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Author: Gregory R. Bock Publisher: John Wiley & Sons ISBN: 0470862629 Category : Science Languages : en Pages : 310
Book Description
A comprehensive review of recent work on chromatin and non-histone proteins, this book arises from the interactions of a multidisciplinary group of scientists involved in the study of acetylation. This area of research opens up new and exciting possibilities for drug design, and so the final chapters in the book examine some of the potential applications in the treatment of various diseases.
Author: Gregory R. Bock Publisher: John Wiley & Sons ISBN: 0470862629 Category : Science Languages : en Pages : 310
Book Description
A comprehensive review of recent work on chromatin and non-histone proteins, this book arises from the interactions of a multidisciplinary group of scientists involved in the study of acetylation. This area of research opens up new and exciting possibilities for drug design, and so the final chapters in the book examine some of the potential applications in the treatment of various diseases.
Author: Publisher: ISBN: Category : Languages : en Pages : 270
Book Description
Protein acetylation has emerged as a major post-translational modification and accumulating evidence indicates that lysine acetylation is a prevalent regulatory mechanism of numerous cellular processes. Mitochondrial proteins, including metabolic enzymes, are among the most highly acetylated and are dynamically altered in response to nutritional status. The mitochondrial nicotinamide adenine dinucleotide dependent deacetylase Sirtuin 3 (SIRT3) is implicated as a possible regulator of the cellular adaptation to nutrient variation and directly deacetylates and regulates the function of proteins involved in lipid metabolism, energy production, and the antioxidant response system. Mitochondrial function has been linked with aging and age-related disease. SIRT3 may play a major role in the maintenance of healthy mitochondria and act as a key molecular regulator of the lifespan benefits of caloric restriction, fasting, and exercise. SIRT3 is postulated to be the main, if only, mitochondrial protein deacetylase, but the extent of its capacity to modulate mitochondrial processes remains unclear. While numerous post-translational modifications exist in the mitochondria, much remains to be understood about the enzymes that add and remove these chemical marks, how this serves to regulate protein function, and how these modifications vary in response to both acute and chronic alterations in nutrient availability. Employing biochemistry and systems biology to test defined hypotheses will allow for exploration of a number of unexplored and unknown questions in the field of mitochondrial biology and will further elucidate the role of mitochondria in aging and disease. This thesis describes the characterization of physiological, site-specific alterations in lysine acetylation in the mitochondria. Using mouse models, quantitative proteomics, detailed biochemistry, and cell culture systems this work stands to elucidate mechanistic details of the regulatory role of protein acetylation.
Author: Alexis Jo Lawton (Ph.D.) Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Reversible protein acetylation is a widespread, regulatory modification that was first discovered on histone proteins, but since has been described throughout the cell in major subcellular compartments. Several functional roles of lysine acetylation have been described including regulating enzymatic activity, protein-protein interactions, protein-DNA interactions, protein stability, and subcellular localization. With the improvements in mass spectrometry technologies, the number of identified acetylated sites has grown exponentially creating a new challenge to determine which sites are functional versus spurious. Furthermore, studies investigating the mechanisms regulating protein acetylation have primarily focused on histone acetylation, but how they may regulate non-histone protein acetylation has not been fully understood. Of importance, one of the main mechanisms through which acetylation may be regulated is through changes in the availability of acetyl-CoA. Since most acetyltransferases, including p300/CBP, have a Km near the estimated cellular concentrations of acetyl-CoA, manipulating concentrations of the substrate through activation of its production can have major effects on the levels of acetylation. Several studies have investigated the effect of knockdowns of acetyl-CoA generating enzymes (such as ACLY or ACSS2), however, few studies have measured the effects on non-histone protein acetylation. My research uses two main approaches to respond to these challenges in the field: 1) a robust, mass spectrometry method to quantify acetylation stoichiometry across the proteome, and 2) investigating dynamics of acetylation in response to changes in acetyl-CoA availability. Understanding both stoichiometry and dynamic responses during cellular stimulation are key features of interrogating the role of protein acetylation at a site- and protein-specific level. My research aims to understand the cellular mechanisms that are controlling dynamic acetylation in response to metabolic changes in acetyl Coenzyme A (acetyl-CoA) availability and production.Here, I examine the dynamics of global protein acetylation and the mechanisms regulating growth-factor stimulated dynamics, with a particular focus on how metabolism and the production and availability of acetyl-CoA is a major regulator of protein acetylation. Chapter 1 introduces protein acetylation and discusses the field's current understanding of acetylation functions, the quantitative techniques for investigating global non-histone