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Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS
Activity by CoQ10 Synthesis
Vera Mugoni, Ruben Postel, Valeria Cantanz...
Background - Terminology Preface
● Ubiad1 = enzyme of interest (UBIAD1 is the associated gene)
● bar mutants = barolo gene...
Background - Epidemiological
● 5.8 million Americans are afflicted with
Heart Failure, with 550,000 new cases
per year
● T...
Background - Biochemical
● Nitric oxide (NO) is synthesized in
endothelial (blood vessel) cells
● Superoxide (O2
-
) is a ...
Background - Big Picture
● Formation of an antioxidant network is
crucial for maintaining cardiovascular
homeostasis - bec...
Hypothesis - Main
● UBIAD1 encodes for Ubiad1, which is
a transmembrane protein with
biosynthetic enzymatic function that
...
Hypothesis - Ancillary
To ensure the veracity of their main conclusion, several accessory experiments
were performed. The ...
Experimental Design - Limitations
● 2 mutant strains in Zebrafish null allele barolo (bar mutants)
○ Bart31131
& Bars847
●...
Experiment 1: ΔUBIAD1 in bar mutants leads to increased
oxidative stress and eventually cardiovascular failure
● Quantifie...
Results - Experiment 1 - Oxidative Outcomes
1E-G: bar exhibits several signs of
increased oxidative damage
1P: treating ba...
Results - Experiment 1 - Zebrafish Anatomy
1A-B: arrows show vascular integrity
defects and arrowheads show collapsed
endo...
Results - Experiments 1 - Immunofluorescence
1H: SNO-Cys is a fluorescent antibody tag of S-
Nitrosylation of proteins - w...
Experiment 2: Ubiad1 produces CoQ10, a biosynthetic
enzyme that modulates the eNOS activity and NO signaling
● Variation o...
Results - Experiment 2 - CoQ10 production
Figures 2C, E-F
2C, E-F: at 72hpf, overall COQ10, and COQ10 and COQ9 labeled wit...
Results - Experiment 2 - 13
C6
Labeling
Figures 2B-F
2A: molecular weights of
CoQ9/10, with/without 13
C6
2B: Mass spectro...
Results - Experiment 2 - Statin Application
Statins are inhibitors of HMG-CoA
reductase, which is crucial in the
mevalonat...
Results - Experiment 2 - Statin Outcomes
Statins inhibit HMG-CoA Reductase, which reduces
CoQ10 synthesis (3D); resulting ...
Results - Experiment 2 - Heterozygotic embryos
Tg reporter shows ubiad1
homozygous (+,+) and ubiad1
heterzygous (+,-) outc...
Experiment 3: Ubiad1, an nonmitodronial enzyme, produces
CoQ10 in the golgi apparatus
● Colocalization studies:
○ Transfec...
Results - Experiment 3 - Ubiad1 localization
Figures 4A-D, S4: confocal
microscopy
4D: Brefeldin A (inhibits ER->Golgi
pro...
Experiment 4: SCCD-related mutations of Ubiad1 are
cardioprotective
● Schnyder crystalline corneal syndrome - a human corn...
Results - Experiment 4 - SCCD
Previous studies have shown that mutations in the Ubiad1 gene is linked with Schnyder
Crysta...
Experiment 5: Ubiad1 (Golgi) & Coq2 (mitochondria)
function independently in CoQ10 production for
cardiovascular protectio...
Results - Experiment 5 - COQ2 Knockout
Lack of COQ2 causes: oxidative stress,
developmental delay, small body, and
severe ...
Results - Experiment 5 - CoQ10 pools
Figure 6
● Mitochondrial CoQ10 and ROS levels do not
correlate with bar phenotype pen...
Experiment 6: Shear stress/Klf2a/eNOS-mediated pathway
is prevented by Ubiad1
● Treatment with N-nitro-L-arginine (L-NAME)...
