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1.
Metabolomics ; 16(10): 106, 2020 10 06.
Article in English | MEDLINE | ID: mdl-33021695

ABSTRACT

INTRODUCTION: Approximately 1% of the world's population is impacted by epilepsy, a chronic neurological disorder characterized by seizures. One-third of epileptic patients are resistant to AEDs, or have medically refractory epilepsy (MRE). One non-invasive treatment that exists for MRE includes the ketogenic diet, a high-fat, low-carbohydrate diet. Despite the KD's success in seizure attenuation, it has a few risks and its mechanisms remain poorly understood. The KD has been shown to improve metabolism and mitochondrial function in epileptic phenotypes. Potassium channels have implications in epileptic conditions as they have dual roles as metabolic sensors and control neuronal excitation. OBJECTIVES: The goal of this study was to explore changes in the lipidome in hippocampal and cortical tissue from Kv1.1-KO model of epilepsy. METHODS: FT-ICR/MS analysis was utilized to examine nonpolar metabolome of cortical and hippocampal tissue isolated from a Kv1.1 channel knockout mouse model of epilepsy (n = 5) and wild-type mice (n = 5). RESULTS: Distinct metabolic profiles were observed, significant (p < 0.05) features in hippocampus often being upregulated (FC ≥ 2) and the cortex being downregulated (FC ≤ 0.5). Pathway enrichment analysis shows lipid biosynthesis was affected. Partition ratio analysis revealed that the ratio of most metabolites tended to be increased in Kv1.1-/-. Metabolites in hippocampal tissue were commonly upregulated, suggesting seizure initiation in the hippocampus. Aberrant mitochondrial function is implicated by the upregulation of cardiolipin, a common component in the mitochondrial membrane. CONCLUSION: Generally, our study finds that the lipidome is changed in the hippocampus and cortex in response to Kv1.1-KO indicating changes in membrane structural integrity and synaptic transmission.


Subject(s)
Epilepsy/metabolism , Lipid Metabolism/physiology , Animals , Diet, Ketogenic/methods , Disease Models, Animal , Epilepsy/diet therapy , Hippocampus/metabolism , Kv1.1 Potassium Channel/genetics , Kv1.1 Potassium Channel/metabolism , Male , Mice , Mice, Inbred ICR , Mice, Knockout
2.
J Proteomics ; 223: 103812, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32418907

ABSTRACT

Epilepsy is a disorder that affects around 1% of the population. Approximately one third of patients do not respond to anti-convulsant drugs treatment. To understand the underlying biological processes involved in drug resistant epilepsy (DRE), a combination of proteomics strategies was used to compare molecular differences and enzymatic activities in tissue implicated in seizure onset to tissue with no abnormal activity within patients. Label free quantitation identified 17 proteins with altered abundance in the seizure onset zone as compared to tissue with normal activity. Assessment of oxidative protein damage by protein carbonylation identified additional 11 proteins with potentially altered function in the seizure onset zone. Pathway analysis revealed that most of the affected proteins are involved in energy metabolism and redox balance. Further, enzymatic assays showed significantly decreased activity of transketolase indicating a disruption of the Pentose Phosphate Pathway and diversion of intermediates into purine metabolic pathway, resulting in the generation of the potentially pro-convulsant metabolites. Altogether, these findings suggest that imbalance in energy metabolism and redox balance, pathways critical to proper neuronal function, play important roles in neuronal network hyperexcitability and can be used as a primary target for potential therapeutic strategies to combat DRE. SIGNIFICANCE: Epileptic seizures are some of the most difficult to treat neurological disorders. Up to 40% of patients with epilepsy are resistant to first- and second-line anticonvulsant therapy, a condition that has been classified as refractory epilepsy. One potential therapy for this patient population is the ketogenic diet (KD), which has been proven effective against multiple refractory seizure types However, compliance with the KD is extremely difficult, and carries severe risks, including ketoacidosis, renal failure, and dangerous electrolyte imbalances. Therefore, identification of pathways disruptions or shortages can potentially uncover cellular targets for anticonvulsants, leading to a personalized treatment approach depending on a patient's individual metabolic signature.


