Mechanisms of protein degradation, oxygen sensing and metabolism
How different polyubiquitin chains are decoded
Specificity in the ubiquitin system is generated by the ability of ubiquitin to form eight different polyubiquitin chain linkages. Each type of ubiquitin linkage must be correctly interpreted to facilitate the desired outcome, and ubiquitin binding proteins (UBPs) provide this critical link between chain recognition and cellular fate. We showed that ubiquitin linkage-selective UBPs can distinguish between the two most abundant ubiquitin chains (lysine-48 and lysine-63) and control the degradation of ubiquitinated substrates. We have also recently shown that the proteasome itself can distinguish between different types of lysine-11 linked polyubiquitin chains, dependent on whether they are pure (homotypic) or mixed with other linkages (heterotypic).
We are currently using biochemical and genetic approaches to examine the function and physiological importance of other chain-specific UBPs. Moreover, we are using forward genetic screens to identify genes required to regulate the degradation of proteins by the proteasome. By the insertion of random mutations into cells expressing proteasome reporters, we can identify genes required for the efficient degradation of these ubiquitinated substrates.
Oxygen sensing and metabolism
A fundamental requirement for cell survival is the ability to respond to the local oxygen and nutrient environment. Central to this process are the hypoxia inducible transcription Factors (HIFs), which are usually rapidly ubiquitinated and degraded by the proteasome when oxygen is abundant. However, findings that HIFs can be stabilised and activated even in aerobic conditions has driven our interest in this field. We have adopted near-haploid and CRISPR/Cas9 human forward genetic screens to identify genes required for the regulation of these key oxygen transcription factors. This approach has uncovered a novel regulatory pathway for oxygen sensing, centred on the 2-oxoglutarate dehydrogenase complex (OGDHC) – a key mitochondrial enzyme required for respiration. Loss of the OGDHC drives the formation of the metabolite L-2-Hydroxyglutarate (L-2-HG), which accumulates in cells and directly inhibits the prolyl hydroxylase (PHD) enzymes that sense oxygen abundance. We have also shown that OGDHC activity can be reduced through decreasing lipoylation of the complex by lipoic acid. These findings highlight the intricate relationship between mitochondrial metabolism, lipoic acid and oxygen sensing enzymes. Understanding the role of L-2-HG in physiological contexts is a current focus of our studies.
Miles AL et al. The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels. eLife http://dx.doi.org/10.7554/eLife.22693 (2017).
Mitochondrial protein lipoylation and the 2-oxoglutarate dehydrogenase complex controls HIF1α stability in aerobic conditions. Burr S.P., Costa A.S.H., Grice G.L., Timms R.T., Lobb I.T., Freisigner P., Dodd R.B., Dougan G., Lehner P.J., Frezza C., and Nathan J.A. Cell Metabolism 2016. http://dx.doi.org/10.1016/j.cmet.2016.09.015
Scholz C.C., Rodriguez J., Pickel C., Burr S.P., Fabrizio J., Nolan K.A., Spielman P.,Cavadas M.A.S., Crifo B., Halligan D.N., Nathan J.A., Von Kreigsheim A., Wenger R.H., Peet D.J., Cummins E.P., and Taylor C.T. FIH regulates cellular metabolism through hydroxylation of the deubiquitinase OTUB1. PLoS Biol. 2016 Jan 11;14. PMC4709136. http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002347
Grice G.L., Lobb I.T., Weekes M.P., Gygi S.P., Antrobus R, and Nathan J.A. (2015). The proteasome distinguishes between heterotypic and homotypic lysine-11 linked polyubiquitin chains. Cell Rep. Jul 28;12(4):545-53. PMC4533228. http://www.cell.com/cell-reports/abstract/S2211-1247(15)00687-7
Boname J.M., Bloor S, Wandel M.P., Nathan J.A., Antrobus R, Dingwell K.S., Thurston T.L., Smith D.L, Smith J.C., Randow F, and Lehner P.J. (2014) Cleavage by Signal Peptide Peptidase is required for the degradation of selected tail-anchored proteins. J Cell Biol. Jun 23;205(6):847-62. PMC4068138. http://jcb.rupress.org/content/205/6/847 Cited by F1000. http://f1000.com/prime/718465519
Van den Boomen D.J.H.,Timms R.T., Grice G.L., Stagg H.R, .Skødt K, Nathan J.A., and Lehner P.J. (2014) TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I. PNAS Aug 5;111(31):11425-30. PMC4128144. http://www.pnas.org/content/111/31/11425.long.
Nathan J.A., Spinnenhirn V, Schmidtke G, Basler M, Groettrup M and Goldberg A.L. (2013). Immuno- and constitutive proteasomes do not differ in ability to degrade ubiquitinated proteins. Cell 152,1184-94. PMC3791394. http://www.cell.com/abstract/S0092-8674(13)00129-3.
Nathan J.A., Kim HT, Ting L, Gygi S, and Goldberg A.L. (2013). Why do cell proteins linked to K63-polyubiquitin chains not associate with proteasomes? EMBO J 32, 552-65. PMC3579138. http://emboj.embopress.org/content/32/4/552. Research highlight in Nature Reviews Molecular Cell Biology. http://www.nature.com/nrm/journal/v14/n3/full/nrm3540.html.
Peth A, Nathan J.A., and Goldberg AL (2013) The ATP costs and time required to degrade ubiquitinated proteins by the 26S proteasome. J Biol Chem 288, 29215-29222. PMC3790020. http://www.jbc.org/content/288/40/29215.full
Deriziotis, P., Andre, R., Smith, D.M., Goold, R., Kinghorn, K.J., Kristiansen, M., Nathan, J.A., Rosenzweig, R., Krutauz, D., Glickman M.H., Collinge J, Goldberg A.L., Tabrizi S.J. (2011). Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry. EMBO J, Jul 8;30(15):3065-77. PMC3160194. http://emboj.embopress.org/content/30/15/3065.long