Results - Experiment 6 - Klf2 MO treatment
Shear stress generates ROS, which signal
for vascular remodeling and angiogenes...
Results - Experiment 6 - nos1 MO treatment
In human cells: KLF2 directly controls eNOS
expression and signaling.
Zebrafish...
Results - Experiment 6 - SNAP treatment
Figure S6D-F
NO donor S-nitroso-N-acetyl-penicillamine
(SNAP) aggravated bar pheno...
Results - Experiment 6 - L-NAME treatment
L-NAME (selective inhibitor of eNOS
activity) decreased bar phenotype
penetrance...
Results - Timeline postfertilization
In bar mutants with no treatment:
24 hr: ubiquitous expression of ubiad1 mRNA
32 hr: ...
Summary of Experiments:
1. Bar mutants exhibit significantly higher rates of oxidative damage and cardiovascular disease
2...
Conclusion - Overview
UBIAD1 does appear to catalyze cytosolic CoQ10 synthesis
The cytosolic CoQ10 pool is necessary to re...
Conclusion - Moving Forward
Oxidative stress is implicated in, or directly the cause of, a large number of
human pathologi...
Limitations and Considerations
Limitations:
Only using zebrafish embryos and human endothelial cells (in vivo)
Early embry...
Special thank you to the Authors! (Some not pictured)
Department of Molecular Biotechnology & Health Sciences, Molecular B...
Bibliography
http://www.hindawi.com/journals/bmri/2012/817341/fig1/
http://circres.ahajournals.org/content/113/6/646.full#...
Questions for Discussion
Any questions/comments?
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  1. 1. Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis Vera Mugoni, Ruben Postel, Valeria Cantanzaro, Elisa De Luca, Emilia Turco, Giuseppe Digilio, Lorenzo Silengo, Michael P. Murphy, Claudio Medana, Didier Y. R. Stainier, Jeroen Bakkers, and Massimo M. Santoro Alexander Barrett-Wilsdon Carrie Bi 11/9/15 NST 190
  2. 2. Background - Terminology Preface ● Ubiad1 = enzyme of interest (UBIAD1 is the associated gene) ● bar mutants = barolo gene mutation in zebrafish (altered UBIAD1 enzyme) ● CoQ10 = antioxidant Coenzyme Q10 (also known as Ubiqunone) ● Coq2 = enzyme previously determined to synthesize CoQ10 in the mitochondria (only) (COQ2 is the associated gene) ● eNOS = endothelial Nitric Oxide Synthase ● SCCD - Schnyder's Crystalline Corneal Dystrophy - disease caused by buildup of cholesterol (which is caused by overactive Ubaid1 gene) ● hpf - Hours post-fertilization: refers to time relative to conception in zebrafish embryos ● Mutant - has a defect in a gene that alters, enhances, or inhibits its function ● Morphant - gene function is knocked down by a morpholino (MO) (base pairs to target RNA)
  3. 3. Background - Epidemiological ● 5.8 million Americans are afflicted with Heart Failure, with 550,000 new cases per year ● The mortality rate is 50% 5 years out and 90% 10 years out ● Oxidative damage is linked to heart failure, and is known to worsen exponentially over time. ● Prevention can save significant costs
  4. 4. Background - Biochemical ● Nitric oxide (NO) is synthesized in endothelial (blood vessel) cells ● Superoxide (O2 - ) is a Reactive Oxygen Species (ROS), and is synthesized when eNOS is uncoupled. ● Oxidative damage is mitigated by a pool of antioxidants, notably CoQ10 ● CoQ10 synthetic intermediates are known, as is the mitochondrial condensation enzyme (COQ2). ● UBIAD1 is proposed to be the equivalent enzyme in the Golgi Compartment membrane. CoQ synthesis pathway (red = UBIAD1 proposed)
  5. 5. Background - Big Picture ● Formation of an antioxidant network is crucial for maintaining cardiovascular homeostasis - because homeostasis has a small margin for deviations, an imbalance give rise to cardiovascular dysfunction and disorders. ● Oxidative stress is an imbalance between the Reactive Oxygen Species (ROS) and the body’s ability to remove the reactive intermediates ● Often, series of antioxidants are coupled in usage work to target specific areas of a tissue that is at risk to damage and degradating.
