Biochemical Studies of ABCE1

Date Issued:: 2012
Abstract/Description:: The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases (>)30 fold from ~30 (&)#181;M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.

Title:	Biochemical Studies of ABCE1.	36 views 24 downloads
Name(s):	Sims, Lynn, Author Igarashi, Robert, Committee Chair Ebert, Steven, Committee Member Self, William, Committee Member Moore, Sean, Committee Member , Committee Member University of Central Florida, Degree Grantor
Type of Resource:	text
Date Issued:	2012
Publisher:	University of Central Florida
Language(s):	English
Abstract/Description:	The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases (>)30 fold from ~30 (&)#181;M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.
Identifier:	CFE0004600 (IID), ucf:49204 (fedora)
Note(s):	2012-12-01 Ph.D. Sciences, Biology Doctoral This record was generated from author submitted information.
Subject(s):	ABC -- ABCE1 -- Rli1 -- ATPase -- ribosome biogenesis -- ribosome maturation -- ribosome recycling -- translation
Persistent Link to This Record:	http://purl.flvc.org/ucf/fd/CFE0004600
Restrictions on Access:	public 2012-12-15
Host Institution:	UCF

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