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-WilsdonCarrie Bi11/9/15NST 190

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)

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

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)

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.

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.

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

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

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

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

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

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

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 13C6-labelled hydroxyl-4-benzoic acid precursor metabolized to CoQ10-13Q6 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

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

Results - Experiment 2 - 13C6 Labeling

Figures 2B-F2A: molecular weights of CoQ9/10, with/without 13C6 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

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

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

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

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-13C6 and comparison of Golgi fractions with

mitochondrial fractions in CoQ10-13Q6 production

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: 13C6 CoQ10 production in fractions

Figure 4A -F Figure S4

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

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: 13C6 labelled CoQ10, total CoQ10, and Cholesterol concentrations, respectively, post injection

Figure 5A-E

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Bibliography

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https://en.wikipedia.org/wiki/UBIAD1

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Questions for Discussion

Any questions/comments?