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Regulation of protein activity by reversible acetylation

An acetylation and activity technology, applied in the field of mitochondrial matrix proteins, can solve the unstudied problems such as the precise function and regulation of AceCS2

Inactive Publication Date: 2009-07-22
THE J DAVID GLADSTONE INST A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J DAVID GLADS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Fujino et al. describe the induction of AceCS2 under certain circumstances, they did not investigate the precise function and regulation of AceCS2 (Fujino et al., 2001, J Biol Chem 276:11420-11426)

Method used

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  • Regulation of protein activity by reversible acetylation
  • Regulation of protein activity by reversible acetylation
  • Regulation of protein activity by reversible acetylation

Examples

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preparation example Construction

[0234] Preparation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, eg, US Patent No. 5,010,175; Furka, 1991, Int J PeptProt Res 37:487-493 (1991) and Houghton et al., 1991, Nature 354:84-88). Other chemistries can also be utilized to generate chemical diversity libraries. These chemicals include, but are not limited to: peptidomimetics (eg, PCT Publication No. WO 91 / 19735), encoded peptides (eg, PCT Publication No. WO 93 / 20242), random biooligomers (eg, PCT Publication No. WO 92 / 00091 ), benzodiazepines Classes (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc Natl Acad Sci USA90:6909-6913), alkene polypeptides (Hagihara et al., 1992, J Amer Chem Soc 114:6568), non-peptidic peptide mimetics containing glucose scaffolds ( Hirschmann et al., 1992, J Amer Chem Soc 11...

Embodiment 1

[0385] Example 1: general method

[0386] A. Cell culture and transfection

[0387] 37°C, 5% CO 2 HEK293, COS-1 and HeLa cells were cultured and grown in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Calcium phosphate transfection was used to transfect HEK293 cells (Chen and Okayama, 1987, MolCell Biol 7:2745-52). Hela and Cos-1 cells were transfected with Fugene from Roche. Stable expression of AceCS2 was generated by selection in complete DME growth medium containing 800 μg / mL Geneticin (Invitrogen). 标志 , SIRT3 标志 、SIRT3-H248Y 标志 or contain empty 标志 - Control vector pcDNA 标志 HEK293 cell line.

[0388] B. Plasmids and mutagenesis

[0389] All expression constructs were generated using standard PCR-based cloning protocols and verified by DNA sequencing. PCR primer 5′-CG GAATTC CATGGCGGCGCGCACCCTGGGC-3' (SEQ ID NO: 15) and 5'-CG GAATTC CTTAGCAGCAGCCTGCTTGTCCTTGC-3' (SEQ ID NO: 16) (each containing an EcoRI rest...

Embodiment 2

[0424] Example 2: Identification of acetyl-CoA synthetase (AceCS2) as a cellular target of SIRT3

[0425] As a protocol to identify lysine acetylated mitochondrial proteins targeted by SIRT3, 4, or 5, the sequence was studied in relation to the acetylated lysine residues found in Salmonella enterica acetyl-CoA synthetase (ACS). Human proteins with similar domain sequences. The acetyl-lysine-containing region of Salmonella enterica ACS was chosen because it is a known substrate of the Sir2-like protein CobB (Starai et al., 2003, Genetics 163:545-55; Starai et al., 2002, Science 298:2390- 2). In the second step, the mitochondrial localization probabilities of human proteins showing high similarity were analyzed using MITOPROT II and PREDOTAR version 1.03 subcellular prediction software (Small et al., 2004, Proteomics 4:1581-90; Claros and Vincens, 1996, Eur J Biochem 241:779-86). This study obtained a human acetyl-CoA synthetase protein with a high probability of localizati...

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Abstract

This invention discloses the first cellular acetylated substrate protein of SIRT3, Acetyl-CoA synthetase 2 (AceCS2), which is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at lysine 642 (Lys642) in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2. Thus, a mammalian sirtuin directly controls the activity of a metabolic enzyme via reversible lysine acetylation. Modulators of the acetylation status or the activity of AceCS2 are useful for the treatment of pathological conditions, such as type II diabetes, hypercholesterolemia, hyperlipidemia, and obesity.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 60 / 813,275, filed June 12, 2006, the contents of which are incorporated herein by reference in their entirety. field of invention [0003] The present invention discloses the first cellular acetylation substrate protein of SIRT3, acetyl-CoA synthetase 2 (AceCS2), which is a mitochondrial matrix protein. The mitochondrial Sir2-like protein (sirtuin) SIRT3 interacts with AceCS2 and directly controls the activity of this metabolic enzyme through reversible lysine acetylation. Regulators of the acetylation status or activity of AceCS2 can be used in the treatment of pathological conditions such as type II diabetes, hypercholesterolemia, hyperlipidemia, and obesity. Background of the invention [0004] Reversible lysine acetylation is a highly regulated post-translational protein modification controlled by protein deacetylases and acetyltransferases ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00
Inventor B·施韦尔E·韦丁
Owner THE J DAVID GLADSTONE INST A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J DAVID GLADS
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