Research Statement

SUMMARY OF GRADUATE RESEARCH AND CURRENT PROJECTS

The focus of my research in Dr. Shih-Hsiung Wu’s lab is a mitochondrial protein called Lon protease, a member of the AAA+ (ATPase-associated with various cellular activities) class of multifunctional proteins. To obtain a clone of human Lon (hLon) I contacted Dr. Carolyn Suzuki, an assistant professor at the University of Medicine and Dentistry in New Jersey. She has since become my second mentor, helping me organize my experimental results into a research paper that was published in Nucleic Acids Research early this year (http://nar.oxfordjournals.org/cgi/content/abstract/gkm1140v1?ck=nck).

Previous project: Biophysical Characterization of Lon Protease and Its DNA-binding Activity

Lon has an indispensible role in mitochondrial metabolism: it degrades abnormal polypeptides as well as certain regulatory proteins and metabolic enzymes, acts as a chaperone, and binds to nucleic acids. However, our understanding of its physiological importance is obscured by its multiple duties. For instance, does Lon bind to mtDNA for replication regulation or for substrate targeting? To determine its sequence specificity for mtDNA binding, I conducted a study of hLon binding to G-quartet-forming oligonucleotides (GFOs) that are identical to the H-strand of mtDNA. A combination of electrophoretic, thermodynamic and spectroscopic data revealed that hLon is not only favorable to sequences that form G-quadruplexes (as compared to double helical and single stranded DNA), but also differentiates among GFOs through different levels of thermal fluctuation reduction. hLon binding to DNA is accompanied by little or no significant change in heat capacity for all except one GFO: a sequence that overlaps mtDNA regulatory region. In sum, our results indicate that hLon recognizes its cognate target(s) by sequence-dependent structural tightening.

Ongoing projects: Proteomics and Interactomics of Mitochondrial Nucleoid & Structural Profiling of Mitochondrial DNA

The fundamental question to Lon is whether it serves as a sensor of environmental stress: for example, a shift from degrading mt-matrix proteins to regulating mtDNA replication/transcription in response to metabolic cues. If true, we may expect its dynamic localization between the matrix and mt-nucleoids within a mitochondrion. My primary job is to investigate the involvement of hLon in mt-nucleods and to discover its regulatory partners as well as substrates.

The basic idea is to separate groups of protein complexes within mt-nucleoids (isolated from rat livers at different ages) in the first step of 2D blue native/SDS PAGE, and the protein components of each group are resolved in the second dimension. We use in-gel digestion and MALDI MS to analyze the resolved spots. In parallel, the purified nucleoids treated by DNaseI are fractionated by sucrose gradient centrifugation, and the bound mtDNA fragments in each fraction are identified by cycle sequencing using appropriate fluorescein-labeled primers. For comparison with the data obtained from 2D PAGE, the sucrose-fractionated protein complexes are analyzed by LC and ESI MS-MS. The experiments to this stage are designed to clarify the interaction network of the nucleoid proteins and their target sequences. As for the real-time observation of a specific nucleoid protein of interest in mitochondria, I invite you to read my proposal at http://niwhu.blogspot.com/2008/10/proposal-summary.html.

On the other hand, I went on to follow up G-quartet formation on mtDNA, a possible scenario that we proposed in the NAR article. I am particularly interested in the connections between mtDNA G-quadruplexes and mt-nucleoid organization, as well as their influences on mtDNA integrity and mitochondrial metabolism. Our experimental scheme is to analyze mtDNA replicative intermediates in association with G-quartet fluorescent markers (e.g. porphyrin or carbazole derivatives) using 2D agarose gel electrophoresis and blotting.

UNDERGRADUATE RESEARCH EXPERIENCE

I learned how to do hands-on research at Dr. Chia-Ching Chang’s molecular biophysics laboratory in my sophomore year at National Dong Hwa University. I was given the responsibility of cloning metallothionein and studied molecular dynamics using Insight II (Accelrys Software Inc.). During vacations I was an intern at the Institute of Chemistry of Academia Sinica under the supervision of Dr. Lou-Sing Kan. There I used thermodynamic and kinetic approaches to study physicochemical properties of triplex DNA formation. I wrote my own grant proposal, and received a research fellowship from the Republic of China National Science Council (NSC). This became my senior undergraduate thesis project: an independent investigation of pyrimidine motif triplex formation that earned an NSC Research Creativity Award. The conference papers that I wrote won financial support for my attendance at the 4th East Asian Biophysical Symposium in Taipei and the 48th Annual Meeting of the Biophysical Society in Baltimore.