FUNCTIONAL SIGNIFICANCE OF THE ACTIN N-TERMINAL REGION
肌动蛋白 N 末端区域的功能意义
基本信息
- 批准号:3283604
- 负责人:
- 金额:$ 16.51万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1984
- 资助国家:美国
- 起止时间:1984-07-01 至 1995-03-31
- 项目状态:已结题
- 来源:
- 关键词:Saccharomyces cerevisiae acetylation actins binding proteins cell free system complementary DNA crosslink cytoskeleton enzyme mechanism gelsolin genetic manipulation laboratory mouse laboratory rabbit myosins peptidases point mutation polymerization posttranslational modifications protein purification protein sequence reticulocytes troponin
项目摘要
The overall goal of our work is to understand the nature of the
interactions controlling the association of actin with myosin and with
various proteins that modulate actin's behavior within the cell. We are
particularly interested in actin's N-terminal region, the site of an
unusual processing reaction involving the posttranslational removal of
acetyl amino acids by a specific processing enzyme. This region of the
actin also forms close contacts with proteins such as myosin, gelsolin,
fragmin, depactin, and troponin I and therefore may be important in
controlling cellular actin function. This grant addresses the importance
of three conserved elements of the 18 amino acid actin N-terminal region:
the acidic amino acids at the N-terminus of mature processed actin; the
conserved Ala-Leu-Val beginning at residue 6; and the conserved Asp-Asn-
Gly-Ser-Gly beginning at position 10 in what otherwise structurally is a
hypervariable part of the actin polypeptide. We will use a yeast
transformation system in which a centromeric plasmid carrying a mutated
intronless yeast actin gene will be introduced into a diploid strain of S.
cerevisiae containing one wild type actin gene and one inactive actin gene.
Using site-directed mutagenesis, mutations will be made in one of the three
structural elements just described or will be inserted between Met-1 and
Asp1 to prevent N-terminal processing. Haploid cells will be tested for
the ability to remain viable with the mutated actin alone. The transformed
diploid will be tested for dominant effects exerted by the mutant actin on
the cell. We will examine affected cells for changes in actin deposition
patterns by immunofluorescent staining techniques. We will also examine
the effects of the mutations on chitin deposition and invertase secretion,
two processes that are dependent on proper actin function. Peptide mapping
of the isolated mutant actin will indicate whether alterations affect
removal of the Met-i by the processing enzyme. We will isolate the mutant
actins in active form and determine the effects of the mutations on actin
polymerization, cation and nucleotide binding, and the interaction of actin
with myosin and gelsolin. These experiments will provide answers for the
role of N-terminal processing and the three conserved structural elements
of the N-terminal region both in vivo and in vitro in actin function and
will allow us to prepare large quantities of mutant actins for biochemical
analysis.
我们工作的总体目标是了解
控制肌动蛋白与肌球蛋白和
调节细胞内肌动蛋白行为的各种蛋白质。 我们
特别感兴趣的是肌动蛋白的N-末端区域,
不寻常的加工反应,涉及翻译后去除
乙酰基氨基酸通过特定的加工酶。 的这个区域
肌动蛋白还与蛋白质如肌球蛋白,凝溶胶蛋白,
因此,可能是重要的
控制细胞肌动蛋白功能。 这项拨款强调了
18个氨基酸的肌动蛋白N-末端区域的三个保守元件:
在成熟的加工过的肌动蛋白的N-末端的酸性氨基酸;
从残基6开始的保守的Ala-Leu-瓦尔;和保守的Asp-Asn-
Gly-Ser-Gly起始于位置10,否则在结构上是
肌动蛋白多肽的高变部分。 我们将使用酵母
转化系统,其中携带突变的
将无内含子酵母肌动蛋白基因导入二倍体S.