protein acetylation, and the mechanisms involved in regulating protein acetylation. Chapter 2 presents an updated method for quantifying lysine acetylation stoichiometry and applies this method to identify serum-stimulated protein acetylation dynamics. Chapter 3 presents an in-progress project that examines the key regulatory nodes controlling growth-factor stimulated acetylation, including the kinase AKT, acetyl-CoA production through ACLY, and citrate export by SLC25A1. Chapter 4 presents the ongoing experiments and future directions for these projects. My thesis work has also allowed me to be a part of some critical collaborations that have investigated the connection between metabolism, acetyl-CoA availability, and protein acetylation in several interesting mouse models. My contributions and our key findings are presented in the Appendixes of this thesis. Appendix 1 investigates the effect of the dysregulation of the ER-localized acetyl-CoA transporter, AT-1, on protein acetylation. Appendix 2 investigates the effect of increased flux of citrate into the cytoplasm and nucleus through overexpression models of the extracellular membrane citrate transporter, SLC13A5, and the mitochondrial citrate transporter, SLC25A1, and their effect on protein acetylation. Appendix 3 investigates the effect of age, diet, and the loss of the mitochondrial localized deacetylase, SIRT3, on mitochondrial protein acetylation. Lastly, Appendix 4 examines the effects of the loss of individual ER-localized acetyltransferases, ATase1 and ATase2, on secretory pathway protein acetylation.
Author: Alexis Jo Lawton (Ph.D.) Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Reversible protein acetylation is a widespread, regulatory modification that was first discovered on histone proteins, but since has been described throughout the cell in major subcellular compartments. Several functional roles of lysine acetylation have been described including regulating enzymatic activity, protein-protein interactions, protein-DNA interactions, protein stability, and subcellular localization. With the improvements in mass spectrometry technologies, the number of identified acetylated sites has grown exponentially creating a new challenge to determine which sites are functional versus spurious. Furthermore, studies investigating the mechanisms regulating protein acetylation have primarily focused on histone acetylation, but how they may regulate non-histone protein acetylation has not been fully understood. Of importance, one of the main mechanisms through which acetylation may be regulated is through changes in the availability of acetyl-CoA. Since most acetyltransferases, including p300/CBP, have a Km near the estimated cellular concentrations of acetyl-CoA, manipulating concentrations of the substrate through activation of its production can have major effects on the levels of acetylation. Several studies have investigated the effect of knockdowns of acetyl-CoA generating enzymes (such as ACLY or ACSS2), however, few studies have measured the effects on non-histone protein acetylation. My research uses two main approaches to respond to these challenges in the field: 1) a robust, mass spectrometry method to quantify acetylation stoichiometry across the proteome, and 2) investigating dynamics of acetylation in response to changes in acetyl-CoA availability. Understanding both stoichiometry and dynamic responses during cellular stimulation are key features of interrogating the role of protein acetylation at a site- and protein-specific level. My research aims to understand the cellular mechanisms that are controlling dynamic acetylation in response to metabolic changes in acetyl Coenzyme A (acetyl-CoA) availability and production.Here, I examine the dynamics of global protein acetylation and the mechanisms regulating growth-factor stimulated dynamics, with a particular focus on how metabolism and the production and availability of acetyl-CoA is a major regulator of protein acetylation. Chapter 1 introduces protein acetylation and discusses the field's current understanding of acetylation functions, the quantitative techniques for investigating global non-histone protein acetylation, and the mechanisms involved in regulating protein acetylation. Chapter 2 presents an updated method for quantifying lysine acetylation stoichiometry and applies this method to identify serum-stimulated protein acetylation dynamics. Chapter 3 presents an in-progress project that examines the key regulatory nodes controlling growth-factor stimulated acetylation, including the kinase AKT, acetyl-CoA production through ACLY, and citrate export by SLC25A1. Chapter 4 presents the ongoing experiments and future directions for these projects. My thesis work has also allowed me to be a part of some critical collaborations that have investigated the connection between metabolism, acetyl-CoA availability, and protein acetylation in several interesting mouse models. My contributions and our key findings are presented in the Appendixes of this thesis. Appendix 1 investigates the effect of the dysregulation of the ER-localized acetyl-CoA transporter, AT-1, on protein acetylation. Appendix 2 investigates the effect of increased flux of citrate into the cytoplasm and nucleus through overexpression models of the extracellular membrane citrate transporter, SLC13A5, and the mitochondrial citrate transporter, SLC25A1, and their effect on protein acetylation. Appendix 3 investigates the effect of age, diet, and the loss of the mitochondrial localized deacetylase, SIRT3, on mitochondrial protein acetylation. Lastly, Appendix 4 examines the effects of the loss of individual ER-localized acetyltransferases, ATase1 and ATase2, on secretory pathway protein acetylation.
