H2S S-sulfhydration of pyruvate carboxylase in gluconeogenesis and its regulation by Trx1
SUPERVISOR: Dr. Rui Wang (formerly with Biology)
ABSTRACT: Hydrogen sulfide (H2S), regarded as the third gasotransmitter, plays diverse physiological and pathological roles in the body along with another two gasotransmitters, including nitric oxide (NO) and carbon monoxide (CO). S-sulfhydration, a novel post-translational modification of proteins, is now considered as an important mechanism of H2S effects under various physiological and pathological conditions. In the liver, cystathionine gamma-lyase (CSE) is the main enzyme to generate H2S. The functions of H2S in the liver are largely unknown. Pyruvate carboxylase (PC), a mitochondrial protein, is an enzyme in the first step of gluconeogenesis in the liver. PC plays a critical role in tricarboxylic acid (TCA) cycle in mitochondria and in gluconeogenesis in the liver. PC is also involved in diverse metabolic pathways, such as lipogenesis and the biosynthesis of neurotransmitters. Here we found that H2S regulates gluconeogenesis through PC S-sulfhydration. First, PC activity and glucose production were decreased in liver of CSE-knockout (KO) mice comparing to the liver of wild type (WT) littermates. PC S-sulfhydration was lower in the liver tissue of CSE-KO mice than WT mice. NaHS treatment induces glucose production and PC activity in the primary liver cells. PC S-sulfhydration was increased by NaHS treatment in a time–dependent manner. Through mutation study, we further confirmed that the cysteine residue 265 is responsible for H2S S-sulfhdyration of PC. In cysteine 265 mutant-transfected cells, PC activity was significantly decreased and PC S-sulfhydration was disappeared. Thioredoxin 1 (Trx1), a well-known redox protein, is reported to be involved in transnitrosylation and/or denitrosylation. Here we first demonstrated that Trx1 acts as a S-desulfhydrase. Overexpressed Trx1 diminished H2S induced S-sulfhydration of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and PC proteins. The S-desulfhydration activity of Trx1 was inhibited by PX12, an inhibitor of Trx1, and DNCB, an inhibitor of thioredoxin reductase (TrxR). Furthermore, the cysteine 32 in Trx1 is required for the direct interaction of Trx1 with GAPDH or PC, and mutation of cysteine 32 abolished the S-desulfhydration activity of Trx1. Taken together, our studies demonstrated that H2S induces the gluconeogenesis through PC S-sulfhydration, and Trx1 acts as a S-desulfhydrase. Our findings will help to understand the mechanisms of H2S regulation of metabolic pathways via S-sulfhydration. These understandings will also extend the knowledge of physiological and pathological roles of H2S in both health and diseases.
H2S as a novel biomarker and therapeutic target for asthma
SUPERVISOR: Dr. Rui Wang (formerly with Biology)
ABSTRACT: Asthma is a chronic inflammatory disease with hyper-responsive bronchoconstriction and airway remodeling, leading to extensive airway narrowing. The pathogenic mechanisms for asthma remain unclear. Studies in the literature have shown that cystathionine gamma lysase (CSE)/ hydrogen sulfide (H2S) system participates in the regulation of airway contractility and immune response. In this PhD thesis study, we found that CSE was a major enzyme responsible for endogenous H2S synthesis in the lung and spleen as CSE gene knock out (CSE-KO) dramatically decreased H2S production rates in these two organs. In asthma model established via ovalbumin (OVA) sensitization and challenge, lung resistance of CSE-KO mice (12-16 weeks old), in response to aerosolized methacholine (MCh) at 12.5 mg/ml, was two times higher than that of wild type (WT) mice (12-16 weeks old). CSE-KO mice also developed more peri-bronchial inflammation and had higher levels of type 2 helper T cell (TH2) cytokines in bronchoalveolar lavage fluid (BALF). As allergic asthma is more prevalent among children than in adults, we next used young (3-4 weeks old) and old (7-8 months old) mice to observed if CSE/H2S were involved in the onset of childhood asthma. With the same intensity and duration of OVA treatments, WT young mice developed much more severe asthma with greater lung resistance, higher levels of eosinophils and TH2 cytokines in BALF, and more peri-bronchial inflammation than did WT old mice. This age-dependent propensity of immunoreaction and asthma development resulted from lower levels of CSE expression and H2S production in splenocytes from young mice, which was reversed by H2S donor treatment. Human umbilical cord blood mononuclear cells also had lower level of CSE proteins than that of peripheral blood mononuclear cell from adult people. CSE-KO mice had more severe asthma than WT mice but without the age-dependent asthma propensity. Lower endogenous level of CSE/H2S in WT young mice and in CSE-KO mice at all ages than in old WT mice promoted the differentiation of splenocytes into type 2 helper T cytokines-generating cells, which was suppressed by H2S donor. CSE/H2S-induced inhibition of type 2 immunity is not mediated by STAT-6 activation. Instead, H2S caused S-sulfhydration of GATA3 in spleen cells and decreased GATA3 nuclear translocation, leading to the inhibition of type 2 immunity. We also found that CSE expression in the airway of WT mice was increased in an age-dependent manner. Lower abundance of CSE in young WT mice or absence of CSE in young/old CSE-KO mice aggravated airway responsiveness to MCh challenge, in the absence of allergen exposure, by more than two times compared to old WT mice. In conclusion, CSE/H2S in peripheral lymph tissues and the lung suppresses allergen-induced type 2 immunity, airway responsiveness and the consequential asthma. Lower activity of CSE/H2S pathway renders higher incidence of allergic asthma in childhood.
Functional imaging of the lungs using magnetic resonance imaging of inert fluorinated gases
SUPERVISOR: Dr. Mitchell Albert (Chemistry)
ABSTRACT: Fluorine-19 (19F) magnetic resonance imaging (MRI) of the lungs using inhaled inert fluorinated gases can potentially provide high quality anatomical and functional images of the lungs. This technique is able to visualize the distribution of the inhaled gas, similar to hyperpolarized (HP) helium-3 (3He) and xenon-129 (129Xe) MRI. Inert fluorinated gases have the advantages of being nontoxic, abundant, and inexpensive compared to HP gases. Due to the high gyromagnetic ratio of 19F, there is sufficient thermally polarized signal for imaging, and averaging within a single breath-hold is possible due to short longitudinal relaxation times. Since inert fluorinated gases do not need to be hyperpolarized prior to their use in MRI, this eliminates the need for an expensive polarizer and expensive isotopes. Inert fluorinated gas MRI of the lungs has been studied extensively in animals since the 1980s, and more recently in healthy volunteers and patients with lung diseases. This thesis focused on the development of static breath-hold inert fluorinated gas MR imaging techniques, as well as the development functional imaging biomarkers in humans and animal models of pulmonary disease. Optimized ultrashort echo time (UTE) 19F MR imaging was performed in healthy volunteers, and images from different gas breathing techniques were quantitatively compared. 19F UTE MR imaging was then quantitatively compared to 19F gradient echo imaging in both healthy volunteers and in a resolution phantom. A preliminary comparison to HP 3He MR imaging is also presented, along with preliminary 19F measurements of the apparent diffusion coefficient (ADC) and iv gravitational gradients of ventilation in healthy volunteers. The potential of inert fluorinated gas MRI in detecting pulmonary diseases was further explored by performing ventilation mapping in animal models of inflammation and fibrosis. Overall, interest in pulmonary 19F MRI of inert fluorinated gases is increasing, and numerous sites around the world are now interested in developing this technique. This work may help to demonstrate that inert fluorinated gas MRI has the potential to be a viable clinical imaging modality that can provide useful information for the diagnosis and management of chronic respiratory diseases.
Bioremediation of contaminated soils from mine sites using native plants in Northwestern Ontario
SUPERVISOR: Dr. Peter Lee (Biology)
ABSTRACT: Practical and scientific importance can be found in this research topic since the results directly apply to remediation of industrial and mined lands in the boreal forest region. Plants suitable for phytostabilization of As, Mo and Sb are identified as well as two hyperaccumulators of Zn. Using phytostabilization practices, metals are immobilized by the below ground components of the plants therefore restricting the flow into the ecosystem and lessening the impacts of metal pollution to the surrounding area. As long as there is little disturbance of the soil physically or chemically, the plants will continue to stabilize the metal in the organic portion of the deceased plants. The ease of replanting a site could incorporate successional ecosystem in the region by focusing on trees and shrubs that are earlier in the revegetation process after a disturbance. The addition of woodbark to the reestablishment of the top soil increases potential nutrients, organic matter, water holding potential as well as diluting potential harmful metal content of the soil and providing a mulching effect. Some concerns exist by using agronomic plant species as the sole part of revegetation as they have the potential to impact the wildlife in the region through excess Mo. Results from this thesis could be helpful for future mine closure plans and in the rehabilitation of other industrial sites.
Hydrogen sulfide modulates gluconeogenesis and mitochanodrial biogenesis in mouse primary hepatocytes
SUPERVISOR: Dr. Lingyun Wu (formerly with Biology)
ABSTRACT: Among many endogenous substances that regulate hepatic energy production is the gasotransmitter hydrogen sulfide (H2S). In the liver, H2S production is largely catalyzed by cystathionine γ-lyase (CSE) and, to a lesser degree, by cystathionine β-synthase. We previously showed that H2S stimulates glucose production in an immortalized carcinoma liver cell line (HepG2 cells) as well as induce ATP generation in isolated vascular smooth muscle cells (VSMCs). Furthermore, we found that H2S upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in rat VSMCs. PGC-1α is a crucial regulator of hepatic gluconeogenesis and mitochondrial biogenesis. Both of these PGC-1α-mediated energy processes are pivotal to maintain whole-body energy homeostasis, whereby their sustained disturbance may lead to the development of type 2 diabetes and metabolic syndrome. Therefore, we investigated the regulation of gluconeogenesis and mitochondrial biogenesis by CSE-generated H2S under physiological conditions in isolated mouse hepatocytes. We found that CSE-knockout (KO) mice had a reduced rate of gluconeogenesis, which was reversed by administration of NaHS (an H2S donor) (i.p.). Interestingly, isolated CSE-KO hepatocytes exhibited a reduced glycemic response to chemical-induced activation of the cAMP/PKA and glucocorticoid pathways compared to wild-type (WT) hepatocytes. Treatment with the inhibitors for PKA (KT5720) or glucocorticoid receptor (RU-486) significantly reduced H2S-stimulated glucose production from both WT and CSE-KO mouse hepatocytes. NaHS treatment upregulated the protein levels of key gluconeogenic transcription factors, such as PGC-1α and CCAAT-enhancer-binding proteins-β (C/EBP-β). Moreover, exogenous H2S augmented the S-sulfhydration of the rate-limiting gluconeogenic enzymes and PGC-1α and increased their activities, which were lower in untreated CSE-KO hepatocytes. Finally, knockdown of PGC-1α, but not C/EBP-β, significantly decreased NaHS-induced glucose production from the primary hepatocytes. After determining that H2S stimulates hepatic glucose production through the PGC-1α signaling pathway, we focused on whether or not H2S induces hepatic mitochondrial biogenesis. We found that CSE-KO hepatocytes produced less mtDNA compared to WT hepatocytes. Mitochondrial content was decreased in CSE-KO hepatocytes compared to normal hepatocytes, which was restored with NaHS treatment. CSE-KO hepatocytes exhibited lower levels of mitochondrial transcription factors and the mitochondrial transcription coactivator, peroxisome proliferator-activated receptor-γ coactivator-related protein (PPRC) compared to WT hepatocytes. Interestingly, NaHS administration upregulated PPRC, yet downregulated PGC-1β protein level in mouse hepatocytes. Moreover, exogenous H2S induced the S-sulfhydration of PPRC, which was lower in untreated CSE-KO hepatocytes, but not that of PGC-β. Finally, knockdown of either PGC-1α or PPRC significantly decreased NaHS-stimulated mitochondrial biogenesis in hepatocytes, where knockdown of both genes were required to completely abolish NaHS-induced mitochondrial biogenesis. Overall this thesis demonstrates the stimulatory effect of endogenous H2S on liver glucose production and reveals four underlying mechanisms. 1) H2S upregulates the expression levels of PGC-1α and PEPCK via glucocorticoid receptor pathway. 2) H2S upregulates the expression level of PGC-1α through the activation of the cAMP/PKA pathway, as well as PGC-1α activity via S-sulfhydration. 3) H2S upregulates the expression and the activities (by S-sulfhydration) of G6Pase and FBPase. 4) H2S augments the protein expression level and activity (via S-sulfhydration) of PPRC. By stimulating the combined activities of PPRC and PGC-1α, H2S induces mitochondrial biogenesis, thereby supplying energy to support its induction of hepatic glucose production. This study may offer clues to the regulation of hepatic energy homeostasis under physiological conditions and its dysregulation in insulin-resistance diseases.
