Challenges to Mitochondrial Protein Homeostasis
Several aspects of mitochondrial biology present difficulties to the maintenance of protein homeostasis including the proximity of the electron transport chain and the generation of reactive oxygen species (ROS). Furthermore, the mitochondrial proteome is composed of components encoded by both the nuclear and mitochondrial genomes, whose transcriptional outputs must be coordinated. The thirteen proteins encoded by the mitochondrial genome, within the matrix, form stoichiometric complexes with many of the mitochondrial-targeted proteins encoded by the nucleus. The nuclear encoded proteins must first be translated in the cytoplasm in an unfolded state and then imported into the mitochondria where they must fold/assemble into a functional protein/complex. Any number of adverse cellular conditions can perturb these processes and affect mitochondrial protein homeostasis including the accumulation of cytosolic aggregates, toxins that generate ROS as well as rapid cellular proliferation.
Impact of Disease States on Mitochondrial Protein Folding Capacity
Aging as well as numerous diseases such as Parkinson's, Friedreich's ataxia and cancer are associated with mitochondrial dysfunction and negatively impact the mitochondrial protein-folding environment. With respect to organelle folding capacity and affected cellular lifestyle, these diseases can be divided into two categories. Aging and age-associated diseases likely represent a slow exhaustion or overwhelming of the organelle folding capacity due to a specific, disease-causing lesion. While rapidly dividing cancer cells likely depend on, or are addicted to pathways that maintain protein homeostasis to sustain the cancerous lifestyle. Hallmarks of malignant cell transformation include the suppression of apoptosis and the physiologic shift from mitochondrial-dependent aerobic respiration to glycolysis. Both represent dramatic alterations in mitochondrial metabolism and are due to both organelle remodeling and mitochondrial dysfunction. Additionally, the proteotoxic stress put on the mitochondrial protein-folding environment by the harsh conditions within the tumor, coupled with the stress associated with increased cellular growth and proliferation indicates a heavy dependence on cellular pathways that protect mitochondrial function.
Many human disease-associated legions cause activation of the UPRmt in C. elegans. In this transgenic line, the promoter from the mitochondrial chaperone gene, hsp-60, regulates expression of GFP. In wildtype worms (left panel), hsp-60 expression remains at baseline levels. However, perturbations in the mitochondrial protein folding environment activates UPRmt signaling and the upregulation of mitochondrial chaperone genes, represented in this case, by increased GFP expression (right panel).
The Mitochondrial Unfolded Protein Response
A mechanism cells utilize to maintain the mitochondrial protein-folding environment is the mitochondrial unfolded protein response (UPRmt). Essentially, a matrix-localized sensor, responsive to mitochondrial chaperone occupancy signals a transcriptional response that selectively upregulates compartment-specific machinery to fold or degrade unfolded/misfolded proteins. The most upstream component of this pathway is a matrix-localized orthologue of the bacterial protease ClpXP, a protease known to have roles in bacterial signal transduction as well as protein quality control. Upon accumulation of unfolded proteins, the percentage of proteins that exceeds the chaperone folding capacity is degraded by the protease ClpXP to peptides. The accumulating peptides are pumped from the mitochondrial matrix to the cytoplasm by an ABC transporter leading to the activation of a cytosol-localized bZiP transcription factor. Activation of the transcription factor promotes nuclear translocation leading to increased transcription of mitochondria-targeted chaperones returning the organelle's protein folding environment to homeostasis. The UPRmt is organized in a manner where changes in chaperone expression are intimately associated with the degradation of a percentage of accumulating unfolded proteins thereby ensuring protein homeostasis at both the level of protein folding/refolding and removal.
A comparison of mitochondrial morphology in healthy muscle cells and muscle cells whose UPRmt is impaired. Healthy mitochondria (top panel, fluorescently labeled) align in an organized manner along the muscle fibers, while mitochondria with reduced folding capacity (lower panel) are swollen with severely altered morphology.
Approaches and Projects
Our lab focuses on the maintenance of mitochondrial protein homeostasis in both C. elegans and mammalian cell culture models taking a variety of genetic and biochemical approaches. Some of the questions in which we are addressing:
- How is protein folding capacity in the mitochondria adjusted and maintained?
- How is proteolysis regulated within the mitochondrial matrix?
- What role does mitochondrial maintenance play in aging and disease manifestation?