10/22/2008
10/19/2008
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.
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.
10/18/2008
Selected Conference Paper
5th East Asian Biophysics Symposium, Okinawa, Japan (2006)
Chen SH and Wu SH, “Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease”
Chen SH and Wu SH, “Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease”
10/16/2008
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.
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.
10/02/2008
Curriculum Vitae
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Curriculum Vitae SHChen
Downloadable PDF at http://niwhu.myweb.hinet.net/curriculum vitae_SHChen.pdf
Curriculum Vitae SHChen
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Downloadable PDF at http://niwhu.myweb.hinet.net/curriculum vitae_SHChen.pdf
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