Author: Josue Baeza Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Reversible N-[epsilon]-acetylation has emerged as a widespread and regulatory modification throughout the cell. First identified on N-terminal histone tails over 50 years ago, acetylation has now been identified on over 36,000 lysine sites on organisms ranging from bacteria to mammals, highlighting the strong, conserved role for this modification. Acetylation is controlled by the opposing action of lysine acetyltransferases and deacetylases and recent evidence supports a role for nonenzymatic acetylation. Lysine acetylation influences many biochemical processes including: protein-protein and protein-DNA interactions, stability, cellular localization and enzymatic activity, which is thought to arise from neutralization of the positive charge on lysine residues at neutral pH. In mitochondria, acetylation generally acts as an inhibitory modification, altering protein interactions, lowered enzymatic activity and mislocalization of mitochondrial proteins. In this case, acetylation acts as a rheostat, modulating the degree of a biochemical process. Therefore, knowing the fraction of the protein that is modified, or stoichiometry, would provide critical information for understanding the regulatory effects of lysine acetylation. The mechanism of mitochondrial protein acetylation remains unknown. While there is a wealth of data supporting a dynamic and regulatory role for acetylation within mitochondria, there is very little evidence for an enzyme-catalyzed reaction. However, the conditions of the mitochondrial matrix: elevated acetyl-CoA levels and alkaline pH, raise the possibility that lysine acetylation can occur nonenzymatically. Throughout my thesis research, I have utilized chemical, biochemical, proteomic, and cellular studies to characterize protein acetylation. Specifically, I developed a mass spectrometry based approach to directly quantify lysine acetylation stoichiometry at the proteome-wide scale. Using this newly developed method, I measured the second order rate constants of nonenzymatic lysine acetylation using mitochondrial and non-mitochondrial proteins, highlighting the wide range of reactivities. The characterization of nonenzymatic rates of native protein demonstrates that the uncatalyzed reaction can account for the levels of acetylation in vivo. I also provide evidence that the majority of mitochondrial acetylation occurs within the mitochondria. Chapter 1 introduces lysine acetylation, the various processes controlling cellular levels including enzymatic and nonenzymatic, and why quantifying acetylation stoichiometry is needed. Chapter 2 discusses the mass spectrometry based method used for quantifying stoichiometry and its application in the model system, Escherichia coli. In chapter 3, I quantify the rates of nonenzymatic acetylation using a panel of mitochondrial and nonmitochondrial proteins, which highlights, for the first time, the wide range of lysine reactivities on native proteins. I then utilize an optimized version of the stoichiometry method to query mammalian lysine acetylation stoichiometry, revealing the wide distribution of acetylation across the cell. Conclusions, ongoing work, and future directions are presented in Chapter 5.
