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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Questions for Discussion
Any questions/comments?