  6. 6. Hypothesis - Main ● UBIAD1 encodes for Ubiad1, which is a transmembrane protein with biosynthetic enzymatic function that catalyzes the non-mitochondrial production of CoQ10. This cytosolic CoQ10 pool then regulates eNOS and mitigates oxidative damage from ROS.
  7. 7. Hypothesis - Ancillary To ensure the veracity of their main conclusion, several accessory experiments were performed. The hypotheses were as follows: ● That barolo mutants exhibit a phenotype of oxidative stress and cardiovascular failure, as a result of the mutation in their UBIAD1 gene. ● That the Ubiad1 enzyme catalyzes the biosynthetic pathway of CoQ10 and is located in the Golgi and ER Membranes ● That SCCD-related mutations of UBIAD1 are cardioprotective ● That mitochondrial and cytosolic pools of CoQ10 perform different roles ● That Ubiad1 mitigates a shear stress/Klfa-mediated oxidative stress pathway
  8. 8. Experimental Design - Limitations ● 2 mutant strains in Zebrafish null allele barolo (bar mutants) ○ Bart31131 & Bars847 ● Short lifetime expectancy: 72 hour timeline ○ Between 60 - 72 hours post fertilization, bar mutants undergo a gradual breakdown of endocardial and myocardial cells leading to cardiac edema and eventually a collapse of the heart and vascular organ failure ● Early embryonic death prevents tracking lifetime damage
  9. 9. Experiment 1: ΔUBIAD1 in bar mutants leads to increased oxidative stress and eventually cardiovascular failure ● Quantified NADP/NADPH ratio and overexpression of antioxidant genes to compare WT sib of redox balance and general oxidative stress deviations ● ROS detector, CellROX used in measuring ROS in ECs of bar mutants ● Antibody against S-nitroso-cysteine (SNO-Cye) for testing levels of S- nitrosylation proteins in heart, blood vessel and pronephros cells ● Immunofluorescence analysis in 8-hydroxy-2’deoxyguanosine and DNA for oxidative lesion of DNA induced by free radical ● Detection fragmentation of myocardial and endothelial cells with TUNEL staining
  10. 10. Results - Experiment 1 - Oxidative Outcomes 1E-G: bar exhibits several signs of increased oxidative damage 1P: treating bar mutants with unmutated ubiad1 rescues them from the bar phenotype 1N: Both have mutated UbiA domains 1O: Ubiad1 expression is minimal in bar mutants, as compared to sibling zebrafish embryos (β-actin is a control) Figures 1E-G, N-P
  11. 11. Results - Experiment 1 - Zebrafish Anatomy 1A-B: arrows show vascular integrity defects and arrowheads show collapsed endocardium and edema 1C: arrows show fragmented endothelium 1D: arrow shows loss of endothelial cells Figures 1A-D C: sib and bar brains at 65 hpf A-B: healthy sibling (sib) and bar mutants at 72 hpf D: Trunk vasculature For reference: zebrafish anatomy
  12. 12. Results - Experiments 1 - Immunofluorescence 1H: SNO-Cys is a fluorescent antibody tag of S- Nitrosylation of proteins - which is a measure of DNA repair 1L: a TUNEL assay assesses DNA fragmentation 1I: 8-OHdG is a type of DNA lesion caused by oxidative stress E: endocardium M: myocardium DA: dorsal aorta PCV: posterior cardinal vein BuA: Bulbus arteriosus PD: Pronepheric ducts Results 1H-I,L
  13. 13. Experiment 2: Ubiad1 produces CoQ10, a biosynthetic enzyme that modulates the eNOS activity and NO signaling ● Variation of CoQ10 formulations (F1, F2, F3) injected in single-cell stage of bar mutants with Ubiad1 deficiency demonstrated intake recovers normal cardiovascular development ● Analysis of 13 C6 -labelled hydroxyl-4-benzoic acid precursor metabolized to CoQ10-13 Q6 by HPLC-MS quantification from lipid extraction (metabolic studies) ● Treatment of statins in zebrafish embryos to block HMG-CoA reductase; Ubiad1 & HMG-CoA reductase in Mevalonate pathway for production of CoQ10
