Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

UbiB proteins regulate cellular CoQ distribution in Saccharomyces cerevisiae.

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8-an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

Dr. Jekyll and Mr. Hyde: Oxidizable phenol-generated reactive oxygen species enhance sulforaphane's antioxidant response element activation, even as they suppress Nrf2 protein accumulation.

The transcription factor Nrf2 is a master regulator of antioxidant and cytoprotective genes, binding to antioxidant response elements (AREs) in their promoter regions. Due to the therapeutic role of the Nrf2/ARE system in oxidative homeostasis, its activation has been investigated in many pre-clinical and clinical trials for common chronic diseases. One of the most promising Nrf2 activators is sulforaphane, the subject of over 50 clinical trials. In this work, we examine the effect of reactive oxygen species (ROS) on sulforaphane's Nrf2/ARE activation in the non-tumorigenic keratinocyte cell line HaCaT, with the non-arylating oxidizable phenol, 2,5-di-tert-butylhydroquinone (dtBHQ), as the source of ROS. We find that, in combination with 2.5 µM sulforaphane, dtBHQ markedly enhances ARE-regulated gene expression, including expression of the cytoprotective proteins aldo-keto reductase family 1 member C1 (AKR1C1) and heme oxygenase-1 (HO-1). Additionally, sulforaphane's therapeutic window is widened by 12.5 µM dtBHQ. Our data suggest that H2O2 generated by dtBHQ oxidation is responsible for these effects, as shown by inclusion of catalase and by co-treatment with sulforaphane and H2O2. While sulforaphane treatment causes Nrf2 protein to accumulate as expected, interestingly, dtBHQ and H2O2 appear to act on targets downstream of Nrf2 protein accumulation to enhance sulforaphane's ARE-regulated gene expression. Inclusion of dtBHQ or H2O2 with sulforaphane does not increase Nrf2 protein levels, and catalase has little effect on Nrf2 protein levels in the presence of sulforaphane and dtBHQ. Surprisingly, dtBHQ suppresses Nrf2 protein synthesis. Inclusion of a superoxide dismutase mimetic with sulforaphane and dtBHQ partly rescues Nrf2 suppression and significantly further increases sulforaphane's efficacy for ARE-reporter expression. Thus, there is a "Dr. Jekyll and Mr. Hyde" effect of ROS: ROS enhance sulforaphane's ARE-regulated gene expression even as they also inhibit Nrf2 protein synthesis. This unexpected finding reveals the degree to which targets in the ARE pathway downstream of Nrf2 protein accumulation contribute to gene expression. The results presented here provide a model system for significant enhancement of sulforaphane's potency with small molecule co-treatment.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling.

Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes, and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide molecular insights into mitochondrial protein functions.

Comparison of human Nrf2 antibodies: A tale of two proteins.

The Nrf2 transcription factor is a master regulator of the cellular defense against oxidative and electrophilic stress. An increase in Nrf2 protein levels and an accumulation of Nrf2 in the nucleus are key parts of the Nrf2 activation mechanism. The western blot technique remains the most widely used method to assess these changes. A well-characterized, specific antibody that is commercially available would greatly enhance these studies in the field. Here, an apparently highly specific Nrf2 monoclonal antibody, EP1808Y from Abcam, is compared with the most widely used Nrf2 antibodies, H-300 and C-20, both from Santa Cruz Biotechnology, in a panel of human cell lines. In addition to detecting Nrf2, EP1808Y avidly detects another protein present in two of the three cell lines tested. This protein can be mistaken for Nrf2 as it co-migrates with verified Nrf2 on two different polyacrylamide gel types. However, unlike Nrf2, its levels and cytoplasmic localization are unaffected by treatment with Nrf2 activators. The possibility that this band corresponds to a form of Nrf2 was excluded by siRNA and immunodepletion experiments. Finally, the monoclonal antibody D1Z9C from Cell Signaling was found to detect Nrf2 with the highest specificity of these four antibodies.

SIRT3 mediates multi-tissue coupling for metabolic fuel switching.

SIRT3 is a member of the Sirtuin family of NAD(+)-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations; however, the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild-type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis.