Research
What is the molecular basis of mitochondrial disease?
My laboratory is investigating the fundamental events that regulate mitochondrial homeostasis in disease (Fig. 1). Our goal is to determine how proteins interact with other biological macromolecules to control these basic processes in healthy cells and go awry to cause disease. We strive to understand these interactions on a physicochemical level, with an eye for gleaning universal principles of protein chemistry including interactions with membrane bilayers that are fundamental to a wide variety of cellular processes. These interactions are likely governed by evolutionarily conserved mechanisms that are still poorly understood. Here we briefly highlight a subset of our past accomplishments with current and future priorities.
Figure 1. Mitochondrial homeostasis is governed by frequent fission and fusion that are central to human health.
Mechanisms of mitochondrial fission and fusion: Implications for neurodegeneration, cardiomyopathies, and death
Frequent mitochondrial fission and fusion events have been appreciated for over a century1, but the genes involved have only recently been identified2. Mutations in fusion genes in humans cause neurodegenerative diseases3 and mutations in fission genes cause neonatal lethality in humans4 and severe cardiomyopathy in mice5,6. These diseases highlight the importance of mitochondrial fission and fusion to human health. Yet how proteins carry out these membrane-remodeling events and respond to their environments to regulate mitochondrial homeostasis is not known. To address these questions, we have initially focused on determining the molecular basis of mitochondrial fission, although we expect our studies will uncover mechanistic connections with other mitochondrial and cellular processes. We investigate these questions using interdisciplinary approaches in yeast7-15, worm16-18, and mammalian19-25 systems in order to clarify which regulatory mechanisms are evolutionarily conserved and most important.
Lethal mutation in fission mechanoenzyme Drp1 impairs its assembly and localization
Only two proteins are conserved for fission in all species that contain mitochondria: the membrane-anchored Fis1 and the dynamin-related mechanoenzyme Drp1. In mammalian cells, we have determined that disease-causing mutations in Drp1 impair its assembly leading to decreased fission and altered cellular distribution of elongated mitochondria23. Using a structure-based design approach, we have identified that Drp1 binding to Fis1 is auto-inhibited7-8,25, consistent with our earlier prediction derived from x-ray crystallography and NMR spectroscopy9,11,19. Identifying what relieves Fis1 auto-inhibition and the role of mito-ER tethering in this process is a current focus. A high priority for the future is to determine how these morphological changes exactly impair mitochondrial function to cause disease. These efforts will benefit from our high-throughput screening of small molecule libraries, which should identify useful chemical biological tools for manipulating mitochondrial morphology and may also identify new therapeutic routes.
Defective regulation of mitochondrial apoptosis
Mitochondria dramatically fragment during a form of programmed cell death called apoptosis, which is regulated by the Bcl-2 (B cell lymphoma 2) proteins that, like Drp1, cycle between cytoplasmic and mitochondrial localizations. Down-regulation of either Fis1 or Drp1 prevents mitochondrial fragmentation and delays the onset of apoptosis, but the mechanistic basis of these observations and their functional importance are unclear26 despite the clear importance of apoptosis to human health. Our work to address these questions has focused on Bcl-2 proteins and the interplay with the fission/fusion machinery.
Bcl-2s do more than regulate apoptosis
We were the first to show that Bcl-2 proteins have two separable functions: one in regulating apoptosis and another in regulating mitochondrial fission and fusion16,17. We started by exploring human Bcl-xL20-22, but a major complication in the mammalian Bcl-2 field is the redundancy of Bcl-2 family members. We bypassed these issues by working with the nematode C. elegans, which contains only one Bcl-2 member, CED-9. We designed two new apoptosis assays in the worm that allowed us to replace the endogenous copy of CED-9 with any variant16. This simple, yet powerful, approach had not been taken before in the apoptosis field. We first tested the role of the C-terminal transmembrane domain that across species is evolutionarily conserved and found it was required for mitochondrial localization, but dispensable for apoptosis. This surprising result suggested an evolutionarily conserved role for Bcl-2 proteins at mitochondria distinct from apoptosis, which we (and others27,28) subsequently showed was to regulate the fission and fusion mechanoenzymes17.