Subject(s)
Epilepsy , Seizures , Anticonvulsants/therapeutic use , Energy Metabolism , Epilepsy/drug therapy , Humans , Oxidation-Reduction , Seizures/drug therapy
3.
PLoS One ; 13(12): e0207161, 2018.
Article in English | MEDLINE | ID: mdl-30540769

ABSTRACT

The success of Staphylococcus aureus as a pathogen is due in part to its ability to adapt to changing environmental conditions using signal transduction pathways, such as metabolite- responsive regulators and two-component systems. S. aureus has a two-component system encoded by the gene pair sav0224 (hptS) and sav0223 (hptR) that regulate the hexose phosphate transport (uhpT) system in response to extracellular glucose-6-phosphate. Glycolytic intermediates such as glucose-6-phosphate are important carbon sources that also modulate the activity of the global metabolite-responsive transcriptional regulator CcpA. Because uhpT has a putative CcpA binding site in its promoter and it is regulated by HptR, it was hypothesized the regulons of CcpA and HptR might intersect. To determine if the regulatory domains of CcpA and HptRS overlap, ccpA was deleted in strains SA564 and SA564-ΔhptRS and growth, metabolic, proteomic, and transcriptional differences were assessed. As expected, CcpA represses hptS and hptR in a glucose dependent manner; however, upon CcpA derepression, the HptRS system functions as a transcriptional activator of metabolic genes within the CcpA regulon. Importantly, inactivation of ccpA and hptRS altered sensitivity to fosfomycin and ampicillin in the absence of exogenous glucose-6-phosphate, indicating that both CcpA and HptRS modulate antibiotic susceptibility.


Subject(s)
Bacterial Proteins/genetics , Staphylococcus aureus/metabolism , Ammonia/analysis , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Binding Sites , Chromatography, High Pressure Liquid , Fosfomycin/pharmacology , Glucose/analysis , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Mutagenesis , Promoter Regions, Genetic , Proteome/analysis , Proteomics , Spectrometry, Mass, Electrospray Ionization , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development , Transcription, Genetic/drug effects
4.
Redox Biol ; 12: 139-149, 2017 08.
Article in English | MEDLINE | ID: mdl-28236766

ABSTRACT

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6h. Exposure of an LC50 dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by ICP-MS. Measurement of the relative ATP and ADP concentrations indicates an initial 3-fold depletion of ATP suggesting an alteration in the ATP:ADP ratio. At the 6h time point the lidocaine exposed population contained ATP concentrations roughly 85% that of the negative control suggesting the surviving population adapted its metabolic pathways to, at least partially restore cellular bioenergetics. Metabolite analysis indicates an increase of intermediates in the pentose phosphate pathway, the preparatory phase of glycolysis, and NADPH. Oxidative stress produced by lidocaine exposure targets aconitase decreasing its activity with an observed decrease in isocitrate and an increase citrate. Similarly, increases in α-ketoglutarate, malate, and oxaloacetate imply activation of anaplerotic reactions. Antioxidant molecule glutathione and its precursor amino acids, cysteine and glutamate were greatly increased at later time points. Phosphatidylserine externalization suggestive of early phase apoptosis was also observed. Genetic studies using metacaspase null strains showed resistance to lidocaine induced cell death. These data suggest lidocaine induces perpetual mitochondrial depolarization, ROS/RNS generation along with increased glutathione to combat the oxidative cellular environment, glycolytic to PPP cycling of carbon generating NADPH, obstruction of carbon flow through the TCA cycle, decreased ATP generation, and metacaspase dependent apoptotic cell death.


