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Mitochondrial fatty acid synthesis coordinates mitochondrial oxidative metabolism

Sara M. Nowinski, Ashley Solmonson, Scott F. Rusin, J. Alan Maschek, Claire L. Bensard, Sarah Fogarty, Mi-Young Jeong, Sandra Lettlova, Jordan A. Berg, Jeffrey T. Morgan, Yeyun Ouyang, Bradley C. Naylor, Joao A. Paulo, Katsuhiko Funai, James E. Cox, Steven P. Gygi, Dennis R. Winge, Ralph J. Deberardinis, Jared Rutter

Preprint posted on 10 May 2020 https://www.biorxiv.org/content/10.1101/2020.05.09.086199v1

Big fat electron transport chain – an unexpected role of mitochondrial fatty acid synthesis in regulating oxidative phosphorylation and muscle differentiation

Selected by Berrak Ugur, Aakriti Jain

Categories: biochemistry, cell biology

Background

Fatty acids are important for a plethora of cellular functions including producing energy, building cell membranes and signaling. Mammalian cells are known to use cytoplasmic fatty acid synthase (FASN) to generate different fatty acids (Smith, 1994). In addition, mitochondria have a specific fatty acid synthesis pathway (mtFAS) that is composed of several distinct enzymes, defects in which are known to cause detrimental phenotypes (Nowinski et al., 2018). For example, tissue specific loss of malonyl CoA-acyl carrier protein transacylase (Mcat) and mitochondrial 2-enoyl thioester reductase (Mecr) have been shown to cause metabolic defects (Smith et al., 2012) and neurodegeneration (Nair et al., 2018) respectively. However, a clear mechanism through which defects in mtFAS affects cellular function remain to be elucidated. The mtFAS pathway produces octanoate, which is converted to lipoic acid and long acyl chains of greater than 14 carbons (Fig1, Nowinski et al., 2018). Lipoic acid regulates the tricarboxylic acid (TCA) cycle activity through its function as a cofactor for multiple dehydrogenases (Solmonson and DeBerardinis, 2018); however, the role of the long acyl chains is unclear.Figure 1: Schematic overview of the role of mtFAS in mitochondrial metabolism. See Nowinski et al., 2018 for more info. DOI: 10.1016/j.cub.2018.08.022

Key Findings:

In this preprint, Nowinski et al. set out to investigate the role of the mtFAS pathway by generating mutants of three key enzymes integral to the pathway – Mcat, Oxsm, and Mecr. Loss of these enzymes was lethal, indicating that mtFAS is an essential pathway. Through glucose labelling over time, the authors showed that mtFAS did not contribute to cellular fatty acid or phospholipid synthesis, which was primarily contributed by cytosolic FASN. These results suggested a possible alternate function for mtFAS. Therefore, the authors used partial loss of function mutations of the three mtFAS enzymes and observed that these mutants had defects in cellular respiration, increased reductive carboxylation of α-ketoglurate (αKG), and overall decreased TCA cycle processivity. These defects were associated with disassembly of components of electron transport chain (ETC) complexes.

The authors conducted unbiased proteomics to probe why mtFAS mutants had issues with ETC assembly, which revealed that a subset of 8 OXPHOS proteins showed a decrease in mtFAS mutants compared to wild-type cells. Two of these proteins (NDUFA6 and SDHB) were shown to have a relationship with a class of proteins known as LYRM (leucine-tyrosine-arginine motif) proteins. This was an exciting observation because LYRM proteins have been shown to have physical interactions with the acyl carrier protein (ACP) in yeast (Angerer et al., 2014). Through co-immunoprecipitation assays, the authors showed that ACP indeed interacts with the LYRM proteins associated with complex I and II of the ETC. Concomitantly, glutamine labelling over time suggested a blockage at the succinate dehydrogenase step (likely due to loss of proper complex II assembly).

Lipoic acid is a known regulator of cellular respiration; therefore, to disentangle the lipoic acid-related phenotypes with those associated with acyl modifications through ACP in mtFAS mutant cells, the authors mutated LiptL, which functions downstream of mtFAS and regulates production of lipoylated enzymes. Interestingly, they observed that although LiptL loss impaired cellular respiration and lipoylation, it did not impair ETC complex assembly. This observation indicated that the ETC impairment observed with mtFAS mutation is separate from that due to lipoylation defects.

This study was done in skeletal myoblasts and interestingly, the proteomics experiment showed that mtFAS mutant cells also had decreased abundance of differentiation markers. Indeed, mtFAS mutant muscle precursors failed to differentiate into muscle cells. These precursor cells are activated by the transcription factor Myogenin (Myog), which controls multiple muscle differentiation genes. The authors found out that the expression of Myog along with other muscle specific proteins were highly decreased or absent in mtFAS mutant cells. The authors hypothesize that the mechanism behind this observation may have to do with the fact that mtFAS cells have a high αKG to succinate ratio, which influences the differentiation cascade because αKG regulates a number of histone demethylases. Overall, these findings support the idea that altered metabolite levels, especially αKG, regulates epigenetic remodeling, which may further impede myogenic differentiation. However, more work will be required to support these claims.

Take home messages:

• Mitochondrial fatty acid synthesis (mtFAS) is an essential pathway that does not contribute to the synthesis of cellular lipid pools
• Mutation of mtFAS results in loss of electron transport chain (ETC) complex assembly, which is associated with respiratory and TCA cycle defects
• mtFAS supports ETC assembly via the post-translational stabilization of LYRM proteins through the acyl carrier protein, and not due to protein lipoylation
• Impairment of mtFAS impedes skeletal myoblast differentiation

What we liked about this story:

Aakriti: I enjoyed reading about the unexpected way that an integral metabolic pathway influences cellular functions. The idea that post-translational regulatory mechanisms through metabolites can confer formation of protein complex structures which has downstream effects on other metabolic pathways is really exciting and leaves a lot of unknown biochemistry to be unraveled.

Berrak: The mysteries of how metabolism is integrated into cellular physiology is a topic that has been a special interest of mine. This story points out to yet another function of a mitochondrial pathway that seems to act differently compared to its cytoplasmic counterpart. I am looking forward to seeing what new biology will come out of mitochondrial FASN pathway.

Remaining Questions

1) Is there any evidence suggesting that mtFAS mutations affect mitochondrial morphology, especially in respect to cristae formation? As ETC assembly is affected, one could foresee a severe morphological defect.

2) Although mtFAS mutant cells have a decrease in some of the TCA cycle intermediates (citrate, fumarate, and malate), αKG levels are highly increased. Do you think that there is a specific regulation on the consumers (oxoglutarate dehydrogenase) or the producers (isocitrate dehydrogenase or glutamate dehydorogenase) by mtFAS to regulate αKG levels specifically?

3) αKG is known to affect plethora of cellular functions including hypoxia inducible factor. Is there evidence suggesting that the HIF pathway is affected as there is also an impairment of oxidative phosphorylation?

4) Are the sites on LYRM proteins that get acylated known? Have you looked into mutating those sites on target LYRM proteins in a wild-type background to see if that could affect ETC complex assembly (similar to mtFAS mutation)?

5) Are LYRM proteins specifically mitochondrial or can they exist in other cellular compartments as well? If so, is it known what other functional modifications can occur on these proteins in other compartments?

References:
Angerer, H., Radermacher, M., Mańkowska, M., Steger, M., Zwicker, K., Heide, H., Wittig, I., Brandt, U., and Zickermann, V. (2014). The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity. Proc. Natl. Acad. Sci. 111, 5207–5212.
Nair, R.R., Koivisto, H., Jokivarsi, K., Miinalainen, I.J., Autio, K.J., Manninen, A., Poutiainen, P., Tanila, H., Hiltunen, J.K., and Kastaniotis, A.J. (2018). Impaired Mitochondrial Fatty Acid Synthesis Leads to Neurodegeneration in Mice. J. Neurosci. Off. J. Soc. Neurosci. 38, 9781–9800.
Nowinski, S.M., Vranken, J.G.V., Dove, K.K., and Rutter, J. (2018). Impact of Mitochondrial Fatty Acid Synthesis on Mitochondrial Biogenesis. Curr. Biol. 28, R1212–R1219.
Smith, S. (1994). The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 8, 1248–1259.
Smith, S., Witkowski, A., Moghul, A., Yoshinaga, Y., Nefedov, M., de Jong, P., Feng, D., Fong, L., Tu, Y., Hu, Y., et al. (2012). Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium. PloS One 7, e47196.
Solmonson, A., and DeBerardinis, R.J. (2018). Lipoic acid metabolism and mitochondrial redox regulation. J. Biol. Chem. 293, 7522–7530.

