THE ROLE OF GRP75 IN SUPERCOMPLEX ASSEMBLY AND NEURODEGENERATION

Emerging evidence supports the proposition that the mitochondrial respiratory chain (MRC) functions viaorganized multicomplex structures called supercomplexes. However the dynamics and regulation ofsupercomplex assembly have not been fully investigated. In particular, hardly any regulatory protein factorsinvolved in supercomplex assembly have been identified. Our long term goal is to understand the dynamics ofmitochondrial respiratory machinery and its underling regulatory mechanism. The objective of this particularapplication is to determine if the mitochondrial chaperon, 75 kDa glucose regulated protein (Grp75) plays a rolein regulating supercomplex assembly and further to identify additional protein factors involved in this importantprocess. The study of mammalian respiratory supercomplex assembly has been difficult since common yeastsystems, which could be utilized as a powerful genetics system to identify putative regulatory factors, lackComplex I an essential component of mammalian supercomplexes. We have previously established anefficient method to isolate cells carrying mitochondrial DNA (mtDNA) mutations and further generated severalcell models with regulated/altered supercomplex assembly, probably due to the enhanced/stabilizedinteractions between supercomplexes and regulative factor(s). Characterizations of these cell lines employingboth molecular and proteomics approaches have implicated the molecular chaperone Grp75 supercomplexassembly. Interesting Grp 75 has previous been implicated in Parkinson's diseases (PD) due to 1). Grp 75mutations have been identified in PD patients; 2) Low Grp 75 expression was found in brains of PD patients; 3).Our preliminary studies showed heterozygous Grp75 mice exhibited lower motor activities associated withdefective supercomplex assembly. The central hypothesis for this application is that Grp75 is an essential partof machinery which regulates the assembly of supercomplexes, and defective of supercomplex assemblyassociated with deficient Grp 75 would lead to neuro-degeneration. To test this hypothesis, we propose topursue the following three specific aims: 1) Characterize the role of Grp75 in supercomplex assembly. Inparticular, we will follow the step-wise assembly and degradation of individual complexes and supercomplexesin presence and absence of Grp75 with newly developed approaches in the lab; 2) Determine the regulatorymechanisms of Grp75 on supercomplex by Identify novel protein factors involved in regulating supercomplexassembly. With proteomic analysis of proteins interacting with Grp75 and Complex I containingsupercomplexes in the cell models with regulated/altered supercomplex assembly, we aim to isolate novelprotein factors involved in regulating supercomplex assembly. 3) Characterize the mouse models with alteredexpression of Grp75. The implications of defective supercomplex dynamics in neuronal degeneration will befurther explored in heterozygous and neuronal-specific Grp75 knockout mouse models. We will investigate theunderlying molecular pathways derived from Grp75 defect to supercomplex deficiency to neuronaldegeneration. The approach is innovative, because it combines our unique cell models exhibiting upregulatedsupercomplex dynamics with newly-developed analytical methods to allow understanding of the complexity ofrespiratory supercomplex assembly. The establishment of novel mouse models with defective supercomplexdynamics should open new possibilities to study bioenergetics in neuronal system and neuro-degeneration.We believe that we are in a strong position to characterize respiratory supercomplex assembly. The researchis significant, because elucidating this mechanism could provide new insights into the regulation of oxidativephosphorylation machinery. In addition, we anticipate our work will also help to identify novel risk genesinvolved in neurodegenerative diseases associated with mitochondrial dysfunction.