Quantum chemistry rules retinoid biology.

This Perspective discusses how retinol catalyzes resonance energy transfer (RET) reactions pivotally important for mitochondrial energy homeostasis by protein kinase C δ (PKCδ). PKCδ signals to the pyruvate dehydrogenase complex, controlling oxidative phosphorylation. The PKCδ-retinol complex reversibly responds to the redox potential of cytochrome c, that changes with the electron transfer chain workload. In contrast, the natural retinoid anhydroretinol irreversibly activates PKCδ. Its elongated conjugated-double-bond system limits the energy quantum absorbed by RET. Consequently, while capable of triggering the exergonic activating pathway, anhydroretinol fails to activate the endergonic silencing path, trapping PKCδ in the ON position and causing harmful levels of reactive oxygen species. However, physiological retinol levels displace anhydroretinol, buffer cyotoxicity and potentially render anhydroretinol useful for rapid energy generation. Intriguingly, apocarotenoids, the primary products of the mitochondrial ß-carotene,9'-10'-oxygenase, have all the anhydroretinol-like features, including modulation of energy homeostasis. We predict significant conceptual advances to stem from further understanding of the retinoid-catalyzed RET.

Retinoic acid regulates pyruvate dehydrogenase kinase 4 (Pdk4) to modulate fuel utilization in the adult heart: Insights from wild-type and ß-carotene 9',10' oxygenase knockout mice.

Regulation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase PDK4 enables the heart to respond to fluctuations in energy demands and substrate availability. Retinoic acid, the transcriptionally active form of vitamin A, is known to be involved in the regulation of cardiac function and growth during embryogenesis as well as under pathological conditions. Whether retinoic acid also maintains cardiac health under physiological conditions is unknown. However, vitamin A status and intake of its carotenoid precursor ß-carotene have been linked to the prevention of heart diseases. Here, we provide in vitro and in vivo evidence that retinoic acid regulates cardiac Pdk4 expression and thus PDH activity. Furthermore, we show that mice lacking ß-carotene 9',10'-oxygenase (BCO2), the only enzyme of the adult heart that cleaves ß-carotene to generate retinoids (vitamin A and its derivatives), displayed cardiac retinoic acid insufficiency and impaired metabolic flexibility linked to a compromised PDK4/PDH pathway. These findings provide novel insights into the functions of retinoic acid in regulating energy metabolism in adult tissues, especially the heart.

ß-carotene improves fecal dysbiosis and intestinal dysfunctions in a mouse model of vitamin A deficiency.

Vitamin A deficiency (VAD) results in intestinal inflammation, increased redox stress and reactive oxygen species (ROS) levels, imbalanced inflammatory and immunomodulatory cytokines, compromised barrier function, and perturbations of the gut microbiome. To combat VAD dietary interventions with ß-carotene, the most abundant precursor of vitamin A, are recommended. However, the impact of ß-carotene on intestinal health during VAD has not been fully clarified, especially regarding the VAD-associated intestinal dysbiosis. Here we addressed this question by using Lrat-/-Rbp-/- (vitamin A deficient) mice deprived of dietary preformed vitamin A and supplemented with ß-carotene as the sole source of the vitamin, alongside with WT (vitamin A sufficient) mice. We found that dietary ß-carotene impacted intestinal vitamin A status, barrier integrity and inflammation in both WT and Lrat-/-Rbp-/- (vitamin A deficient) mice on the vitamin A-free diet. However, it did so to a greater extent under overt VAD. Dietary ß-carotene also modified the taxonomic profile of the fecal microbiome, but only under VAD. Given the similarity of the VAD-associated intestinal phenotypes with those of several other disorders of the gut, collectively known as Inflammatory Bowel Disease (IBD) Syndrome, these findings are broadly relevant to the effort of developing diet-based intervention strategies to ameliorate intestinal pathological conditions.

The mitochondrial PKCδ/retinol signal complex exerts real-time control on energy homeostasis.

The review focuses on the role of vitamin A (retinol) in the control of energy homeostasis, and on the manner in which certain retinoids subvert this process, leading potentially to disease. In eukaryotic cells, the pyruvate dehydrogenase complex (PDHC) is negatively regulated by four pyruvate dehydrogenase kinases (PDKs) and two antagonistically acting pyruvate dehydrogenase phosphatases (PDPs). The second isoform, PDK2, is regulated by an autonomous mitochondrial signal cascade that is anchored on protein kinase Cδ (PKCδ), where retinoids play an indispensible co-factor role. Along with its companion proteins p66Shc, cytochrome c, and vitamin A, the PKCδ/retinol complex is located in the intermembrane space of mitochondria. At this site, and in contrast to cytosolic locations, PKCδ is activated by the site-specific oxidation of its cysteine-rich activation domain (CRD) that is configured into a complex RING-finger. Oxidation involves the transfer of electrons from cysteine moieties to oxidized cytochrome c, a step catalyzed by vitamin A. The PKCδ/retinol signalosome monitors the internal cytochrome c redox state that reflects the workload of the respiratory chain. Upon sensing demands for energy PKCδ signals the PDHC to increase glucose-derived fuel flux entering the KREBS cycle. Conversely, if excessive fuel flux surpasses the capacity of the respiratory chain, threatening the release of damaging reactive oxygen species (ROS), the polarity of the cytochrome c redox system is reversed, resulting in the chemical reduction of the PKCδ CRD, restoration of the RING-finger, refolding of PKCδ into the inactive, globular form, and curtailment of PDHC output, thereby constraining the respiratory capacity within safe margins. Several retinoids, notably anhydroretinol and fenretinide, capable of displacing retinol from binding sites on PKCδ, can co-activate PKCδ signaling but, owing to their extended system of conjugated double bonds, are unable to silence PKCδ in a timely manner. Left in the ON position, PKCδ causes chronic overload of the respiratory chain leading to mitochondrial dysfunction. This review explores how defects in the PKCδ signal machinery potentially contribute to metabolic and degenerative diseases.

ß-apo-10'-carotenoids support normal embryonic development during vitamin A deficiency.

Vitamin A deficiency is still a public health concern affecting millions of pregnant women and children. Retinoic acid, the active form of vitamin A, is critical for proper mammalian embryonic development. Embryos can generate retinoic acid from maternal circulating ß-carotene upon oxidation of retinaldehyde produced via the symmetric cleavage enzyme ß-carotene 15,15'-oxygenase (BCO1). Another cleavage enzyme, ß-carotene 9',10'-oxygenase (BCO2), asymmetrically cleaves ß-carotene in adult tissues to prevent its mitochondrial toxicity, generating ß-apo-10'-carotenal, which can be converted to retinoids (vitamin A and its metabolites) by BCO1. However, the role of BCO2 during mammalian embryogenesis is unknown. We found that mice lacking BCO2 on a vitamin A deficiency-susceptible genetic background (Rbp4-/-) generated severely malformed vitamin A-deficient embryos. Maternal ß-carotene supplementation impaired fertility and did not restore normal embryonic development in the Bco2-/-Rbp4-/- mice, despite the expression of BCO1. These data demonstrate that BCO2 prevents ß-carotene toxicity during embryogenesis under severe vitamin A deficiency. In contrast, ß-apo-10'-carotenal dose-dependently restored normal embryonic development in Bco2-/-Rbp4-/- but not Bco1-/-Bco2-/-Rbp4-/- mice, suggesting that ß-apo-10'-carotenal facilitates embryogenesis as a substrate for BCO1-catalyzed retinoid formation. These findings provide a proof of principle for the important role of BCO2 in embryonic development and invite consideration of ß-apo-10'-carotenal as a nutritional supplement to sustain normal embryonic development in vitamin A-deprived pregnant women.

Vitamin A as PKC Co-factor and Regulator of Mitochondrial Energetics.

For the past century, vitamin A has been considered to serve as a precursor for retinoids that facilitate vision or as a precursor for retinoic acid (RA), a signaling molecule that modulates gene expression. However, vitamin A circulates in plasma at levels that far exceed the amount needed for vision or the synthesis of nanomolar levels of RA, and this suggests that vitamin A alcohol (i.e. retinol) may possess additional biological activity. We have pursued this question for the last 20 years, and in this chapter, we unfold the story of our quest and the data that support a novel and distinct role for vitamin A (alcohol) action. Our current model supports direct binding of vitamin A to the activation domains of serine/threonine kinases, such as protein kinase C (PKC) and Raf isoforms, where it is involved in redox activation of these proteins. Redox activation of PKCs was first described by the founders of the PKC field, but several hurdles needed to be overcome before a detailed understanding of the biochemistry could be provided. Two discoveries moved the field forward. First, was the discovery that the PKCδ isoform was activated by cytochrome c, a protein with oxidoreduction activity in mitochondria. Second, was the revelation that both PKCδ and cytochrome c are tethered to p66Shc, an adapter protein that brings the PKC zinc-finger substrate into close proximity with its oxidizing partner. Detailed characterization of the PKCδ signalosome complex was made possible by the work of many investigators. Our contribution was determining that vitamin A is a vital co-factor required to support an unprecedented redox-activation mechanism. This unique function of vitamin A is the first example of a general system that connects the one-electron redox chemistry of a heme protein (cytochrome c) with the two-electron chemistry of a classical phosphoprotein (PKCδ). Furthermore, contributions to the regulation of mitochondrial energetics attest to biological significance of vitamin A alcohol action.

Retinol as electron carrier in redox signaling, a new frontier in vitamin A research.

Nature uses carotenoids and retinoids as chromophores for diverse energy conversion processes. The key structural feature enabling the interaction with light and other manifestations of electro-magnetism is the conjugated double-bond system that all members of this superfamily share in common. Among retinoids, retinaldehyde alone was long known as the active chromophore of vision in vertebrates and invertebrates, as well of various light-driven proton and ion pumps in Archaea. Until now, vitamin A (retinol) was solely regarded as a biochemical precursor for bioactive retinoids such as retinaldehyde and retinoic acid (RA), but recent results indicate that this compound has its own physiology. It functions as an electron carrier in mitochondria. By electronically coupling protein kinase Cδ (PCKδ) with cytochrome c, vitamin A enables the redox activation of this enzyme. This review focuses on the biochemistry and biology of the PCKδ signaling system, comprising PKCδ, the adapter protein p66Shc, cytochrome c and retinol. This complex positively regulates the conversion of pyruvate to acetyl-coenzyme A (CoA) by the pyruvate dehydrogenase enzyme. Vitamin A therefore plays a key role in glycolytic energy generation. The emerging paradigm of retinol as electron-transfer agent is potentially transformative, opening new frontiers in retinoid research.

Retinol as a cofactor for PKCδ-mediated impairment of insulin sensitivity in a mouse model of diet-induced obesity.

We previously defined that the mitochondria-localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl-coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet-induced insulin resistance. We crossed mice overexpressing human retinol-binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ(-/-) strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ(+/+)hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ(-/-)hRBP and PKCδ(-/-) mice. Therefore, in addition to the well-defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole-body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.

Two protein kinase C isoforms, δ and ε, regulate energy homeostasis in mitochondria by transmitting opposing signals to the pyruvate dehydrogenase complex.

Energy production in mitochondria is a multistep process that requires coordination of several subsystems. While reversible phosphorylation is emerging as the principal tool, it is still unclear how this signal network senses the workloads of processes as different as fuel procurement, catabolism in the Krebs cycle, and stepwise oxidation of reducing equivalents in the electron transfer chain. We previously proposed that mitochondria use oxidized cytochrome c in concert with retinol to activate protein kinase Cδ, thereby linking a prominent kinase network to the redox balance of the ETC. Here, we show that activation of PKCε in mitochondria also requires retinol as a cofactor, implying a redox-mechanism. Whereas activated PKCδ transmits a stimulatory signal to the pyruvate dehdyrogenase complex (PDHC), PKCε opposes this signal and inhibits the PDHC. Our results suggest that the balance between PKCδ and ε is of paramount importance not only for flux of fuel entering the Krebs cycle but for overall energy homeostasis. We observed that the synthetic retinoid fenretinide substituted for the retinol cofactor function but, on chronic use, distorted this signal balance, leading to predominance of PKCε over PKCδ. The suppression of the PDHC might explain the proapoptotic effect of fenretinide on tumor cells, as well as the diminished adiposity observed in experimental animals and humans. Furthermore, a disturbed balance between PKCδ and PKCε might underlie the injury inflicted on the ischemic myocardium during reperfusion. dehydrogenase complex.

Hiding in plain sight: uncovering a new function of vitamin A in redox signaling.

The protein kinase Cδ signalosome modulates the generation of acetyl-Coenzyme A from glycolytic sources. This module is composed of four interlinked components: PKCδ, the signal adapter p66Shc, cytochrome c, and vitamin A. It resides in the intermembrane space of mitochondria, and is at the center of a feedback loop that senses upstream the redox balance between oxidized and reduced cytochrome c as a measure of the workload of the respiratory chain, and transmits a forward signal to the pyruvate dehydrogenase complex to adjust the flux of fuel entering the tricarboxylic acid cycle. The novel role of vitamin A as co-activator and potential electron carrier, required for redox activation of PKCδ, is discussed. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

Are zinc-finger domains of protein kinase C dynamic structures that unfold by lipid or redox activation?

Protein kinase C (PKC) is activated by lipid second messengers or redox action, raising the question whether these activation modes involve the same or alternate mechanisms. Here we show that both lipid activators and oxidation target the zinc-finger domains of PKC, suggesting a unifying activation mechanism. We found that lipid agonist-binding or redox action leads to zinc release and disassembly of zinc fingers, thus triggering large-scale unfolding that underlies conversion to the active enzyme. These results suggest that PKC zinc fingers, originally considered purely structural devices, are in fact redox-sensitive flexible hinges, whose conformation is controlled both by redox conditions and lipid agonists.

Regulation of intermediary metabolism by the PKCdelta signalosome in mitochondria.

PKCδ has emerged as a novel regulatory molecule of oxidative phosphorylation by targeting the pyruvate dehydrogenase complex (PDHC). We showed that activation of PKCδ leads to the dephosphorylation of pyruvate dehydrogenase kinase 2 (PDK2), thereby decreasing PDK2 activity and increasing PDH activity, accelerating oxygen consumption, and augmenting ATP synthesis. However, the molecular components that mediate PKCδ signaling in mitochondria have remained elusive so far. Here, we identify for the first time a functional complex, which includes cytochrome c as the upstream driver of PKCδ, and uses the adapter protein p66Shc as a platform with vitamin A (retinol) as a fourth partner. All four components are necessary for the activation of the PKCδ signal chain. Genetic ablation of any one of the three proteins, or retinol depletion, silences signaling. Furthermore, mutations that disrupt the interaction of cytochrome c with p66Shc, of p66Shc with PKCδ, or the deletion of the retinol-binding pocket on PKCδ, attenuate signaling. In cytochrome c-deficient cells, reintroduction of cytochrome c Fe(3+) protein restores PKCδ signaling. Taken together, these results indicate that oxidation of PKCδ is key to the activation of the pathway. The PKCδ/p66Shc/cytochrome c signalosome might have evolved to effect site-directed oxidation of zinc-finger structures of PKCδ, which harbor the activation centers and the vitamin A binding sites. Our findings define the molecular mechanisms underlying the signaling function of PKCδ in mitochondria.

Control of oxidative phosphorylation by vitamin A illuminates a fundamental role in mitochondrial energy homoeostasis.

The physiology of two metabolites of vitamin A is understood in substantial detail: retinaldehyde functions as the universal chromophore in the vertebrate and invertebrate eye; retinoic acid regulates a set of vertebrate transcription factors, the retinoic acid receptor superfamily. The third member of this retinoid triumvirate is retinol. While functioning as the precursor of retinaldehyde and retinoic acid, a growing body of evidence suggests a far more fundamental role for retinol in signal transduction. Here we show that retinol is essential for the metabolic fitness of mitochondria. When cells were deprived of retinol, respiration and ATP synthesis defaulted to basal levels. They recovered to significantly higher energy output as soon as retinol was restored to physiological concentration, without the need for metabolic conversion to other retinoids. Retinol emerged as an essential cofactor of protein kinase Cdelta (PKCdelta), without which this enzyme failed to be activated in mitochondria. Furthermore, retinol needed to physically bind PKCdelta, because mutation of the retinol binding site rendered PKCdelta unresponsive to Rol, while retaining responsiveness to phorbol ester. The PKCdelta/retinol complex signaled the pyruvate dehydrogenase complex for enhanced flux of pyruvate into the Krebs cycle. The baseline response was reduced in vitamin A-deficient lecithin:retinol acyl transferase-knockout mice, but this was corrected within 3 h by intraperitoneal injection of vitamin A; this suggests that vitamin A is physiologically important. These results illuminate a hitherto unsuspected role of vitamin A in mitochondrial bioenergetics of mammals, acting as a nutritional sensor. As such, retinol is of fundamental importance for energy homeostasis. The data provide a mechanistic explanation to the nearly 100-yr-old question of why vitamin A deficiency causes so many pathologies that are independent of retinoic acid action.

Vitamin A depletion causes oxidative stress, mitochondrial dysfunction, and PARP-1-dependent energy deprivation.

A significant unresolved question is how vitamin A deprivation causes, and why retinoic acid fails to reverse, immunodeficiency. When depleted of vitamin A, T cells undergo programmed cell death (PCD), which is enhanced by the natural competitor of retinol, anhydroretinol. PCD does not happen by apoptosis, despite the occurrence of shared early events, including mitochondrial membrane depolarization, permeability transition pore opening, and cytochrome c release. It also lacks caspase-3 activation, chromatin condensation, and endonuclease-mediated DNA degradation, hallmarks of apoptosis. PCD following vitamin A deprivation exhibits increased production of reactive oxygen species (ROS), drastic reductions in ATP and NAD(+) levels, and activation of poly-(ADP-ribose) polymerase (PARP) -1. These latter steps are causative because neutralizing ROS, imposing hypoxic conditions, or inhibiting PARP-1 by genetic or pharmacologic approaches prevents energy depletion and PCD. The data highlight a novel regulatory role of vitamin A in mitochondrial energy homeostasis.

Retinoylserine and retinoylalanine, natural products of the moth Trichoplusia ni.

Insect cells convert vitamin A into a number of retinoids that are evolutionarily conserved with those of mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic acid peptides, N-trans-retinoylserine (1) and N-trans-retinoylalanine (2), have been isolated from a cell line of the common cabbage looper, Trichoplusia ni. These are the first examples of naturally occurring retinoic acid linked to amino acids through an amide bond; the amino acid moieties are depicted in the more common l-configuration, although the absolute configuration was not determined due to the minuscule sample amount.

Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing.

Multicellular organisms initiate adaptive responses when oxygen (O(2)) availability decreases, but the underlying mechanism of O(2) sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1 alpha and HIF-2 alpha and that an increase in reactive oxygen species (ROS) links this complex to HIF-alpha stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1 alpha stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O(2) sensors and signal hypoxic HIF-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.

Location and functional significance of retinol-binding sites on the serine/threonine kinase, c-Raf.

Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-alpha C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the alphaC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of alphaC1B, Thr-7 and Tyr-22, to conform to alphaC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.

Regulation of the cardiac mitochondrial membrane potential by retinoids.

Cardiomyocytes suffering irreversible damage under oxidative stress during ischemia activate their suicide program. Mitochondria play a key role in this process, while they themselves are subject to regulation by a number of signaling pathways. We demonstrate here that retinoids influence mitochondrial function in cardiomyocytes. Depending on their chemical nature, retinoids can either ameliorate or exacerbate stress-related damage. Thus, vitamin A, retinol, was protective because retinol deprivation enhanced oxidative damage, as indicated by rapid loss of mitochondrial membrane potential. Supplementation with a physiological concentration of retinol reversed this effect. Anhydroretinol (AR), a known antagonist, which works by displacing retinol from the common binding sites on serine/threonine kinases, also caused mitochondrial membrane depolarization. The AR effect was both Ca(2+)-dependent and cyclosporin-sensitive, suggesting an upstream signaling mechanism rather than direct membrane effect. Our results agree with a model where retinol supports mitochondrial integrity by enabling upstream signaling processes. The consequences of disrupting these processes by AR are opening of the permeability transition pore, release of cytochrome c, and activation of the suicide program.

Zinc release from protein kinase C as the common event during activation by lipid second messenger or reactive oxygen.

Zinc is a structural component of many regulatory molecules including transcription factors and signaling molecules. We report that two alternate signaling pathways of protein kinase C (PKC) activation involving either the lipid second messengers (diacylglycerol and its mimetics, the phorbol esters) or reactive oxygen converge at the zinc finger of the regulatory domain. They all trigger the release of zinc ions. An increase in intracellular free Zn(2+) was observed by confocal microscopy in intact cells treated with phorbol ester or by mild oxidation. The source of liberated Zn(2+) was traced to PKC and particularly the zinc finger domains. The activated form of native PKCalpha contained significantly less Zn(2+) than the resting form. Furthermore, purified recombinant PKC protein fragments shed stoichiometric amounts of Zn(2+) upon reaction with diacylglycerol, phorbol ester, or reactive oxygen in vitro. Our results offer new insight into the regulation of PKC. Far from cementing rigid structures, zinc actually is the linchpin that orchestrates dynamic changes in response to specific signals, allowing kinase activity to be turned on or off.