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Biochemical and pharmacological char...

Varner, Amanda Susan.

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Biochemical and pharmacological characterization of protein palmitoylation.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Biochemical and pharmacological characterization of protein palmitoylation./
作者:	Varner, Amanda Susan.
面頁冊數:	188 p.
附註:	Source: Dissertation Abstracts International, Volume: 64-07, Section: B, page: 3211.
Contained By:	Dissertation Abstracts International64-07B.
標題:	Health Sciences, Pharmacology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3097060

Biochemical and pharmacological characterization of protein palmitoylation.
Varner, Amanda Susan.

Biochemical and pharmacological characterization of protein palmitoylation. - 188 p.

Source: Dissertation Abstracts International, Volume: 64-07, Section: B, page: 3211.

Thesis (Ph.D.)--The Pennsylvania State University, 2003.

Protein palmitoylation is a post-translational modification in which 16-carbon palmitate groups are attached to cysteine residues of cellular proteins via thioester bonds. This lipid modification is necessary for plasma membrane targeting of several important signaling proteins, including Ras proteins and certain Src-related tyrosine kinases. Although protein palmitoylation is essential for plasma membrane localization and, thus, function of such proteins, the mechanism of protein palmitoylation is unknown since the enzymes responsible for this modification remain unidentified. To study palmitoyl acyl transferase (PAT) activity towards distinct protein substrates, we developed an HPLC-based in vitro palmitoylation (IVP) assay with fluorescence detection using two NBD-labeled substrate peptides mimicking either the N-terminal palmitoylation motif of proteins such as Src-related tyrosine kinases or the C-terminal palmitoylation motif of proteins such as H-/N-Ras. Enzymatic palmitoylation of both peptides by PAT was differentially affected by treatment with N-ethylmaleimide and by thermal denaturation. Furthermore, MyrGS(tBu)-OMe, a peptidomimetic of the MyrGCK(NBD) peptide substrate, caused selective inhibition of enzymatic FarnCNRas(NBD) palmitoylation with no inhibitory effect on MyrGCK(NBD) palmitoylation. Overall, these data demonstrate that the enzymatic palmitoylation of farnesyl- or myristoyl-containing substrates can be differentially inhibited under enzymatically challenging conditions. Therefore, differential PAT activities and, perhaps, multiple PAT enzymes exist for the palmitoylation of proteins containing specific palmitoylation motifs.Subjects--Topical Terms:

1017717
Health Sciences, Pharmacology.

Biochemical and pharmacological characterization of protein palmitoylation.
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100 1 $a Varner, Amanda Susan. $3 1948411
245 1 0 $a Biochemical and pharmacological characterization of protein palmitoylation.
300 $a 188 p.
500 $a Source: Dissertation Abstracts International, Volume: 64-07, Section: B, page: 3211.
500 $a Adviser: Charles D. Smith.
502 $a Thesis (Ph.D.)--The Pennsylvania State University, 2003.
520 $a Protein palmitoylation is a post-translational modification in which 16-carbon palmitate groups are attached to cysteine residues of cellular proteins via thioester bonds. This lipid modification is necessary for plasma membrane targeting of several important signaling proteins, including Ras proteins and certain Src-related tyrosine kinases. Although protein palmitoylation is essential for plasma membrane localization and, thus, function of such proteins, the mechanism of protein palmitoylation is unknown since the enzymes responsible for this modification remain unidentified. To study palmitoyl acyl transferase (PAT) activity towards distinct protein substrates, we developed an HPLC-based in vitro palmitoylation (IVP) assay with fluorescence detection using two NBD-labeled substrate peptides mimicking either the N-terminal palmitoylation motif of proteins such as Src-related tyrosine kinases or the C-terminal palmitoylation motif of proteins such as H-/N-Ras. Enzymatic palmitoylation of both peptides by PAT was differentially affected by treatment with N-ethylmaleimide and by thermal denaturation. Furthermore, MyrGS(tBu)-OMe, a peptidomimetic of the MyrGCK(NBD) peptide substrate, caused selective inhibition of enzymatic FarnCNRas(NBD) palmitoylation with no inhibitory effect on MyrGCK(NBD) palmitoylation. Overall, these data demonstrate that the enzymatic palmitoylation of farnesyl- or myristoyl-containing substrates can be differentially inhibited under enzymatically challenging conditions. Therefore, differential PAT activities and, perhaps, multiple PAT enzymes exist for the palmitoylation of proteins containing specific palmitoylation motifs.
520 $a Using the IVP assay, we demonstrated that a synthetic analog of cerulenin, termed 16C, inhibits palmitoylation of proteins with different palmitoylation motifs. Therefore, we used 16C to study the biological consequences of palmitoylation inhibition in intact cells. Both cerulenin and 16C are shown to cause dose-dependent inhibitions of overall protein palmitoylation in NIH/3T3 fibroblasts, which subsequently affects critical signaling pathways and, therefore, cellular function in these cells. Specifically, cerulenin and 16C inhibit signaling through the cell proliferative MAPK and cell survival PI3K pathways. Additionally, cerulenin and 16C induced apoptosis, reduced the fraction of cells in the S phase of the cell cycle, and inhibited fibroblast proliferation in response to fetal bovine serum, bFGF, and PDGF. Therefore, inhibition of protein palmitoylation markedly affects signal transduction through multiple pathways, ultimately resulting in apoptosis, inhibited cell proliferation, and inhibited cell cycle progression.
590 $a School code: 0176.
650 4 $a Health Sciences, Pharmacology. $3 1017717
650 4 $a Chemistry, Biochemistry. $3 1017722
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710 2 0 $a The Pennsylvania State University. $3 699896
773 0 $t Dissertation Abstracts International $g 64-07B.
790 1 0 $a Smith, Charles D., $e advisor
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791 $a Ph.D.
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