Tags: etc, fatty acid, lipid metabolism, mitochondria

Posted on: 25 May 2020 , updated on: 26 May 2020

doi: https://doi.org/10.1242/prelights.20950

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Author's response

The author team shared

Thank you for your interest in our work and your thoughtful commentary! We share your enthusiasm about how this ancient, conserved metabolic pathway has evolved to control cellular functions in unexpected ways, and are excited to further explore the regulatory functions of this pathway in mammalian cells.

In response to your questions:

1) We did not perform electron microscopy to analyze mitochondrial cristae structure, but we share your opinion that there are likely to be morphological defects in mtFAS mutants, especially in light of the ETC assembly defects we observed. When performing mitoTracker microscopy we did note a slightly more fragmented mitochondrial network, especially in mutants with more severe membrane potential phenotypes, but we did not quantify this phenomenon.

2) Great observation. The TCA cycle effects of mtFAS loss were at first difficult to parse because the downstream effects of mtFAS are so pleiotropic in nature and affect TCA cycle metabolism at several points. We think that the increase in αKG abundance can most reasonably be attributed to decreased oxoglutarate dehydrogenase (OGDH) activity. The E2 subunit of OGDH, DLST, requires lipoylation for its catalytic activity (Christensen et al., 2007) and this lipoylation is undetectable via western blot in mtFAS mutants (Figure 1B).

3) The idea that HIF pathways might be activated in mtFAS mutants is extremely interesting. We are continuing to explore how mtFAS impairment mechanistically connects to changes in cellular signaling pathways that control cell fate, and this pathway is high on our list of those to examine.

4) The nature of the physical interactions between acyl-ACP and the LYRM proteins is fascinating. Structural studies have beautifully captured a few of these physical interactions and given us a window into their unusual nature (Cory et al., 2017; Fiedorczuk et al., 2016). In each of the structures, the LYRM protein is comprised of a 3-helix bundle, with the conserved LYR motif near the N-terminus of the first α-helix. Intriguingly, the acyl chain is threaded into the middle of the 3-helix bundle, while the LYR residues seem to be important for making contacts with the 4’-phosphopantetheine cofactor that covalently links the acyl chain to a conserved serine on ACP. In yeast, we have shown that if you mutate the LYR motif on the various LYRM proteins, their complex assembly function is impaired (Van Vranken et al., 2018). However, we don’t actually know how LYRMs “sense” the presence of an acyl chain, and/or how acylation affects the confirmation and function of the LYRM proteins and their ability to bind their targets, and perform their complex assembly functions. This would be a great area for further structure/function-based studies.

5) Great question! As you can imagine, MANY proteins have Leu-Tyr-Arg motifs, but that does not necessarily mean that they are bona fide LYRM family members. To make matters more complicated, the motif is actually degenerate, with several family members containing LYK rather than the more common LYR. Therefore, we believe there are very likely to be novel LYRMs that have not been discovered yet. As to their existence outside mitochondria, the jury is out – but we’d be keen to know the answer too!

Thanks again for your outstanding summary and questions! We hope that this work lays the foundation for future studies and therapeutic approaches aimed at harnessing mtFAS to improve mitochondrial function in metabolic disease settings.

Best,
Sara and Jared

REFERENCES

Christensen, C.E., Kragelund, B.B., von Wettstein-Knowles, P., and Henriksen, A. (2007). Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase. Protein science : a publication of the Protein Society 16, 261-272.
Cory, S.A., Van Vranken, J.G., Brignole, E.J., Patra, S., Winge, D.R., Drennan, C.L., Rutter, J., and Barondeau, D.P. (2017). Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions. Proceedings of the National Academy of Sciences of the United States of America 114, E5325-e5334.
Fiedorczuk, K., Letts, J.A., Degliesposti, G., Kaszuba, K., Skehel, M., and Sazanov, L.A. (2016). Atomic structure of the entire mammalian mitochondrial complex I. Nature 538, 406-410.
Van Vranken, J.G., Nowinski, S.M., Clowers, K.J., Jeong, M.Y., Ouyang, Y., Berg, J.A., Gygi, J.P., Gygi, S.P., Winge, D.R., and Rutter, J. (2018). ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly. Molecular cell 71, 567-580.e564.

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