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Coenzyme Q (CoQ) is an essential player in the respiratory electron transport chain and is the only lipid-soluble antioxidant synthesized endogenously in mammalian and yeast cells. In humans, genetic mutations, pathologies, certain medical treatments, and aging, result in CoQ deficiencies, which are linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The only strategy available for these patients is CoQ supplementation. CoQ supplements benefit a small subset of patients, but the poor solubility of CoQ greatly limits treatment efficacy. Consequently, the efficient delivery of CoQ to the mitochondria and restoration of respiratory function remains a major challenge. A better understanding of CoQ uptake and mitochondrial delivery is crucial to make this molecule a more efficient and effective therapeutic tool. In this study, we investigated the mechanism of CoQ uptake and distribution using the yeast Saccharomyces cerevisiae as a model organism. The addition of exogenous CoQ was tested for the ability to restore growth on non-fermentable medium in several strains that lack CoQ synthesis (coq mutants). Surprisingly, we discovered that the presence of CoQ biosynthetic intermediates impairs assimilation of CoQ into a functional respiratory chain in yeast cells. Moreover, a screen of 40 gene deletions considered to be candidates to prevent exogenous CoQ from rescuing growth of the CoQ-less coq2Δ mutant, identified six novel genes (CDC10 , RTS1 , RVS161 , RVS167, VPS1 , and NAT3) as necessary for efficient trafficking of CoQ to mitochondria. The proteins encoded by these genes represent essential steps in the pathways responsible for transport of exogenously supplied CoQ to its functional sites in the cell, and definitively associate CoQ distribution with endocytosis and intracellular vesicular trafficking pathways conserved from yeast to human cells. Image 1 • CoQ biosynthetic intermediates impair assimilation of exogenously supplied CoQ. • C DC10 , RTS1 , RVS161 , RVS167 , NAT3, and VPS1 genes are necessary for CoQ trafficking. • These proteins mediate transport of exogenous CoQ to functional sites in the cell. • CoQ transport is associated with endocytosis and intracellular vesicular trafficking. [ABSTRACT FROM AUTHOR]

Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4′ of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is a redox-active lipid essential for mitochondrial energy generation, cellular redox regulation, antioxidant defense, apoptosis and cell death. Despite this, it is largely unknown what regulates the cellular CoQ content, how optimal CoQ content in different cellular membranes is regulated, and how this information is ‘fed back’ to the CoQ synthesis machinery. Understanding CoQ homeostasis is critical as CoQ deficiency is implicated in numerous diseases such as heart failure, and aging is associated with a decrease in tissue CoQ. Currently, CoQ supplements are essentially the only strategy available to overcome CoQ deficiencies. This strategy can be effective in cases of severe CoQ deficiency but the low bioavailability of supplemental CoQ for most tissues represents a significant limitation. Thus, a better understanding of key steps regulating CoQ content may provide the basis for novel strategies to enrich cellular CoQ content. To investigate CoQ regulation, we combined genetic and biochemical methods using the model organism S. cerevisiae. Carrying out a genome-wide screen (~6,000 mutants) to discover genes critical for CoQ content, over 100 genes were identified. Furthermore, the global transcriptional responses to changes in cellular CoQ content were determined by microarray analyses of WT and select CoQ biosynthesis mutants (coq1, 3, 7, 8 and 9). From the microarrays, coq1, 3 and 7 had similar transcriptional profiles, while coq8 and 9 clustered together indicating distinct transcriptional responses depending on how CoQ biosynthesis is disrupted. Interestingly, CoQ biosynthetic genes were not differentially expressed between the WT and coq mutants indicating that CoQ deficiency is not transcriptionally signaled. Similarly, antioxidant defense genes were not differentially expressed suggesting that in yeast CoQ is not required for steady-state maintenance of redox homeostasis. These and other results from the genome-wide screen will be presented, with the data providing novel insights into cellular CoQ regulation and function. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t -butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D 1 -Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D 2 -Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function. [ABSTRACT FROM AUTHOR]

Abstract: Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D2-Lin is 12.8±0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast. [Copyright &y& Elsevier]

Abstract: The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D2-linoleic or 11,11,14,14-D4-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is an essential redox-active lipid, and its deficiency is implicated in numerous diseases. However, it is largely unknown what regulates CoQ content. To investigate cellular CoQ regulation, we performed a genome-wide screen (~5,000 S. cerevisiae single gene mutants) to isolate genes critical for CoQ homeostasis, and measured CoQ content in each mutant using HPLC-EC detection. Some 30 knockout mutants were identified with significantly higher CoQ content compared to WT including the cho2 mutant. CHO2 encodes a phosphatidylethanolamine (PE) methyltransferase that catalyzes the conversion of PE to phosphatidylcholine (PC). Homologs of CHO2 are found in mammals (PEMT) indicating there may be role for this gene in CoQ regulation in other species. The cho2 mutant contained concentrations of CoQ five times greater than WT, and a significantly increased rate of CoQ synthesis. To investigate if Pemt–/– mice also display elevated CoQ content akin to yeast cho2 mutants, we analyzed the CoQ content in liver and adipose tissue from Pemt+/+ and Pemt–/– mice. In these tissues, CoQ content was approximately double in Pemt–/– compared to Pemt+/+. Taken together, our data suggests a novel role for phosphatidylethanolamine methyltransferases in CoQ content regulation. [ABSTRACT FROM AUTHOR]