Hill Lab

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.

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 mice^5,6. 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 yeast^7-15, worm^16-18, and mammalian^19-25 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-inhibited^7-8,25, consistent with our earlier prediction derived from x-ray crystallography and NMR spectroscopy^9,11,19. 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 localization's. 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 fusion^16,17. We started by exploring human Bcl-xL^20-22, 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 others^27,28) subsequently showed was to regulate the fission and fusion mechanoenzymes¹⁷.

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.