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

Glucosamine Supplementation in Premating Drinking Water Improves Within-Litter Birth Weight Uniformity of Rats Partly through Modulating Hormone Metabolism and Genes Involved in Implantation

Within-litter birth weight variation in multiparous animals has become a big issue due to high incidence of low birth weight neonates, which gives rise to high preweaning mortality and morbidity. Foetus with various birth weights is the outcome of diverse embryos competence which is affected by oocyte quality. Glucosamine (GlcN) has been reported to be involved in oocyte maturation; however, its effect on pregnant outcomes remains unknown. The present study was conducted to investigate the effects of premating GlcN supplementation via drinking water on within-litter birth weight variation and its underlying mechanism. Fifty eight Sprague-Dawley female rats were randomly assigned to one of two groups with normal drinking water or drinking water supplemented with 0.5 mM GlcN from six to eight weeks old. Variation of within-litter birth weight in the GlcN group was 5.55%, significantly lower compared with 8.17% in the control group. Birth weight was significantly increased in the GlcN group (2.27 ± 0.06) compared with the control group (2.08 ± 0.04). Both absolute and relative weights of the ovary at the end of GlcN treatment were higher in the GlcN group than in the control group (P<0.05). In the GlcN group, there were more successfully implanted blastocysts (13.38 ± 0.63 and 15.75 ± 0.59 in the control and treatment group, respectively) with more uniform distribution along the two uterine horns compared with the control group. Besides, gene expressions of Alk3 and Bmp2 were increased in the implantation sites, while IGF-1 and Mucin-1 were decreased significantly in rats administrated with GlcN. Maternal progesterone, estradiol, and IGF-1 concentrations on D 19.5 were significantly increased, while insulin and total cholesterol levels were significantly decreased in contrast with control dams. In summary, the administration of 0.5 mM GlcN solution before mating reduced within-litter birth weight variation, accompanied with increased fetal weight. Further investigation indicated that the improved outcome of pregnancy results at least partly from the increased ovary weights of the rats, the homogeneous embryo developmental competence, the enhanced receptivity of the uterine environment, and the adjusted maternal hormone levels

An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells

Succinylation is a novel post-translational modification identified on many proteins and is involved in multiple biological processes. Succinylation levels are dynamically regulated, balanced by succinylation and desuccinylation processes, and are closely connected to metabolic state in vivo. Sirtuins have been shown to possess NAD+-dependent desuccinylation activity in vitro and in vivo, among which the desuccinylation activity of SIRT5 is most extensively studied. Our understanding of the response of succinylation levels to different metabolic conditions, is hampered by the lack of a fast NAD+-dependent desuccinylation assay in a physiological context. In the present study, we therefore optimized and validated a fluorescence-based assay for measuring NAD+-dependent desuccinylation activity in cell lysates. Our results demonstrated that shorter and stricter reaction time was critical to approach the initial rate of NAD+-dependent desuccinylation activity in crude cell lysate systems, as compared to the desuccinylation reaction of purified His-SIRT5. Analysis of desuccinylation activity in SIRT5 knockout HEK293T cells confirmed the relevance of SIRT5 in cellular desuccinylation activity, as well as the presence of other NAD+-dependent desuccinylase activities. In addition, we were able to analyse desuccinylation and deacetylation activity in multiple cell lines using this assay. We showed a remarkably higher desuccinylase activity, but not deacetylase activity, in proliferative cultured muscle and adipose cells in comparison with their differentiated counterparts. Our results reveal an alteration in NAD+-dependent desuccinylation activity under different metabolic states.</p

Single‐cell transcriptomics reveals distinct cell response between acute and chronic pulmonary infection of Pseudomonas aeruginosa

Abstract Knowledge of the changes in the immune microenvironment during pulmonary bacterial acute and chronic infections is limited. The dissection of immune system may provide a basis for effective therapeutic strategies against bacterial infection. Here, we describe a single immune cell atlas of mouse lungs after acute and chronic Pseudomonas aeruginosa infection using single‐cell transcriptomics, multiplex immunohistochemistry, and flow cytometry. Our single‐cell transcriptomic analysis revealed large‐scale comprehensive changes in immune cell composition and high variation in cell–cell interactions after acute and chronic P. aeruginosa infection. Bacterial infection reprograms the genetic architecture of immune cell populations. We identified specific immune cell types, including Cxcl2+ B cells and interstitial macrophages, in response to acute and chronic infection, such as their proportions, distribution, and functional status. Importantly, the patterns of immune cell response are drastically different between acute and chronic infection models. The distinct molecular signatures highlight the importance of the highly dynamic cell–cell interaction process in different pathological conditions, which has not been completely revealed previously. These findings provide a comprehensive and unbiased immune cellular landscape for respiratory pathogenesis in mice, enabling further understanding of immunologic mechanisms in infection and inflammatory diseases

A non-invasive 25-Gene PLNM-Score urine test for detection of prostate cancer pelvic lymph node metastasis

Background: Prostate cancer patients with pelvic lymph node metastasis (PLNM) have poor prognosis. Based on EAU guidelines, patients with &gt;5% risk of PLNM by nomograms often receive pelvic lymph node dissection (PLND) during prostatectomy. However, nomograms have limited accuracy, so large numbers of false positive patients receive unnecessary surgery with potentially serious side effects. It is important to accurately identify PLNM, yet current tests, including imaging tools are inaccurate. Therefore, we intended to develop a gene expression-based algorithm for detecting PLNM. Methods: An advanced random forest machine learning algorithm screening was conducted to develop a classifier for identifying PLNM using urine samples collected from a multi-center retrospective cohort (n = 413) as training set and validated in an independent multi-center prospective cohort (n = 243). Univariate and multivariate discriminant analyses were performed to measure the ability of the algorithm classifier to detect PLNM and compare it with the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram score. Results: An algorithm named 25 G PLNM-Score was developed and found to accurately distinguish PLNM and non-PLNM with AUC of 0.93 (95% CI: 0.85–1.01) and 0.93 (95% CI: 0.87–0.99) in the retrospective and prospective urine cohorts respectively. Kaplan–Meier plots showed large and significant difference in biochemical recurrence-free survival and distant metastasis-free survival in the patients stratified by the 25 G PLNM-Score (log rank P &lt; 0.001 and P &lt; 0.0001, respectively). It spared 96% and 80% of unnecessary PLND with only 0.51% and 1% of PLNM missing in the retrospective and prospective cohorts respectively. In contrast, the MSKCC score only spared 15% of PLND with 0% of PLNM missing. Conclusions: The novel 25 G PLNM-Score is the first highly accurate and non-invasive machine learning algorithm-based urine test to identify PLNM before PLND, with potential clinical benefits of avoiding unnecessary PLND and improving treatment decision-making