  14. 14. Results - Experiment 2 - CoQ10 production Figures 2C, E-F 2C, E-F: at 72hpf, overall COQ10, and COQ10 and COQ9 labeled with “heavy Carbon”, respectively
  15. 15. Results - Experiment 2 - 13 C6 Labeling Figures 2B-F 2A: molecular weights of CoQ9/10, with/without 13 C6 2B: Mass spectrometry of de novo synthesized CoQ10 - peak at m/z=885. 67 (Coq10-Na+ ) 2D: Administration of exogenous CoQ10 rescues bar mutants; Vit K2 administration does not
  16. 16. Results - Experiment 2 - Statin Application Statins are inhibitors of HMG-CoA reductase, which is crucial in the mevalonate pathway to produce the polyprenyl-PP pool for CoQ10 synthesis. WT embryos are treated with DMSO and statin at 72 hpf; statin treatment embryos characterized by bar-like phenotype (3A-C). CoQ10 supplement treatment before statin treatment prevents cardiac edema (*) and brain hemorrhages (^) (3K). Figure 3A-B, K
  17. 17. Results - Experiment 2 - Statin Outcomes Statins inhibit HMG-CoA Reductase, which reduces CoQ10 synthesis (3D); resulting lack of CoQ10 caused increased oxidative stress in the cardiovascular oxidative phenotype (3L). Injecting exogenous CoQ10 reverses this effect (3L). MEV, SIM, and MEN are statins Most importantly: statins cause bar-like phenotype to develop in WT embryos, and CoQ10 administration reverses the effect (3C, 3L, 3J). Figure 3
  18. 18. Results - Experiment 2 - Heterozygotic embryos Tg reporter shows ubiad1 homozygous (+,+) and ubiad1 heterzygous (+,-) outcomes Embryo trunk vasculature - (+,-) shows collapsed Se and DA morphology (3H- I). Heterozygous embryos are more prone to develop bar-like phenotypes (*,^) when treated when statin (3F,G). Figures 3F-I
  19. 19. Experiment 3: Ubiad1, an nonmitodronial enzyme, produces CoQ10 in the golgi apparatus ● Colocalization studies: ○ Transfected Golgi-specific marker TGN46, GM-130 and ɣ-adaptin in asymmetric perinuclear region like Golgi membranes to demonstrate that Ubiad1 is in the proximity of the Golgi compartments ○ Transfected GFP in human ECs and treated with brefeldin ● Subcellular fractionation experiments: ○ Incubation of ECs with hydroxy-4-benzoic acid-13 C6 and comparison of Golgi fractions with mitochondrial fractions in CoQ10-13 Q6 production
  20. 20. Results - Experiment 3 - Ubiad1 localization Figures 4A-D, S4: confocal microscopy 4D: Brefeldin A (inhibits ER->Golgi protein transport) added 4E: Western blot - cells were fractionated, then tested 4F: 13 C6 CoQ10 production in fractions Figure 4A -F Figure S4
  21. 21. Experiment 4: SCCD-related mutations of Ubiad1 are cardioprotective ● Schnyder crystalline corneal syndrome - a human corneal dystrophy has been linked to Ubiad1 by previous studies. ● Levels of expression and CoQ10 synthesis tested in Ubiad1-N102S and Ubiad1-D112 mutation (two common mutations). ● Protein level analysis by Western Blot in WT and SCCD isoforms (N102S & D112) to determine if variant positively regulate CoQ10 synthesis
  22. 22. Results - Experiment 4 - SCCD Previous studies have shown that mutations in the Ubiad1 gene is linked with Schnyder Crystalline Corneal Syndrome. The authors reproduced two common mutations (N102S and D112G), created constructs of them (with promoter and stop sequences). 5A: effect of injecting UBIAD isoforms on bar mutants 5B: Western blot, post UBIAD isoform injection 5C-E: 13 C6 labelled CoQ10, total CoQ10, and Cholesterol concentrations, respectively, post injection Figure 5A-E
