Assistant Professor (male)
X3-106Δ
Organic Chemistry and Biochemistry
Τμήμα Χημείας
Πανεπιστήμιο Ιωαννίνων
Πανεπιστημιούπολη Ιωαννίων
e-mail: paschalisdoulias@uoi.gr
Tel: (26510) 08428
Fax:
https://www.linkedin.com/in/paschalis-thomas-doulias-90694458/
Βιοχημεία
My research interests involve basic and translational research focusing on the following thematic areas.
Basic Research
We are interested in investigating the molecular mechanisms that regulate the biochemical pathway of mitochondrial fatty acid oxidation (β-oxidation). Beta oxidation represents the main pathway of fatty acid catabolism in mammalian cells. It utilizes long, medium and short chain fatty acids as a substrate for the production of acetyl coenzyme A (acetyl-CoA) and reducing equivalents. The heart, under physiological conditions, derives nearly 70% of ATP molecules that are required for contractile, ionic and other functions through the oxidation of long chain fatty acids (LC-FAs). Energy generation in exercising muscle is accomplished via the catabolism of fatty acid. Finaly, the adaptive response to the physiological stress of fasting is achieved by the augmented rate of LC-FAO in the liver. The acetyl-CoA that is generated feeds into the TCA cycle whereas part of it is converted to ketone bodies. The latter are released into the circulation and are used to regenerate acetyl-CoA in the brain and kidney, two organs that cannot oxidize fatty acids.
Despite its physiological importance the molecular mechanisms regulating mFAO in health and disease remain incompletely understood.
Our working hypothesis is that energy demands activate signaling cascades that intergrade on the enzymes participating to mFAO pathway in the form of post translational modifications (PTMs). These signals control enzymatic activity and coordinate pathway performance in accordance with energy demands.
This hypothesis is tested in cells and animal models. Available models include mice with genetic deletion of the mitochondrial enzymes sirtuin-3 or sirtuin-5 as well as mice lacking the endothelial nitric oxide synthase (eNOS-/-). These genotypes display biochemical, metabolic and phenotypic changes consistent with impaired oxidation of LC-FAs. Sirt-3 is the major lysine deacetylase in the mitochondria. Its genetic deletion leads to hyperacetylation of metabolic enzymes including the proteins participating in mFAO. Sirtuin 5- (Sirt5) is the major lysine de-succinylase in the mitochondria. Succinylation is the posttranslational modification of lysine by succinate. Genetic deletion of Sirt5 in mice results in augmented levels of protein succinylation in mitochondria. Again, metabolic enzymes and specifically proteins participating in the mFAO pathway display augmented succinylation in the absence of Sirt5. Our published work in eNOS-/-mice documents reduced levels of NO and protein S-nitrosylation in the circulation and in several organs including the heart, liver and skeletal muscle. S-nitrosylation is a selective and reversible NO-derived PTM occurring on cysteine residues. We have also generated a mouse model harboring the Cys238A mutation on very long chain acyl-CoA dehydrogenase (VLCAF), the first enzyme in the mFOA pathway. Published work documents that cysteine 238 is necessary for the regulation of enzymatic activity by S-nitrosylation. This model will facilitate our understanding regarding the coordinated regulation of enzyme activity by PTMs.
Finally, the functional consequences of PTMs on enzymatic activity will be investigated in cell models using site directed mutagenesis.
Translational Research
This research area focused on long chain fatty acid oxidation disorders (LC-FAODs), a family of rare inherited autosomal recessive diseases that present with cardiomyopathy, intermittent muscle breakdown and liver failure. Treatments for LC-FAODs have largely involved nutritional and symptomatic management, thus there is an unmet need to develop therapies for these inborn errors of metabolism. The prevailing mechanism for the clinical phenotypes relates to the insufficient production of acetyl-CoA from the oxidation of LC-FAs leading to depletion of tricarboxylic acid cycle (TCA) intermediates and inadequate generation of ATP to meet metabolic demands.
We have conceptualized an innovative therapeutic strategy towards LC-FAODs. Our working hypothesis stems from the discovery that nitric oxide signaling regulates the mFAO pathway through selective and reversible post-translational modification of cysteine residues to form S-nitrosocysteine. We have documented that: (1) S-nitrosylation of cysteine residue 237 in human (238 in mouse) in VLCAD lowered the KM by 5-fold and increased the catalytic efficiency by 29-fold8. (2) Long term administration of bioactive NO in eNOS-/- mice restored the capacity to metabolize fatty acids and metabolic homeostasis10. (3) Pharmacological delivery of nitric oxide to human VLCAD-deficient fibroblasts restored the catalytic efficiency of the mutant protein and corrected the acylcarnitine profile, a diagnostic marker of the disease9. (3) Treatment of VLCAD-deficient cells with 2-mononitrate-1,3-diheptanoin (MNDH), an innovative hybrid molecule that simultaneously delivers NO and metabolic substrate in the form of heptanoic acid, restored the catalytic efficiency of mutant VLCAD and increased the rate of palmitate oxidation.
Therefore, based on this evidence as well as the knowledge derived from the use of nitric oxide medications in humans, we propose to test nitric oxide (NO) as a potential therapeutic for LC-FA oxidation disorders. Currently, we are trying to achieve the following goals. (1) Comprehensive evaluation and characterization of small molecules to augment nitric oxide signaling. We are testing existing FDA approved NO-delivering molecules as well as patent protected hybrid compositions that simultaneously deliver NO and metabolic substrate. (2) Determine how augmented NO signaling restores LC-FAO and corrects the metabolic insufficiency in common LC-FAODs. The compounds with favorable properties are tested in cells from patients with LC-FAODs, (3) Test the efficacy of NO signaling to correct VLCAD-deficiency in vivo. VLCAD-deficiency represents the most common LC-FAOD presenting with the most severe and life-threatening symptoms. These studies involve the generation of a mouse model expressing human VLCAD harboring the pathologic mutations V283A/V283A and its comprehensive biochemical, metabolic, and phenotypic assessment.
CV
Metrcis
Publications: 49 Citations: 3251 H-index: 28
Recent Publications (2018-2023)
1. Doulias PT, Yang H, Andreyev AY, Dolatabadi N, Scott H, K Raspur C, Patel PR, Nakamura T, Tannenbaum SR, Ischiropoulos H, Lipton SA. (2023). S-Nitrosylation-mediated dysfunction of TCA cycle enzymes in synucleinopathy studied in postmortem human brains and hiPSC-derived neurons. Cell Chem Biol 30: 965-975
2. Khare S, Kim LC, Lobel G, Doulias PT, Ischiropoulos H, Nissim I, Keith B, Simon MC. (2021) ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism. Cancer Metab 9: 40.
