wildtype and SIRT5 knockout liver did not reveal any clear pattern of SIRT5 target sites in the peroxisome after DC feeding. However, SIRT5 co-immunoprecipitated with 15 peroxisomal proteins, including the key peroxisomal FAO enzymes acyl-CoA oxidase-1 and enoyl-CoA/3-hydroxyacyl-CoA dehydrogenase (EHHADH). In vitro, recombinant SIRT5 partially desuccinylated chemically modified recombinants ACOX1a, ACOX1b, and EHHADH. Desuccinylation by SIRT5 had no effect on enzyme activity for ACOX1a and EHHADH. For ACOX1b, SIRT5-mediated desuccinylation decreased activity by ~15%. Possible interpretations of these data are discussed.
已知赖氨酸琥珀酰化及其由沉默调节蛋白5（SIRT5）介导的逆转过程可调节线粒体脂肪酸β-氧化（FAO）。我们最近发现，给小鼠喂食十二烷二酸（一种12碳二羧酸（DC），可经四轮链缩短生成琥珀酰辅酶A）会导致过氧化物酶体中高水平的蛋白质超琥珀酰化，尤其是过氧化物酶体FAO酶上。然而，SIRT5逆转DC诱导的过氧化物酶体琥珀酰化的能力，或在此背景下调节过氧化物酶体FAO的能力，仍未得到探索。在此，我们表明喂食DC会强烈促使SIRT5进入肝脏过氧化物酶体。敲除SIRT5会损害过氧化物酶体FAO，肝脏匀浆中C-DC通量降低以及尿液中部分缩短的DC分解代谢物水平升高证明了这一点。此外，质谱分析显示SIRT5基因敲除肝脏中过氧化物酶体蛋白琥珀酰化有减少的趋势。这与DC通过过氧化物酶体FAO途径的通量降低一致，从而减少了与赖氨酸残基发生化学反应以产生蛋白质琥珀酰化的琥珀酰辅酶A的产生。对野生型和SIRT5基因敲除肝脏中位点水平琥珀酰化的质谱比较未发现喂食DC后过氧化物酶体中SIRT5靶位点的任何明确模式。然而，SIRT5与15种过氧化物酶体蛋白共免疫沉淀，包括关键的过氧化物酶体FAO酶酰基辅酶A氧化酶-1和烯酰辅酶A/3-羟酰基辅酶A脱氢酶（EHHADH）。在体外，重组SIRT5使化学修饰的重组体ACOX1a、ACOX1b和EHHADH部分去琥珀酰化。SIRT5介导的去琥珀酰化对ACOX1a和EHHADH的酶活性没有影响。对于ACOX1b，SIRT5介导的去琥珀酰化使活性降低了约15%。我们讨论了这些数据的可能解释。