  23. 23. Experiment 5: Ubiad1 (Golgi) & Coq2 (mitochondria) function independently in CoQ10 production for cardiovascular protection ● To differentiate between the role of Ubiad1 and Coq2 in CoQ10 production, gene expression was silenced separately (to test if one could rescue the other by an upregulation of the active gene) and also silenced simultaneously to measure efficiency of each producer and pool ● By using MitoQ, a specific mitochondrial targeted antioxidant, it is demonstrated that the two subcellular pools in CoQ10 are separated in their ability to mediate oxidative damage, each affecting the embryonic phenotypes differently
  24. 24. Results - Experiment 5 - COQ2 Knockout Lack of COQ2 causes: oxidative stress, developmental delay, small body, and severe hindbrain edema Lack of COQ2 does not cause: compromised vascular integrity or survival. These phenotypes differ from and do not occur in bar or SCCD mutants Figures S5A-B: coq2 morphants at 48hpf (B) and 72 hpf (C) Figure 6A
  25. 25. Results - Experiment 5 - CoQ10 pools Figure 6 ● Mitochondrial CoQ10 and ROS levels do not correlate with bar phenotype penetrance ● Knocking out/silencing both UBIAD1 and COQ2 lowers [CoQ10] more than inhibiting just one ● UBIAD1 and COQ2 have specific roles
  26. 26. Experiment 6: Shear stress/Klf2a/eNOS-mediated pathway is prevented by Ubiad1 ● Treatment with N-nitro-L-arginine (L-NAME), the selective inhibitor of eNOS activity to test morphological and cardiovascular functionality in the bar mutants ● Treatment with S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor, on the bar phenotype to test for level of reactive oxidative species ● Silencing of Ubiad1 for NO-forming activity in both zebrafish embryos and in human ECs; DAF-2DA staining for NO levels
  27. 27. Results - Experiment 6 - Klf2 MO treatment Shear stress generates ROS, which signal for vascular remodeling and angiogenesis. Kruppel-like factor 2 (Klf2) - mediates hemodynamic forces; blocking this pathway (by knocking out Klf2a or preventing blood flow by knocking out troponin T2 (tnnt2) in bar mutants significantly delays bar phenotypic expression (e.g. heart problems) and leads to a partial recovery from EC oxidative stress. Figures S6A-C
  28. 28. Results - Experiment 6 - nos1 MO treatment In human cells: KLF2 directly controls eNOS expression and signaling. Zebrafish embryos have a blood-flow-dependent klf2a-NO signaling cascade. Inhibition of zebrafish nos1 expression significantly rescued bar phenotype. Problem: inhibiting nos interferes with cell development and blood flow. Solution: use selective nos inhibitor L-NAME Figures 7A-B
  29. 29. Results - Experiment 6 - SNAP treatment Figure S6D-F NO donor S-nitroso-N-acetyl-penicillamine (SNAP) aggravated bar phenotype and increased oxidative species This may be due a shift in the equilibrium, such that eNOS is more likely to uncouple and produce ROS if there is a high [NO] bar mutants show a marked drop in NO synthesis
  30. 30. Results - Experiment 6 - L-NAME treatment L-NAME (selective inhibitor of eNOS activity) decreased bar phenotype penetrance (i.e. bar + L-NAME and WT-sib are morphologically more similar) (7C). L- NAME also enables functional cardiovascular recovery, where oxidative stress is significantly reduced and redox balance is restored (7D, E, F). In silencing eNOS in human ECs, ROS levels are regulated (7G). In silencing Ubiad1, NOS activity was measured, ΔUbiad1 reduced formation of NO Figures 7 C-H