Impacting fields beyond mitochondrial homeostasis
Devising a high-throughput screen to dissect complex proteins-proteins interactions
In thinking about complex protein-protein interactions such as those that govern mitochondrial fission and apoptosis, we realized a need to quickly identify alleles that could parse out different interactions and possibly separate different functions. To accomplish this, we conceived a new screen to identify critical residues of one protein that interacts with several others. We have applied this technology to Fis1, rapidly screened >3000 mutations, and simultaneously identified >300 alleles of Fis1 that selectively disrupt yeast two-hybrid interactions between three binding partners13. Our analysis has identified new, functionally important residues of Fis1 that will be critical for dissecting its role in fission. More generally, we think the application of this new screening technology, which we call hotspot (for identifying protein-protein interaction hotspots), can help address how amino acid sequence specifies certain interactions over others. We anticipate that this technology will also gain widespread use to define critical residues between protein complexes in vivo.
Protein amphitropism in mitochondrial biology
For proteins described above, a key feature is the structural transformation from soluble to membrane-bound conformations of proteins, a phenomenon referred to as amphitropism27. Associating with or dissociating from a membrane (i.e. amphitropism) has significant functional consequences for numerous biological processes: it can affect enzymatic activity (CCT, PLC), can promote changes in organelle and cell morphology (minD, dynamins), or can act as a regulatory switch in various signaling cascades (PKC, ESCRTs). However, neither what drives proteins to reversibly interact with membranes nor how this function controls biological outcomes are clearly understood. We anticipate that many more soluble proteins exhibit functional amphitropism and have devised proteomic-based screens to identify these proteins, which will allow us to begin to formulate predictive algorithms as we continue to establish this nascent field.
Figure 2. Proteins that reversibly interact with membranes are amphitropic, which can be assisted (lipid- or cation-dependent) or unassisted (oligomerization- or pH-dependent). The different mechanisms of unassisted aphitropism and how they are regulated are not well known, but will be aided by two proteomic screens we are currently pursuing.
Literature cited
1. MR Lewis and WH Lewis, Mitochondria in Tissue Culture. Science 39, 330-333 (1914).
2. DC Chan, Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241-1252 (2006).
3. H Chen and DC Chan, Mitochondrial dynamics--fusion, fission, movement, and mitophagy--in neurodegenerative diseases, Hum Mol Genet 18:R169-76 (2009).
4. HR Waterham et al. A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736-1741 (2007).
5. H Ashrafian et al., A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy, PLoS Genet 6, e1001000 (2010) doi:10.1371/journal.pgen.1001000
6. SB Ong et al., Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury, Circulation 121, 2012-22 (2010).
7. Y Fannjiang, WC Cheng, SJ Lee, B Qi, J Pevsner, JM McCaffery, RB Hill, G Basañez, and JM Hardwick, Mitochondrial fission proteins regulate programmed cell death in yeast., Genes Dev 18, 2785-97 (2004).
8. RC Wells, LK Picton, SCP Williams, FJ Tan, and RB Hill, Direct binding of the dynamin-like GTPase, Dnm1, to mitochondrial dynamics protein Fis1 is negatively regulated by the Fis1 N-terminal arm, J Biol Chem 282, 33769-75 (2007).
9. LK Picton†, S Casares†, AC Monahan, A Majumdar, and RB Hill, Evidence for conformational heterogeneity of fission protein Fis1 from S. cerevisiae, Biochemistry 21, 6598-609 (2009). †co-first authors
10. RC Wells and RB Hill, The Fis1 cytosolic domain reversibly clusters lipid vesicles, PLoS-1, 6, e21384 (2011).
11. JE Tooley, V Khangulov, JP Lees, JL Schlessman, M Bewley, A Héroux, J Bosch*, and RB Hill*, 1.75 Å crystal structure of fission protein Fis1 from Saccharomyces cerevisiae reveals elusive interactions of the autoinhibitory domain, Acta Cryst F (in press). *co-corresponding authors
12. JP Lees, LK Picton, CM Manlandro, AZE Tan, S Casares, JM Flanagan, KG Fleming, and RB Hill, The N-terminal arm prevents homodimerization of fission protein Fis1 from S. cerevisiae, in process of addressing reviewers’ concerns.