Subject(s)
Anesthetics, Local/adverse effects , Lidocaine/adverse effects , Metabolomics/methods , Saccharomyces cerevisiae/drug effects , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Energy Metabolism/drug effects , Glutathione/metabolism , Humans , Microbial Viability/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/metabolism , Tandem Mass Spectrometry
5.
Int J Biol Macromol ; 96: 817-832, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28057574

ABSTRACT

Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a healthy sample with an affected sample have been widely used for disease biomarker discovery. Although proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a multitude of structural and functional processes. The correct association between metal ions and proteins is essential because this binding can significantly interfere with normal protein function. Employment of a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic treatment with insulin and non-diabetic rats. Proteins were detected by ESI-MS/MS. Seventy-five different proteins were found with alterations in the metal ions of interest. The most prominent pathways affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen storage, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treatment can modify some metal bonds in proteins, and therefore affect their mechanism of action and function.


Subject(s)
Diabetes Mellitus/metabolism , Liver/metabolism , Metalloproteins/metabolism , Metals/metabolism , Proteomics , Amino Acids/metabolism , Animals , Carbohydrate Metabolism , Energy Metabolism , Male , Oxidative Stress , Rats , Rats, Wistar
6.
OMICS ; 20(12): 711-726, 2016 12.
Article in English | MEDLINE | ID: mdl-27849439

ABSTRACT

Diabetes mellitus type 1 (DM1) is a major public health problem that continues to burden the healthcare systems worldwide, costing exponentially more as the epidemic grows. Innovative strategies and omics system diagnostics for earlier diagnosis or prognostication of DM1 are essential to prevent secondary complications and alleviate the associated economic burden. In a preclinical study design that involved streptozotocin (STZ)-induced DM1, insulin-treated STZ-induced DM1, and control rats, we characterized the insulin-dependent and -independent changes in protein profiles in liver samples. Digested proteins were subjected to LC-MSE for proteomic data. Progenesis QI data processing and analysis of variance were utilized for statistical analyses. We found 305 proteins with significantly altered abundance among the control, DM1, and insulin-treated DM1 groups (p < 0.05). These differentially regulated proteins were related to enzymes that function in key metabolic pathways and stress responses. For example, gluconeogenesis appeared to return to control levels in the DM1 group after insulin treatment, with the restoration of gluconeogenesis regulatory enzyme, FBP1. Insulin administration to DM1 rats also restored the blood glucose levels and enzymes of general stress and antioxidant response systems. These observations are crucial for insights on DM1 pathophysiology and new molecular targets for future clinical biomarkers, drug discovery, and development. Additionally, we underscore that proteomics offers much potential in preclinical biomarker discovery for diabetes as well as common complex diseases such as cancer, dementia, and infectious disorders.


Subject(s)
Insulin/metabolism , Liver/metabolism , Precision Medicine/methods , Proteome/metabolism , Animals , Biomarkers/metabolism , Computational Biology , Male , Proteomics , Rats
7.
Proteomics ; 16(13): 1889-903, 2016 07.
Article in English | MEDLINE | ID: mdl-27193513

ABSTRACT

Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.


Subject(s)
Anesthetics, Local/adverse effects , Lidocaine/adverse effects , Oxidative Stress/drug effects , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Two-Dimensional Difference Gel Electrophoresis/methods , Carbohydrate Metabolism/drug effects , Protein Interaction Maps/drug effects , Proteome/metabolism , Proteomics , Saccharomyces cerevisiae/metabolism
8.
Nat Cell Biol ; 14(6): 614-24, 2012 May 06.
Article in English | MEDLINE | ID: mdl-22561345

ABSTRACT

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore-spindle binding and chromosome segregation are mediated by the multi-copy KNL1(Spc105), MIS12(Mtw1) and NDC80(Ndc80) complexes that form the so-called KMN network. KMN-spindle attachment is regulated by the Aurora B(Ipl1) and MPS1(Mps1) kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.


Subject(s)
Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Anaphase , Cell Cycle Proteins/genetics , Chromosomes, Fungal/metabolism , Microtubule-Associated Proteins/genetics , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus/metabolism
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