  31. 31. Results - Timeline postfertilization In bar mutants with no treatment: 24 hr: ubiquitous expression of ubiad1 mRNA 32 hr: Onset of oxidative stress 36 hr: trunk blood vessels start failing (bar phenotype starts) 48 hr: distinct expression of ubiad1 mRNA in heart 60-72 hr: heart failure due to gradual breakdown of endocardial and myocardial cells 72 hr: complete heart and vascular organ failure; death
  32. 32. Summary of Experiments: 1. Bar mutants exhibit significantly higher rates of oxidative damage and cardiovascular disease 2. Ubiad1 is the cytosolic equivalent of Coq2: performing the crucial enzymatic condensation necessary to produce CoQ10; statins block production by inhibiting precursor synthesis 3. Ubiad1 is a transmembrane protein, located in the Golgi Compartment 4. SCCD mutations cause unusually high expression of Ubiad1; this is therapeutic in bar mutants 5. Ubiad1 and Coq2 produce CoQ10 in different organelles; those CoQ10 pools then serve different purposes. Ubiad1 and Coq2 expression are not strongly correlated 6. The Klf2 pathway produces ROS; inhibiting it rescues the bar phenotype, but can have other effects
  33. 33. Conclusion - Overview UBIAD1 does appear to catalyze cytosolic CoQ10 synthesis The cytosolic CoQ10 pool is necessary to regulate eNOS UBIAD1 mitigates multiple ROS sources (eNOS and Klf2) and associated damage The mitochondrial CoQ10 pool is produced by Coq2 and is less involved in free radical detoxification
  34. 34. Conclusion - Moving Forward Oxidative stress is implicated in, or directly the cause of, a large number of human pathologies. Cytosolic CoQ10 mitigates at least two ROS sources and prevents degenerative damage. Upregulating Ubiad1 could be beneficial
  35. 35. Limitations and Considerations Limitations: Only using zebrafish embryos and human endothelial cells (in vivo) Early embryonic death prevents tracking lifetime consequences Implications: What would a drug made from this info do? Would it be taken for a lifetime to prevent oxidative damage (large group of people) or for those with life-threatening oxidation (small pool)? Possible implications of hyperactive Ubiad1 (e.g. SCCD)? Necessity of ROS, NO, cholesterol? Statin use is very prevalent in humans; statins and metabolites then accumulate in the environment. Statins are toxic to zebrafish - and likely are in other fish
  36. 36. Special thank you to the Authors! (Some not pictured) Department of Molecular Biotechnology & Health Sciences, Molecular Biotechnology Center Department of Chemistry & Department of Developmental Genetics University of Torino, Hubrecht Institute, Medical Research Council, Max Planck Institute
  37. 37. Bibliography http://www.hindawi.com/journals/bmri/2012/817341/fig1/ http://circres.ahajournals.org/content/113/6/646.full#ref-1 http://www.ncbi.nlm.nih.gov/pubmed/21949114 http://ajpheart.physiology.org/content/301/6/H2181 https://en.wikipedia.org/wiki/UBIAD1 http://www.ncbi.nlm.nih.gov/pubmed?linkname=gene_pubmed&from_uid=29914 http://weeksmd.com/2012/05/statins-polluting-the-environment/ http://www.ncbi.nlm.nih.gov/pubmed/21571039 http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitotheory.html http://www.ncbi.nlm.nih.gov/pubmed/22779765
  38. 38. Questions for Discussion Any questions/comments?

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