13. CM Manlandro, BJ Zimmerman, M Christie, and RB Hill, hotspot, an efficient yeast two-hybrid based screen to identify disruptive residues at multiple protein interfaces, manuscript in preparation.
14. RB Hill, TJ Barfield, JP Lees, D Toptygin, L Brand and KR MacKenzie, Reversible kinetic stabilization of a latent, but functional, state, manuscript in preparation.
15. LK Picton, JP Lees, and RB Hill, Autoinhibition of Fis1 regulates mitochondrial fission, manuscript in preparation.
16. FJ Tan, JE Zuckerman, AZ Fire, and RB Hill, Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain, Cell Death Differ 14, 1925-35 (2007).
17. FJ Tan, M Husain, CM Manlandro, M Koppenol, AZ Fire, and RB Hill, CED-9 and mitochondrial homeostasis in C. elegans muscle. J Cell Sci 102, 3373-82 (2008).
18. FJ Tan, JE Zuckerman, RC Wells, and RB Hill, The C. elegans B-cell lymphoma 2 (bcl-2) homolog Cell death abnormal 9 (CED-9) associates with and remodels lipid membranes, Protein Sci 20, 62-74 (2011).
19. JA Dohm, SJ Lee, JM Hardwick, RB Hill*, and AG Gittis*, The cytosolic domain of the human mitochondrial fission protein Fis1 adopts a TPR fold, Proteins 54, 153-6 (2004). * co-corresponding authors
20. GR Thuduppathy and RB Hill, Acid destabilization of the solution conformation of Bcl-xL does not drive its pH-dependent insertion into membranes, Protein Sci 15, 248-57 (2006).
21. GR Thuduppathy, JW Craig, V Kholodenko, A Schon, and RB Hill, Evidence that membrane insertion of the cytosolic domain of Bcl-xL is governed by an electrostatic mechanism, J. Mol. Biol 359,1045-58 (2006).
22. GR Thuduppathy, O Terrones, JW Craig, G Basañez, and RB Hill, The N-terminal domain of Bcl-xL reversibly binds membranes in a pH-dependent manner, Biochemistry 45, 14533-42 (2006).
23. CR Chang†, CM Manlandro†, D Arnoult, J Stadler, AE Posey, RB Hill*, and C Blackstone*, A lethal de novo mutation in the middle domain of the dynamin-related GTPase Drp1 impairs higher-order assembly and mitochondrial division, J Biol Chem 285, 32494-503 (2010). †co-first authors * co-corresponding authors
24. DV Jeyaraju, HM McBride, RB Hill*, L Pellegrini*, A new proteolytic cascade converts mitochondrial Parl into an archetypal bacterial rhomboid, Cell Death Differ 18, 1531-9 (2011). * co-corresponding authors
25. CM Manlandro, D Arnoult, J Stadler, C Blackstone*, and RB Hill*, Fis1 directly recruits the fission mechanoenyme Drp1 to sites of scission, manuscript in preparation. * co-corresponding authors
26. ME Soriano and L Scorrano, The interplay between BCL-2 family proteins and mitochondrial morphology in the regulation of apoptosis, Adv Exp Med Biol 687, 97-114 (2010).
27. JE Johnson and RB Cornell, Amphitropic proteins: regulation by reversible membrane interactions, Mol Membr Biol 16, 217-35 (1999).
28. SG Rolland, Y Lu, CN David, and B Conradt, The BCL-2-like protein CED-9 of C. elegans promotes FZO-1/Mfn1,2- and EAT-3/Opa1-dependent mitochondrial fusion, J Cell Biol 186:525-40 (2009).
29. Y Lu, SG Rolland, and B Conradt, A molecular switch that governs mitochondrial fusion and fission mediated by the BCL2-like protein CED-9 of Caenorhabditis elegans, Proc Natl Acad Sci 108:E813-22 (2011).