Author: Sumana Venkat Publisher: ISBN: Category : Languages : en Pages : 376
Book Description
The tricarboxylic acid (TCA) cycle is a central route for the energy production via oxidative phosphorylation and is ubiquitous throughout nature. Apart from playing a major role in energy provision, it is also essential for a wide range of physiological functions in any organism. Although an increasing emphasis is laid on the interlinking role of the cycle, its regulation and control are less very less understood. As the field progresses, the consequences of TCA cycle anomalies are shown to be tied to various cancers and other disorders. The matter becomes more complex when considering the varying functions of the isozymes present in the cycle. The repertoire of twenty amino acids that is present in almost all organisms makes up for the hundreds of proteins responsible for the basic functioning of an organism. In order to synthesize a wide spectrum of viable proteins, one of the methods that the cells utilize is post-translational modifications. These modifications can be reversible depending on the conditions and they render conformational changes to the protein, thereby influencing its enzyme activity or labeling them for transportation or degradation. One such post-translational modification is acetylation, which is the main focus of this project. Acetylation is predominantly seen across all domains of life, and in spite of the prevalence of this modification, it is only recently that with the advances in technology the basic role of acetylation is being understood. However, there are still many questions that need to be answered to recognize the complete role of protein acetylation. Recently, a newer technique has been adopted to study the impact of post-translational modifications. The genetic code expansion strategy helps in the incorporation of a noncanonical amino acid into a distinct position, thereby giving rise to a customized protein. It uses stop codon suppression to insert the unnatural amino acid. The pyrrolysyl-tRNA synthetase and its cognate tRNA were used in this project to create TCA cycle enzymes with acetyllysine. In this dissertation, I examine the changes caused in the enzymes, malate dehydrogenase, isocitrate dehydrogenase, and citrate synthase, of the TCA cycle in response to acetylation of specific lysine residues and deduce the potential mechanisms regulating these changes. In doing so in E. coli, a greater understanding of protein acetylation is achieved and its implications on modulating functions are examined.
Author: Yiming Qin (Ph.D.) Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
A key challenge in biology is to understand how organisms adjust their cellular machinery in response to environmental cues, and how that affects metabolic operation and leads to different physiological and pathological manifestations. One way this may occur is through reversible lysine acetylation, which has been recognized as a key regulatory mechanism for protein function across diverse cellular processes. Lysine acetylation is catalyzed by lysine acetyltransferases or non-enzymatically, whereas lysine deacetylation is carried out by lysine deacetylases. The regulation of both acetylation and deacetylation are intimately intertwined with metabolism that allows cells to sense the internal and external environment and modulate their activities accordingly. Thus, the understanding of lysine acetylation provides critical insights into the cellular adaptation under the etiological process and opens doors to the development of novel therapeutic strategies. In this dissertation, I present two independent studies, one involving the deacetylase SIRT3 and one involving the acetyltransferase P300, to examine the metabolic conditions that modulate cellular acetylation and functional consequences of altered acetylation. Chapter 1 introduces protein acetylation, describes the chemistry of (de)acetylation reactions, and provides a detailed discussion about two regulatory enzymes, SIRT3 and P300, in terms of their regulation and function. Chapter 2 together with Chapter 3 discuss a series of discoveries about the role of SIRT3 in calorie-restricted (CR)-mediated longevity, mitochondrial health, and aerobic fitness. Chapter 2 covers the quantitative evaluation of mitochondrial acetylome and functional assessment of mitochondrial and whole-body oxidative capacity in response to the loss of SIRT3, calorie restriction, and aging. Chapter 3 further describes the metabolic consequences associated with loss of SIRT3, including an unexpected lifespan extension in SIRT3 knockout (Sirt3-/-) CR mice compared to their wild type (WT) CR counterparts, and proposes a working model that connects longevity with prolonged fasting state of Sirt3-/- CR mice. Chapter 4 switches gears to discuss P300 autoacetylation and offers a rapid, high yield, low-cost purification protocol for the hypoacetylated P300 histone acetyltransferase (HAT) domain for the ongoing project that investigates the kinetics of short-chain fatty acid induced P300 activation. Lastly, Chapter 5 concludes this dissertation, and provides future directions and preliminary results for both SIRT3 and P300-related acetylation regulation and metabolic significance.
Author: Gregory R. Bock
Author: Gregory R. Bock
Author: Andrew Rouble
Author: 
Author: Alexis Jo Lawton (Ph.D.)
Author: Alexis Jo Lawton (Ph.D.)
Author: Eric Verdin
Author: Josue Baeza
Author: Sumana Venkat
Author: Yiming Qin (Ph.D.)
Author: