Proposal Summary

Dynamic Structures and Composition of Human Mitochondrial Nucleoids: Biochemical and Biophysical Studies

人類粒線體類核結構動態: 生化及生物物理之研究


This proposal is centered on: (1) the proteomics of mitochondrial nucleoid; (2) characterization of protein-protein interactions between the nucleoid components; (3) the composition of mitochondrial nucleoid in connection with changes of cellular metabolic state as well as oxidative pressure, and how it is related to the regulation of mtDNA replication/transcription; (4) the dynamics of mitochondrial metabolic enzymes found in the nucleoid.

Mitochondrion not only is the cellular powerhouse but also acts as a critical trigger of programmed cell death and maintains the homeostasis of metabolites and calcium. Mitochondrial abnormalities typically have manifestations in brain, eye and muscle, and are thus termed mitochondrial encephalomyopathies. For example, patients with MERRF syndrome (myoclonic epilepsy associated with ragged-red fibers) suffer from ataxia, epilepsy and myoclonus. On the other hand, LHON (Leber hereditary optical neuropathy) is related to optic atrophy. However, mitochondrial diseases have genetic and clinical complications, thus the symptoms of a particular disease may different between individuals even if they are members of the same family. This phenomenon can be partly explained by the differences in mitochondrial loads and threshold effects among different tissues. While there is hundreds of nucleoids located within a single mitochondrion, the exact number of mitochondria and nucleoids varies from tissue to tissue. A nucleoid possibly contains 2-15 mtDNA molecules, and pathogenic mtDNA molecules that contain mutations may be transmitted and accumulated in daughter mitochondria. As the number of pathogenic mtDNA goes beyond a threshold, mitochondrial dysfunction becomes apparent. Clarifying the dynamics of nucleoid composition may help us understand the fate of pathogenic mtDNA (e.g. transfer and repair) and its relevance to the onset of mitochondrial abnormalities.

Mitochondria constantly fuse and divide in response to cellular requirements, and the nucleoid may also change to adjust mtDNA distribution. Recent studies have found that mitochondrial nucleoid contains some metabolic enzymes in addition to conventional DNA transaction proteins. However, the physiological significance of these metabolic proteins locating at the nucleoid remains to be clarified. It is tantalizing to suggest that these enzymes may be involved in mtDNA regulation coupling with their metabolic functions. In this proposed project, we plan to characterize the protein components of mitochondrial nucleoid by using multidimensional liquid chromatography (MDLC) and ESI MS/MS. In addition, the protein-protein interactions between nucleoid components are analyzed by blue-native/SDS 2D PAGE in combination with MALDI MS analysis. The database of nucleoid composition and protein interaction network can be established from the above results (Figure 1). To further investigate the extent to which the metabolic proteins may affect nucleoid integrity, we (1) manipulate the expression of selected enzyme with siRNA, and then analyze the nucleoid constituents as well as the mtDNA stability. Alternatively, we (2) profile the changes in nucleoid composition and its protein interaction network under oxidative stress.

To examine the roles of nucleoid proteins with the relevance of mtDNA transaction, we analyze mtDNA fragments bound by the protein complexes. Digested by DNaseI, the mtDNA sequences in complex with nucleoid proteins are fractionated by sucrose gradient centrifugation and analyzed by PCR sequencing. Here we are interested in the fractions that contain consensus sequences, which may include the mtDNA non-coding region and G-quadruplex-forming sequences. We in turn analyze the nucleoid proteins in the fractions of interest by immunoblotting, and compare the results with those from 2D electrophoresis. We may then specify any metabolic protein involved in nucleoid regulation for further analysis. It is of special interest to identify members of AAA family (ATPase-associated with various cellular activities), a class of multifunctional proteins that possibly regulate mtDNA replication/transcription in response to cellular demands.

Finally, we may also choose a nucleoid protein of interest (identified from the above mentioned methods) for investigating its functional dynamics in a mitochondrion. Here we take hLon as an example. By expressing the plasmid containing hLon fused with fluorescence protein in cultured cells, the dynamics of the fluorescent hLon can be observed by using FCS (fluorescence correlation spectroscopy). We can compare the differences in diffusion coefficient and retention time among selected subensembles in mitochondria (Figure 2). As a motivation to study the structural dynamics of mtDNA-binding proteins, we label mtDNA with SYTOXR Blue and examine interactions between mtDNA and the fluorescent hLon in a separate experiment using FRET-FLIM (fluorescence resonance energy transfer coupled with fluorescence lifetime imaging microscopy). Taken together, this proposal is aimed to address genetic and age-related mitochondrial abnormalities by the way of better understanding the connections between the mt-nucleoid proteins dynamics and metabolic cues.





Figure 1. Expected result of nucleoid composition and the interaction network of these proteins



Figure 2. Scheme of human mitochondrial Lon (or other specified protein) dynamics in mitochondria. FCS stands for fluorescence correlation spectroscopy; D means diffusion coefficient and τdiff corresponds to diffusion time. FRET-FLIM is the abbreviation for the instrument of fluorescence resonance energy transfer coupled with fluorescence lifetime imaging microscopy, where τφ is fluorescence phase lifetime, τm is fluorescence modulation lifetime, Da is the diffusion constant of FRET acceptor, τr is its diffusion time, and the time constants are obtained by fitting of the relative quantity of donor (or receptor) along the time axis.