Modeling the molecular mechanisms of biocompatibility of artificial materials
SUPERVISOR: Dr. Oleg Rubel (formerly with Chemistry)
ABSTRACT: One of the most common reasons for implant failure is immune rejection. Implant rejection leads to additional surgical intervention and, ultimately, increases health cost as well as recovery time. Within a few hours after implantation, the implant surface is covered with host proteins. Adsorption of fibrinogen, a soluble plasma glycoprotein, is responsible in triggering the immune response to a given material and, subsequently, in determining its biocompatibility. The work presented here is focused on modeling the interaction between artificial surfaces and plasma proteins at the microscopic level by taking into account the physico-chemical properties of the surfaces. Carbon-based nanomaterials are chosen as a model system due to their unique bioadhesive and contradictory biocompatible properties as well as the possibility of functionalization for specific applications. Graphene and its derivatives, such as graphene oxide and reduced graphene oxide, demonstrate controversial toxicity properties in vitro as well as in vivo. In this study, by covalently adding chemical groups, the wettability of graphene surfaces and the subsequent changes in its biocompatibility are being examined. An empirical force field potential (AMBER03) molecular dynamic simulation code implemented in the YASARA software package was utilized to model graphene/biomolecule interactions. The accuracy of the force field choice was verified by modeling the adsorption of individual amino acids to graphene surface in a vacuum. The obtained results are in excellent agreement with previously published ab initio findings. In order to mimic the natural protein environment, the interaction of several amino acids with graphene in an explicit solvent was modeled. The results show that the behaviour of amino acids in aqueous conditions is drastically different from that in vacuum. This finding highlights the importance of the host environment when biomaterial-biomolecule interfaces are modeled. The surface of Graphene Oxide (GO) has been shown to exhibit properties that are useful in applications such as biomedical imaging, biological sensors and drug delivery. An assessment of the intrinsic affinity of amino acids to GO by simulating their adsorption onto a GO surface was performed. The emphasis was placed on developing an atomic charge model for GO that was not defined before. Next, the simulation of a fibrinogen fragment (D-domain) at the graphene surface in an explicit solvent with physiological conditions was performed. This D-domain contains the hidden (not expressed to the solvent) motifs (PI 7190-202 and P2 7377-395, and specifically P2-C portion 7383-395) that were experimentally found to be responsible for attracting inflammatory cells through CDllb/CD18 (Mac-1) leukocyte integrin and, consequently, promoting the cascade of immune reactions. It was hypothesized that the hydrophobic nature of graphene would cause critical changes in the fibrinogen D-domain structure, thus exposing the sequences and result in the foreign body reaction. To further study this issue, molecular mechanics was used to stimulate the interactions between fibrinogen and a graphene surface. The atomistic details of the interactions that determine plasma protein affinity modes on surfaces with high hydrophobicity were studied. The results of this work suggest that graphene is potentially pro-inflammatory surface, and cannot be used directly (without alterations) for biomedical purposes. A better understanding of the molecular mechanisms underlying the interaction between synthetic materials and biological systems will further the ultimate goal of understanding the biocompatibility of existing materials as well as design of new materials with improved biocompatibility.
Cardiac oxidative stress and antioxidant status in response to radiation and monocrotaline-induced cardiac dysfunction
SUPERVISOR: Dr. Neelam Khaper (NOSM, Biology adjunct)
ABSTRACT: Cardiovascular disease is the leading cause of death and disability worldwide. Oxidative stress has been implicated in many types of cardiovascular disease. Chronic cardiac stress conditions have been shown to be associated with an increase in myocardial oxidative stress following myocardial infarction, which in turn may lead to depressed contractile function, myocardial remodeling and heart failure. Antioxidants play a protective role against oxidative stress damage through the removal of free radical intermediates and inhibition of oxidation reactions. An imbalance of free radicals and antioxidants results in cellular and sub-cellular damage. Therefore, treatment with non-enzymatic antioxidants may provide protection against high levels of free radicals. We investigated, in three different studies, if treatment with antioxidants can protect the heart under conditions of oxidative stress. In the first study, a complex dietary supplement composed of numerous antioxidants and anti-inflammatory components was given to C57BL/6 mice that received a whole body radiation dose of 5 Gy to investigate potential cardioprotective effects by measuring cardiac antioxidant status and apoptosis. In the second study, the same complex dietary supplement was given to Thy1-GFP mice that received a radiation dose of 10 Gy to the head to investigate the abscopal effect on the heart by measuring cardiac inflammation and fibrosis. In the final study, the potential cardioprotective effects of secoisolariciresinol diglucoside, a compound found in flaxseed, was investigated in a Wistar rat model of pulmonary arterial hypertension by correlating cardiac functions with oxidative stress. We have shown that treatment with antioxidants may offer some protection to the heart in these models of oxidative stress though it is important to consider the extent of oxidative stress and when developing a antioxidant treatment protocol.