Lon蛋白酶是屬於AAA+家族的多功能蛋白質，泛存於各物種中，其中人類Lon具有兩同源體(homologue)，一於粒線體(本論文探討對象)，另一則位於過氧化酶體。它是ATP驅動的分子機器，同時具有蛋白酶、chaperone及DNA結合特性。正因為它具有多樣功能，繫於細胞的存亡，我們對Lon的瞭解也模糊於其多種功能之間，例如它的DNA結合能力，究竟是為了引導Lon到DNA所在位置以便其水解與DNA調控相關蛋白，或是Lon實際上直接參與DNA複製轉錄之調控，無法釐清。為確定Lon對DNA結合的特異性，我們在此篇論文詳細分析Lon對不同DNA序列的熱力學驅動力(thermodynamic driving forces)，以及焓、亂度對溫度的依賴性，以探討在生物合理溫度範圍內是由何種參數主導反應發生，以及機制為何。
首先我們以一系列的電泳與圓二色光譜實驗，就競爭量(mass competition)多寡粗略推斷Lon對單股、雙股及四股DNA的結合能量層級有顯著差異，並證實人類Lon蛋白酶對G-quartet四重螺旋DNA有特殊偏好，這項發現看似吸引人卻潛藏著誤估其生理意義的可能性，原因在於Lon是相當龐大的蛋白質，它的單體約100kDa，且傾向六到七個組合為多體，這樣的蛋白質可能碰巧表面有相當面積的正價胺基酸，而得與負價的聚合物(如G-quartet DNA)作用，剖析之間的熱力學得以讓我們瞭解Lon與G-quartet作用是否真具生理意義。
結果顯示，人類Lon對粒線體DNA(mtDNA)上諸多G-quartet序列有著相似的結合強度與自由能變化，與這些序列的結合並伴隨著極微弱或可忽略的比熱變化，惟存在一個例外－那即是座落於mtDNA複製/轉錄主要調控區段的序列，我們稱為LSP(light strand promoter region)。Lon與LSP序列的結合在20℃以上均由焓變化主導，並導致比熱的遽減，其程度約莫相當51個胺基酸殘基參與摺疊反應，表示Lon與LSP的結合造成蛋白質區域性的結構變化(輔以圓二色光譜證實)，比較LSP之G-quartet核心序列與Lon的結合，其反應在生物合理溫度範圍間均為亂度主導，並且無明顯之比熱變化。我們提出一套理論解釋這樣的現象－Lon傾向於結合至mtDNA上的G-quartet形成區域，換句話說這些序列為招募Lon的熱點(hotspot)，然而Lon可以結合這些大部分的序列並很輕易的離開，惟當它找尋到像LSP這般特殊標的，它們的結合造成整體結構變得緊密(依比熱變化程度判斷為熱擾亂度的降低)，這樣具有序列特異性的結合致使結構改變，我們認為跟Lon直接參與mtDNA調控有莫大關係。
本篇論文以發表於去年三月份的核酸研究期刊(Nucleic Acids Research)，但事實上我們的故事還沒完全說完，在這當中發現一些前人未曾指出的有趣現象，像是mtDNA存在G-quartet的生理意義為何?我們只知道mtDNA序列歧異，即一股為guanine-rich，我們稱之為H股，另一為互補的L股，經由G-quartet預測程式分析，H股上佈有約二十個G-quartet高發生區，大部分並座落於結構鬆散的”D-loop”區域，因此就序列組成與微環境許可度來看，mtDNA確實可能發生G-quartet這樣的結構，並且除了本文的Lon蛋白酶外，粒線體存有其他潛在的G-quartet結合蛋白，只是相關文獻的可見度不高，多年來一直被忽略，如拓譜酶I型(粒線體具有同源體)與端粒反轉錄酶(視情況轉移至粒線體)，我們期望在未來能進一步探討這些粒線體G-quartet結合蛋白，以及G-quartet這樣區域性DNA結構功能為何，它們的結合與mtDNA類核的組織或複製轉錄的關聯將是非常值得關注的題目。
12/19/2008
Statement of Purpose
My early interest in biophysics can be traced back to my sophomore year at National Dong Hwa University. There I studied triplex DNA under the supervision of Dr. Chia-Ching Chang and Dr. Lou-Sing Kan of Academia Sinica. 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.
During the past four years I have been very fortunate in having Dr. Shih-Hsiung Wu as my mentor for both my master’s thesis at National Taiwan University and my current research at Academia Sinica. Dr. Wu has given me a great deal of latitude in choosing topics, designing experiments, and writing research articles—that is, I have held full responsibility for all aspects of the publication process. He has consistently encouraged me with such advice as, “At most I can show you the road signs, but please remember that we are all finding our own way.” He has made the experience more than simply learning how to do technical research; I learned a lot about myself and about working with others to achieve a goal.
The focus of my research in Dr. Wu’s lab is a mitochondrial protein called Lon protease, a member of the AAA+ class of multifunctional proteins. In brief, I conducted a study of the physicochemical properties of hLon binding to G-quartet-forming oligonucleotides (GFOs). A combination of 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. This is a very interesting finding in two contexts. In biological terms, our results provide direct evidence that hLon recognizes a sequence of mtDNA regulatory region coupled with structural tightening. In biophysical terms, the same results indicate that a new variable—DNA local structures—must be introduced to current theories of how DNA-binding proteins search for target sequences. For more detail, I invite you to read my report at http://nar.oxfordjournals.org/cgi/content/abstract/gkm1140v1?ck=nck.
In Dr. Wu’s lab I have continued working on several ideas emerging from my graduate research—for example, protein dynamics involved in the mitochondrial nucleoids and G-quartet formation on mtDNA (please see the research summary in the my CV at http://niwhu.blogspot.com/2008/10/rsum.html and my research statement at http://niwhu.blogspot.com/2008/10/research-statement.html). I am particularly interested in finding out whether G-quartet structures of mtDNA interact with potential G-quartet-binding proteins that reside in mitochondria (e.g., Lon, mtTop1) or translocate to mitochondria (e.g., TERT). I have spent time developing several possible methods for investigating these questions, including proteomics and fluorescence spectroscopy. Still, I recognize a need for further training and practice using such techniques as single-molecule fluorescence and cryo-electron microscopy, which allow for the detection of biomolecular energetics and dynamics in a scale-free manner. This year I wrote a proposal describing my Ph.D. research intentions and received a Republic of China Government Fellowship that will support my study in the US to develop these skills (http://niwhu.blogspot.com/2008/10/proposal-summary.html).
During the past four years I have been very fortunate in having Dr. Shih-Hsiung Wu as my mentor for both my master’s thesis at National Taiwan University and my current research at Academia Sinica. Dr. Wu has given me a great deal of latitude in choosing topics, designing experiments, and writing research articles—that is, I have held full responsibility for all aspects of the publication process. He has consistently encouraged me with such advice as, “At most I can show you the road signs, but please remember that we are all finding our own way.” He has made the experience more than simply learning how to do technical research; I learned a lot about myself and about working with others to achieve a goal.
The focus of my research in Dr. Wu’s lab is a mitochondrial protein called Lon protease, a member of the AAA+ class of multifunctional proteins. In brief, I conducted a study of the physicochemical properties of hLon binding to G-quartet-forming oligonucleotides (GFOs). A combination of 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. This is a very interesting finding in two contexts. In biological terms, our results provide direct evidence that hLon recognizes a sequence of mtDNA regulatory region coupled with structural tightening. In biophysical terms, the same results indicate that a new variable—DNA local structures—must be introduced to current theories of how DNA-binding proteins search for target sequences. For more detail, I invite you to read my report at http://nar.oxfordjournals.org/cgi/content/abstract/gkm1140v1?ck=nck.
In Dr. Wu’s lab I have continued working on several ideas emerging from my graduate research—for example, protein dynamics involved in the mitochondrial nucleoids and G-quartet formation on mtDNA (please see the research summary in the my CV at http://niwhu.blogspot.com/2008/10/rsum.html and my research statement at http://niwhu.blogspot.com/2008/10/research-statement.html). I am particularly interested in finding out whether G-quartet structures of mtDNA interact with potential G-quartet-binding proteins that reside in mitochondria (e.g., Lon, mtTop1) or translocate to mitochondria (e.g., TERT). I have spent time developing several possible methods for investigating these questions, including proteomics and fluorescence spectroscopy. Still, I recognize a need for further training and practice using such techniques as single-molecule fluorescence and cryo-electron microscopy, which allow for the detection of biomolecular energetics and dynamics in a scale-free manner. This year I wrote a proposal describing my Ph.D. research intentions and received a Republic of China Government Fellowship that will support my study in the US to develop these skills (http://niwhu.blogspot.com/2008/10/proposal-summary.html).
11/26/2008
Personal History Statement
I have a special reason for appreciating every opportunity I have to obtain an education and explore my talents: both of my parents had to leave school at the age of thirteen and work full-time to support themselves and my grandparents. This was at the very beginning of the period that eventually came to be known as the “economic miracle” of Taiwan. My father has at various times worked as a mechanic and as a worker in a rubber production facility, and my mother continues to work long hours as a spa therapist at a small beauty salon.
In contrast, I was born during an era when my country was undergoing enormous change. Forty years of martial law came to an end when I was five, and a new government administration included a group of technical bureaucrats who successfully pushed for public funding of basic science research and education. I benefited from those decisions in high school and as an undergraduate; unlike students in many other developing countries, I am very fortunate in having several options for my future. My decision is influenced by recent advancements in the biological sciences that include animal cloning and human genome mapping.
One of my best adolescent experiences was attending the 2001 Wu Chien-Shiung Science Camp. I had some minor surgery just before the camp, but the physical pain was not going to stop me from meeting Nobel laureates Steve Chu, Douglas Osheroff, and Yuan-Tseh Lee. You can imagine my excitement when I was selected to explain my proposed solution to HIV on a stage in front of hundreds of experienced scientists, despite not having any laboratory experience. I was very nervous, but I was also running on adrenaline from an all-night brainstorming session that I had shared with my teammates. The source of my energy that night was the spirit of exploration, and I was hooked by the experience.
Every creative mind must find its niche, and mine is in biophysics. I enjoy working with the big picture of biomolecule binding energetics, structural dynamics, and motion in living cells. However, as my undergraduate mentor Dr. Chia-Ching Chang says, “Straight A’s do not guarantee a successful research career.” I try to keep in mind my commitment and the work ethic that my parents taught me. Accordingly, I was willing to work on my master’s thesis research into the early morning hours, excited with anticipation at reading data compilation and calculation results at 2 a.m. Moments like those have cemented my interest in becoming a biophysicist, using my energy and enthusiasm to explore the hidden rhythms of biological phenomena.
In contrast, I was born during an era when my country was undergoing enormous change. Forty years of martial law came to an end when I was five, and a new government administration included a group of technical bureaucrats who successfully pushed for public funding of basic science research and education. I benefited from those decisions in high school and as an undergraduate; unlike students in many other developing countries, I am very fortunate in having several options for my future. My decision is influenced by recent advancements in the biological sciences that include animal cloning and human genome mapping.
One of my best adolescent experiences was attending the 2001 Wu Chien-Shiung Science Camp. I had some minor surgery just before the camp, but the physical pain was not going to stop me from meeting Nobel laureates Steve Chu, Douglas Osheroff, and Yuan-Tseh Lee. You can imagine my excitement when I was selected to explain my proposed solution to HIV on a stage in front of hundreds of experienced scientists, despite not having any laboratory experience. I was very nervous, but I was also running on adrenaline from an all-night brainstorming session that I had shared with my teammates. The source of my energy that night was the spirit of exploration, and I was hooked by the experience.
Every creative mind must find its niche, and mine is in biophysics. I enjoy working with the big picture of biomolecule binding energetics, structural dynamics, and motion in living cells. However, as my undergraduate mentor Dr. Chia-Ching Chang says, “Straight A’s do not guarantee a successful research career.” I try to keep in mind my commitment and the work ethic that my parents taught me. Accordingly, I was willing to work on my master’s thesis research into the early morning hours, excited with anticipation at reading data compilation and calculation results at 2 a.m. Moments like those have cemented my interest in becoming a biophysicist, using my energy and enthusiasm to explore the hidden rhythms of biological phenomena.
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”
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