Small change, big impact: functional characterization of human SIRT5 variants

Mitochondria play a central role in cellular function and ultimate in organism health through their distinct bioenergetic, biosynthetic, and intracellular signaling functions. A number of human diseases are associated with mitochondrial dysfunctions, among them, genetic mitochondrial diseases. These disorders are caused by genetic variants in mitochondrial or nuclear DNA that encode the mitochondrial proteins. Sirtuin 5 (SIRT5) is a mitochondrial protein with a role in post-translational modifications homeostasis. It is a versatile NAD+-dependent desuccinylase, demalonylase and deglutarylase, with robust activities removing lysine succinyl-, malonyl- and glutaryl- groups on many metabolic enzymes. Using genetically modified mouse models, it was shown that SIRT5 is involved in various major metabolic pathways and that it plays an important role in metabolic response to environmental stress. Inconsistency in its regulatory roles of enzymatic activities and the fact that tissue specific SIRT5 knockout mice failed to produce the phenotypes observed in whole-body knockout mice illustrate the complexity of SIRT5 biology. More importantly, all studies addressing the biological function of SIRT5 were performed in animals or cell lines. It is therefore unclear, whether or not SIRT5 has an essential role in maintianing human metabolic health.In this thesis, I reported two novel homozygous SIRT5 variants in two unrelated patients with clinical mitochondrial disease manifestations. Using primary skin fibroblasts from the patients with the SIRT5 variants, I studied the physiopathological relevance of SIRT5 in humans and gained insights in the SIRT5 biological functon in humans. In addition, I also investigated a novel analytic method for better understanding NAD+-dependent desuccinylase activity in crude cell lysates.In Chapter 2 of this thesis, I optimized a fluorescence (fluor de lys) based desuccinylase activity assay to analyse the intracellular NAD+-dependent desuccinylase activity in whole cell lysates. I discovered distinct NAD+-dependent desuccinylase activities, for which SIRT5 was partially responsible. In addition, SIRT5 protein level was higher in differentiated C2C12 and 3T3-L1 cells as compared to their proliferative counterparts, whereas the desuccinylase activity was lower. Thus, NAD+-dependent desuccinylase activity in crude cell lysates varied between different metabolic conditions. The mismatch between SIRT5 protein level and the desuccinylase activity highlights the importance of analysing NAD+-dependent desuccinylase activity, over only analysing expression of proteins. In Chapter 3, I aimed to understand whether the SIRT5 variants identified in patients were associated with biological defects by studying the biochemistry of the SIRT5 variants as well as their biological impacts. Enzymatic kinectis of recombinant SIRT5 variants were mildly affected as compared to SIRT5 wild-type (WT), and thermal stabilities of SIRT5 variants were evidently decreased. It reflected at cellular level with strikingly reduced SIRT5 protein levels in the patients-derived SIRT5 variant fibroblasts as compared to controls. As a result, global succinylation levels were significantly increased in the patients’ fibroblasts. Among an array of mitochondrial functional analyses under different stress conditions, increased proton leak rates were the most consistent and pronounced phenotype under galactose culture medium, a metabolic challenging condition where fibroblasts were forced to strongly rely on mitochondrial respiration and consequently led to higher cellular ROS level. Further analysis showed that redox couple NADPH/NADP+ was decreased by 50% in patients under galactose condition. Although no difference was oberved under basal condition, intracellular ROS accumulation rates were significantly higher in the SIRT5 variant fibroblasts upon exogenous H2O2 challenge. Also, the H2O2-induced intracelluar ROS accumulation rates were positively related to intracellular NADPH/NADP+ ratios (R2 = 0.9476), suggesting a mechanistic link between the two phenotypes. To conclude, this study showed that each of the two SIRT5 variants was a loss-of-function variant and that redox homeostasis was significantly perturbated in the SIRT5 variant human fibroblasts. It highlights the important role for SIRT5 in maintaining human redox homeostasis. Next, in Chapter 4, I further looked into redox system to understand how the redox balance was affected by the SIRT5 variants. In the SIRT5 variant fibroblasts, intracellular GSH levels were significantly higher, 1.5-fold of controls, while glutathion reductase (GR) was surprisingly 2-fold lower than controls at gene expression as well as enzymatic activity level. In contrast, glutathione peroxidase 4 (GPx4) was significantly increased by 1.5-fold at transcriptional level in the SIRT5 variant fibroblasts. These changes showed an impairment in the GSH-dependent antioxidant system, which, in turn, made the patients’ fibroblasts less dependent on GSH-antixoidant defense system, as supported by the observation of blunted response to BSO-induced GSH deprivation. In contrast to evident decrease in cell viability associated with increased ROS levels observed in control fibroblasts subjected to BSO, no apparent changes were detected in the SIRT5 variant fibroblasts. In addition, longer time was needed for the SIRT5 variant fibroblasts to show BSO-induced cell morphological damage as compared to controls. As a result of decreased dependence on impaired GSH antioxidant system, the SIRT5 fibroblasts were more sensitive to inhibition of thioreodxin system, as evidenced by the morphological changes and increased ROS levels upon auranofin treatment, a well-established thioredoxin reductases inhibitor. Collectively, this study showed that SIRT5 variant fibroblasts displayed an imparied GSH-dependent antioxidant system associated with a disrupted glutathione-thioreodxin balance. In Chapter 5, a novel Bayesian hierarchical model was described to analyse Seahorse data which is featured with complex data structure.This thesis highlights the critical role for SIRT5 in regulating human redox homeostasis and provides evidence supporting that SIRT5 loss-of-function variants are associated with mitochondrial disease.

Bibliographic Details

Main Author:	Yuan, Taolin
Other Authors:	Keijer, J.
Format:	Doctoral thesis biblioteca
Language:	English
Published:	Wageningen University
Subjects:	Life Science,
Online Access:	https://research.wur.nl/en/publications/small-change-big-impact-functional-characterization-of-human-sirt
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