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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: 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: 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: John McMurry Publisher: Roberts and Company Publishers ISBN: 9780974707716 Category : Medical Languages : en Pages : 524
Book Description
Intended for advanced undergraduates and graduate students in all areas of biochemistry, The Organic Chemistry of Biological Pathways provides an accurate treatment of the major biochemical pathways from the perspective of mechanistic organic chemistry.
Author: Zeljko Simic Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
Mitochondria represent cellular organelles with a high level of acetylated proteins in comparison to other cellular compartments but mitochondrial residential lysine acetyltransferases have not been recognized. Here we show that acetylation, as well as other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of short peptide substrate derived from carbamoyl phosphate synthetase 1, dicarboxylic acyl groups show highest level of reactivity. Reactivities of the CoA-thioesters decrease with extending the acyl chain. These results were confirmed by using the protein substrate cyclophilin A. Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl group from cyclophilinA, whereas SIRT5 is an efficient protein desuccinylase. Considering that we have not found convincing evidence about existence of mitochondrial lysine acetyltransferase so far, it leads to conclusion that acylation landscape on the lysine residues might largely depend on the enzymatic activities of sirtuins and availability and/or reactivity of certain acyl-CoA thioesters.
Author: Zeljko Simic Publisher: ISBN: Category : Languages : en Pages :
Book Description
Mitochondria represent cellular organelles with a high level of acetylated proteins in comparison to other cellular compartments but mitochondrial residential lysine acetyltransferases have not been recognized. Here we show that acetylation, as well as other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of short peptide substrate derived from carbamoyl phosphate synthetase 1, dicarboxylic acyl groups show highest level of reactivity. Reactivities of the CoA-thioesters decrease with extending the acyl chain. These results were confirmed by using the protein substrate cyclophilin A. Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl group from cyclophilinA, whereas SIRT5 is an efficient protein desuccinylase. Considering that we have not found convincing evidence about existence of mitochondrial lysine acetyltransferase so far, it leads to conclusion that acylation landscape on the lysine residues might largely depend on the enzymatic activities of sirtuins and availability and/or reactivity of certain acyl-CoA thioesters.
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: Gregory Randall Wagner Publisher: ISBN: Category : Acetylation Languages : en Pages : 252
Book Description
Friedreich's Ataxia (FRDA) is a rare and poorly understood autosomal recessive disease caused by a pathological deficiency of the mitochondrial protein frataxin. Patients suffer neurodegeneration, ataxia, diabetes, and heart failure. In an effort to understand the mechanisms of heart failure in FRDA, we investigated the role of the protein modification acetylation, which is highly abundant on mitochondrial proteins and has been implicated in regulating intermediary metabolism. Using mouse models of FRDA, we found that cardiac frataxin deficiency causes progressive hyperacetylation of mitochondrial proteins which is correlated with loss of respiratory chain subunits and an altered mitochondrial redox state. Mitochondrial protein hyperacetylation could be reversed by the mitochondria-localized deacetylase SIRT3 in vitro, suggesting a defect in endogenous SIRT3 activity. Consistently, frataxin-deficient cardiac mitochondria showed significantly decreased rates of fatty acid oxidation and complete oxidation to carbon dioxide. However, the degree of protein hyperacetylation in FRDA could not be fully explained by SIRT3 loss. Our data suggested that intermediary metabolites and perhaps acetyl-CoA, which is required for protein acetylation, are accumulating in frataxin-deficient mitochondria. Upon testing the hypothesis that mitochondrial protein acetylation is non-enzymatic, we found that the minimal chemical conditions of the mitochondrial matrix are sufficient to cause widespread non-enzymatic protein acetylation in vitro. These data suggest that mitochondrial protein hyperacetylation in FRDA cardiomyopathy mediates progressive post-translational suppression of mitochondrial oxidative pathways which is caused by a combination of SIRT3 deficiency and, likely, an accumulation of unoxidized acetyl-CoA capable of initiating non-enzymatic protein acetylation. These findings provide novel insight into the mechanisms underlying a poorly understood and fatal cardiomyopathy and highlight a fundamental biochemical mechanism that had been previously overlooked in biological systems.
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: F.U. Hartl Publisher: Elsevier ISBN: 0080877001 Category : Science Languages : en Pages : 377
Book Description
A large number of newly-synthesized polypeptides must cross one or several intracellular membranes to reach their functional locations in the eukaryotic cell. The mechanisms of protein trafficking, in particular the post-translational targeting and membrane translocation of proteins, are of fundamental biological importance and are the focus of intensive research world-wide. For more than 15 years, mitochondria have served as the paradigm organelle system to study these processes. Although key questions, such as how precisely proteins cross a membrane, still remain to be answered, exciting progress has been made in understanding the basic pathways of protein import into mitochondria and the components involved. In addition to a fascinating richness and complexity in detail, the analysis of mitochondrial protein import has revealed mechanistic principles of general significance: Major discoveries include the demonstration of the requirement of an unfolded state for translocation and of the essential role of molecular chaperones on both sides of the membranes in maintaining a translocation-competent conformation and in protein folding after import. It is becoming clear how a polypeptide chain is "reeled" across the membrane in an ATP-dependent process by the functional cooperation of membrane proteins, presumably constituting part of a transmembrane channel, with peripheral components at the trans-side of the membrane.In this volume, eminent experts in the field take the time to review the central aspects of mitochondrial biogenesis. The logical order of the 16 chapters is determined by the sequence of steps during protein import, starting with the events taking place in the cytosol, followed by the recognition of targeting signals, the translocation of precursor proteins across the outer and inner membranes, their proteolytic processing and intramitochondrial sorting, and finally their folding and oligomeric assembly. In addition, the mechanisms involved in the export of mitochondrially encoded proteins as well as recent advances in understanding the division and inheritance of mitochondria will be discussed.
Author: Josue Baeza
Author: Josue Baeza
Author: 
Author: Raviprasadh Rajasekaran
Author: John McMurry
Author: Zeljko Simic
Author: Zeljko Simic
Author: Alexis Jo Lawton (Ph.D.)
Author: Gregory Randall Wagner
Author: Alexis Jo Lawton (Ph.D.)
Author: F.U. Hartl