UbiB proteins regulate cellular CoQ distribution

AbstractCoenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for many core metabolic processes in mitochondria, including oxidative phosphorylation1-3. While lesser appreciated, CoQ also serves as a key membrane-embedded antioxidant throughout the cell4. 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 yeast genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally regulate 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 (PUFAs), 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 new protein machinery central to CoQ trafficking in yeast and lend new insights into the broader interplay between mitochondrial and cellular processes.

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UbiB proteins regulate cellular CoQ distribution in Saccharomyces cerevisiae

Nature Communications ◽

10.1038/s41467-021-25084-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zachary A. Kemmerer ◽

Kyle P. Robinson ◽

Jonathan M. Schmitz ◽

Mateusz Manicki ◽

Brett R. Paulson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Membrane ◽

Coenzyme Q ◽

Mitochondrial Proteins ◽

Inner Mitochondrial Membrane ◽

Lipid Profiling ◽

Enhanced Resistance ◽

Biochemical Fractionation ◽

Process Loss ◽

Sites Of Action

AbstractBeyond 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.

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Lipocalin 2 as Putative Modulator of Local Inflammatory Processes in the Spinal Cord and Component of Organ Cross-talk After Spinal Cord Injury

10.21203/rs.3.rs-513109/v2 ◽

2021 ◽

Author(s):

Victoria Behrens ◽

Clara Voelz ◽

Nina Müller ◽

Weiyi Zhao ◽

Tim Clarner ◽

...

Keyword(s):

Spinal Cord ◽

Spinal Cord Injury ◽

Blood Serum ◽

Negative Influence ◽

Lipocalin 2 ◽

Cellular Processes ◽

Cord Injury ◽

Inflammatory Processes ◽

Underlying Mechanisms ◽

Sites Of Action

Abstract Lipocalin 2 (Lcn2), an immunomodulator, regulates various cellular processes such as iron transport and defense against bacterial infection. Under pathological conditions, Lcn2 promotes neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes. Although it seems to have a negative influence on the functional outcome in spinal cord injury (SCI), the extent of its involvement in SCI and the underlying mechanisms are not yet fully known. In this study, using a SCI contusion mouse model, we first investigated the expression pattern of Lcn2 in different parts of the CNS (spinal cord and brain), blood serum and in the liver. Interestingly, we could note a significant increase in Lcn2 throughout the whole spinal cord, in the brain, liver and in blood serum. This demonstrates the diversity of its possible sites of action in SCI. Further, genetic deficiency of Lcn2 (Lcn2-/-) significantly reduced certain aspects of gliosis in the SCI-mice. Taken together, our studies provide first valuable hints, suggesting that Lcn2 is involved in the local and systemic effects post SCI, and might modulate the impairment of different peripheral organs after injury.

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What does the commonly used DCF test for oxidative stress really show?

Biochemical Journal ◽

10.1042/bj20100208 ◽

2010 ◽

Vol 428 (2) ◽

pp. 183-190 ◽

Cited By ~ 172

Author(s):

Markus Karlsson ◽

Tino Kurz ◽

Ulf T. Brunk ◽

Sven E. Nilsson ◽

Christina I. Frennesson

Keyword(s):

Oxidative Stress ◽

Cytochrome C ◽

Enzymatic Oxidation ◽

Short Exposure ◽

Outer Side ◽

Inner Mitochondrial Membrane ◽

Fluorescent Compound ◽

Redox Active ◽

J774 Cells ◽

Active Transition

H2DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate ‘cellular oxidative stress’. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H2DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2′,7′-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H2DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H2DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H2O2, directly oxidizes H2DCF. Consequently, oxidation of H2DCF requires the presence of either cytochrome c or of both redox-active transition metals and H2O2. Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H2DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H2O2, showing that by itself it is unable to oxidize H2DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

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Lipocalin 2 as Putative Modulator of Local Inflammatory Processes in the Spinal Cord and Component of Organ Cross-talk After Spinal Cord Injury

10.21203/rs.3.rs-513109/v1 ◽

2021 ◽

Author(s):

Victoria Behrens ◽

Clara Voelz ◽

Nina Müller ◽

Weiyi Zhao ◽

Tim Clarner ◽

...

Keyword(s):

Spinal Cord ◽

Spinal Cord Injury ◽

Blood Serum ◽

Negative Influence ◽

Lipocalin 2 ◽

Cellular Processes ◽

Cord Injury ◽

Inflammatory Processes ◽

Underlying Mechanisms ◽

Sites Of Action

Abstract Lipocalin 2 (Lcn2), an immunomodulator, regulates various cellular processes such as iron transport and defense against bacterial infection. Under pathological conditions, Lcn2 promotes neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes. Although it seems to have a negative influence on the functional outcome in spinal cord injury (SCI), the extent of its involvement in SCI and the underlying mechanisms are not yet fully known. In this study, using a SCI contusion mouse model, we first investigated the expression pattern of Lcn2 in different parts of the CNS (spinal cord and brain), blood serum and in the liver. Interestingly, we could note a significant increase in Lcn2 throughout the whole spinal cord, in the brain, liver and in blood serum. This demonstrates the diversity of its possible sites of action in SCI. Further, genetic deficiency of Lcn2 (Lcn2−/−) significantly reduced certain aspects of gliosis in the SCI-mice. Taken together, our studies provide first valuable hints, suggesting that Lcn2 is involved in the local and systemic effects post SCI, and might modulate the impairment of different peripheral organs after injury.

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Structure and functionality of a multimeric human COQ7:COQ9 complex

10.1101/2021.11.15.468694 ◽

2021 ◽

Author(s):

Mateusz Manicki ◽

Halil Aydin ◽

Luciano A. Abriata ◽

Katherine A. Overmyer ◽

Rachel M. Guerra ◽

...

Keyword(s):

Antioxidant Defense ◽

Binding Capacity ◽

Coenzyme Q ◽

Lipid Binding ◽

Mitochondrial Inner Membrane ◽

Access Channel ◽

Redox Active ◽

Oligomeric Complex ◽

Dynamics Simulations ◽

Functional Analyses

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined 'complex Q' metabolon. Here we present a structure and functional analyses of a substrate- and NADH-bound oligomeric complex comprised of two complex Q subunits: the hydroxylase COQ7, which performs the penultimate step in CoQ biosynthesis, and the prenyl lipid-binding protein COQ9. We reveal that COQ7 adopts a modified ferritin-like fold with an extended hydrophobic access channel whose substrate binding capacity is enhanced by COQ9. Using molecular dynamics simulations, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for CoQ intermediates to translocate from within the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer, closed like a capsid, with lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors and coordinate subsequent synthesis steps toward producing mature CoQ.

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Pseudomonas aeruginosa Pyocyanin Is Critical for Lung Infection in Mice

Infection and Immunity ◽

10.1128/iai.72.7.4275-4278.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 4275-4278 ◽

Cited By ~ 211

Author(s):

Gee W. Lau ◽

Huimin Ran ◽

Fansheng Kong ◽

Daniel J. Hassett ◽

Dimitri Mavrodi

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Respiratory Tract ◽

Tissue Damage ◽

Lung Infection ◽

Lung Epithelial Cells ◽

Cellular Processes ◽

Redox Active ◽

Lung Epithelial

ABSTRACT Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection. PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells. Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.

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A proximity biotinylation map of a human cell

10.1101/796391 ◽

2019 ◽

Cited By ~ 25

Author(s):

Christopher D. Go ◽

James D.R. Knight ◽

Archita Rajasekharan ◽

Bhavisha Rathod ◽

Geoffrey G. Hesketh ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Cell ◽

Large Scale ◽

Mitochondrial Dynamics ◽

Hek293 Cells ◽

Fine Grained ◽

Cellular Processes ◽

Biochemical Fractionation ◽

Cellular Compartments ◽

Better Than

Compartmentalization is an essential characteristic of eukaryotic cells, ensuring that cellular processes are partitioned to defined subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2-6 have helped to define the proteomes of multiple organelles and macromolecular structures. However, many compartments have remained refractory to such methods, partly due to lysis and purification artefacts and poor subcompartment resolution. Recently developed proximity-dependent biotinylation approaches such as BioID and APEX provide an alternative avenue for defining the composition of cellular compartments in living cells (e.g. 7-10). Here we report an extensive BioID-based proximity map of a human cell, comprising 192 markers from 32 different compartments that identifies 35,902 unique high confidence proximity interactions and localizes 4,145 proteins expressed in HEK293 cells. The recall of our localization predictions is on par with or better than previous large-scale mass spectrometry and microscopy approaches, but with higher localization specificity. In addition to assigning compartment and subcompartment localization for many previously unlocalized proteins, our data contain fine-grained localization information that, for example, allowed us to identify proteins with novel roles in mitochondrial dynamics. As a community resource, we have created humancellmap.org, a website that allows exploration of our data in detail, and aids with the analysis of BioID experiments.

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A potential novel pathway to regulate cellular concentrations of the redox-active lipid coenzyme Q

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2018.04.236 ◽

2018 ◽

Vol 120 ◽

pp. S71

Author(s):

Anita Ayer ◽

Ghassan J. Maghzal ◽

Jelske N. van der Veen ◽

Ian W. Dawes ◽

Dennis E. Vance ◽

...

Keyword(s):

Coenzyme Q ◽

Redox Active

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What Regulates the Cellular Content of the Redox-Active Lipid Coenzyme Q?

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2016.10.185 ◽

2016 ◽

Vol 100 ◽

pp. S72

Author(s):

Anita Ayer ◽

Ghassan J Maghzal ◽

Catherine F Clarke ◽

Ian W Dawes ◽

Roland Stocker

Keyword(s):

Coenzyme Q ◽

Cellular Content ◽

Redox Active

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Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/3904905 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Manuel J. Acosta Lopez ◽

Eva Trevisson ◽

Marcella Canton ◽

Luis Vazquez-Fonseca ◽

Valeria Morbidoni ◽

...

Keyword(s):

Vanillic Acid ◽

Coenzyme Q ◽

Mitochondrial Respiratory Chain ◽

Human Cell Line ◽

Food Additive ◽

Large Set ◽

Promising Alternative ◽

Atp Production ◽

Redox Active ◽

Genes Encoding

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

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