3. Lau B, Fazelinia H, Mohanty I, Raimo S, Tenopoulou M, Doulias PT, Ischiropoulos H. (2021). Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways. Redox Biol 47: 102153
4. Doulias PT, Tenopoulou M, Zakopoulos I, Ischiropoulos H. (2021). Organic mercury solid phase chemoselective capture for proteomic identification of S-nitrosated proteins and peptides. Nitric Oxide 117: 1-6
5. Zamani P, Proto EA, Wilson N, Fazelinia H, Ding H, Spruce LA, Davila A Jr, Hanff TC, Mazurek JA, Prenner SB, Desjardins B, Margulies KB, Kelly DP, Arany Z, Doulias PT, Elrod JW, Allen ME, McCormack SE, Schur GM, D’Aquilla K, Kumar D, Thakuri D, Prabhakaran K, Langham MC, Poole DC, Seeholzer SH, Reddy R, Ischiropoulos H, Chirinos JA. (2021). Multimodality assessment of heart failure with preserved ejection fraction skeletal muscle reveals differences in the machinery of energy fuel metabolism. ESC Heart Fail 8: 2698-2712
6. Doulias PT, Nakamura T, Scott H, McKercher SR, Sultan A, Deal A, Albertolle M, Ischiropoulos H, Lipton SA (2021). TCA cycle metabolic compromise due to an aberrant S-nitrosoproteome in HIV-associated neurocognitive disorder with methamphetamine use. J Neurovirol. 27: 379
7. Ramick MG, Kirkman DL, Stock JM, Muth BJ, Farquhar WB, Chirinos JA, Doulias PT, Ischiropoulos H, Edwards DG. (2021). The effect of dietary nitrate on exercise capacity in chronic kidney disease: a randomized controlled pilot study Nitric Oxide – Biology and Chemistry 106: 17-23
8. Umanah GKE, Ghasemi M, Yin X, Chang M, Kim JW, Zhang J, Ma E, Scarffe LA, Lee YI, Chen R, Tangella K, McNamara A, Abalde-Atristain L, Dar MA, Bennett S, Cortes M, Andrabi SA, Doulias PT, Ischiropoulos H, Dawson TM, Dawson VL. (2020). AMPA Receptor Surface Expression Is Regulated by S-Nitrosylation of Thorase and Transnitrosylation of NSF. Cell Rep. 33:108329
9. Vartika Mishra V, Diane B. Re DB, Le Verche V, Alvarez MJ, Alessandro Vasciaveo A, Jacquier A, Doulias PT, Greco TM, Nizzardo M, Papadimitriou D, Nagata T, Rinchetti P, Perez-Torres E, Politi K, Ikiz B, Clare K, Than ME, Corti S, Ischiropoulos H, Lotti F, Califano A, Przedborski S (2020). Systematic elucidation of neuron-astrocyte interaction in ALS using multi-modal integrated bioinformatics. Nat. Commun. 11: 5579-5598
10. Elshenawy S, Pinney SE, Stuart T, Doulias PT, Zura G, Parry S, Elovitz MA, Bennett MJ, Bansal A, Strauss JF III, Ischiropoulos H, Simmons RA (2020). The Metabolomic Signature of the Placenta in Spontaneous Preterm Birth. Int J Mol Sci. 21:1043
11. Urbano R., Karlinsey J.E. , Stephen J. Libby S.J., Doulias PT, Harry Ischiropoulos H., Helen I. Warheit-Niemi H.I., Liggitt D.H., Alexander R. Horswill A.R., Ferric C. Fang F.C. (2018). Host Nitric Oxide Disrupts Microbial Cell-to-Cell Communication to Inhibit Staphylococcal Virulence. Cell Host and Microbe 23:594-606
12. Guan D, Xiong Y, Borck PC, Jang C, Doulias PT, Papazyan R, Fang B, Jiang C, Zhang Y, Briggs ER, Hu W, Steger D, Ischiropoulos H, Rabinowitz JD, Lazar MA (2018). Diet-Induced Circadian Enhancer Remodeling Synchronizes Opposing Hepatic Lipid Metabolic Processes. Cell 174: 831-842
13. Frawley ER, Karlinsey JE, Singhal A, Libby SJ, Doulias PT, Ischiropoulos H, Fang FC (2018). Nitric Oxide Disrupts Zinc Homeostasis in Salmonella enterica Serovar Typhimurium. MBio. 9: pii: e01040-18.
14. Doulias PT, Tenopoulou M, Nakamoto K, Berrios K, Zura G, Li C, Faust M, Yakovishina V, Evans P, Tan L, Bennett MJ, Snyder NW, Quinn WJ 3rd, Baur JA, Atochin DN, Huang PL, Ischiropoulos H (2018). Oral nitrite restores age-dependent phenotypes in eNOS-null mice. JCI Insight. 3. pii: 122156
15. Doulias PT, Gould NS (2018). Analysis of Cysteine Post Translational Modifications Using Organic Mercury Resin. Curr Protoc Protein Sci. 94:e69
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