酿酒酵母含有一个野生型肌动蛋白基因和一个失活肌动蛋白基因。
使用定点诱变,将在三个基因中的一个中进行突变。
刚刚描述的或将插入Met-1和Met-2之间的结构元件,
Asp 1防止N-末端加工。 将对单倍体细胞进行检测,
单独使用突变的肌动蛋白保持活力的能力。 经变换的
二倍体将测试突变肌动蛋白对
牢房 我们将检查受影响的细胞中肌动蛋白沉积的变化
免疫荧光染色技术。 我们亦会研究
突变对几丁质沉积和转化酶分泌的影响,
这两个过程依赖于肌动蛋白的正常功能。 肽图谱
分离的突变肌动蛋白将表明是否改变影响
通过加工酶去除Met-1。 我们会隔离变种人
活性形式的肌动蛋白,并确定突变对肌动蛋白的影响
聚合,阳离子和核苷酸结合,以及肌动蛋白的相互作用
含有肌球蛋白和凝溶胶蛋白。 这些实验将为
N-末端加工和三个保守结构元件的作用
N-末端区域在体内和体外的肌动蛋白功能,
将使我们能够制备大量的突变肌动蛋白,
分析.
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Peter A. Rubenstein其他文献
Highlights of the Molecular Interactions of Actin Meeting, Hawaii, 1–5 April 1997
- DOI:
10.1023/a:1005381201156 - 发表时间:
1998-04-01 - 期刊:
- 影响因子:1.700
- 作者:
Peter A. Rubenstein;Larry S. Tobacman - 通讯作者:
Larry S. Tobacman
Enzymatic Synthesis of Cytidine Diphosphate 3,6-Dideoxyhexoses: IX. PURIFICATION AND PROPERTIES OF THE CYTIDINE DIPHOSPHATE-<span class="small-caps">d</span>-GLUCOSE PYROPHOSPHORYLASE FROM <em>PASTEURELLA PSEUDOTUBERCULOSIS</em>, TYPE V
- DOI:
10.1016/s0021-9258(19)42543-x - 发表时间:
1974-06-25 - 期刊:
- 影响因子:
- 作者:
Peter A. Rubenstein;Jack L. Strominger - 通讯作者:
Jack L. Strominger
Teaching Biochemistry to Students of Dentistry, Medicine, and Pharmacy: 7th International Conference of the Association of Biochemistry Educators (ABE) Tucson, AZ, USA, May 5–9, 2019
- DOI:
10.1007/s40670-019-00851-w - 发表时间:
2019-11-15 - 期刊:
- 影响因子:1.800
- 作者:
Susan D. Cline;Jana M. Simmons;Eric C. Niederhoffer;Danielle L. Cruthirds;Sage C. Arbor;David S. Franklin;Emine E. Abali;Robert C. Bateman;Joseph D. Fontes;Janet E. Lindsley;Peter A. Rubenstein;Karen Symes;Susan M. Viselli - 通讯作者:
Susan M. Viselli
Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells
表皮生长因子控制 BC3H1 细胞中平滑肌 α-异肌动蛋白的表达
- DOI:
- 发表时间:
1988 - 期刊:
- 影响因子:7.8
- 作者:
Yung;Peter A. Rubenstein - 通讯作者:
Peter A. Rubenstein
Enzymatic Synthesis of Cytidine Diphosphate 3,6-Dideoxyhexoses: VII. MECHANISTIC ROLES OF ENZYME E1 AND PYRIDOXAMINE 5′-PHOSPHATE IN THE FORMATION OF CYTIDINE DIPHOSPHATE-4-KETO-3,6-DIDEOXY-<span class="small-caps">d</span>-GLUCOSE FROM CYTIDINE DIPHOSPHATE-4-KETO-6-DEOXY-<span class="small-caps">d</span>-GLUCOSE
- DOI:
10.1016/s0021-9258(19)42541-6 - 发表时间:
1974-06-25 - 期刊:
- 影响因子:
- 作者:
Peter A. Rubenstein;Jack L. Strominger - 通讯作者:
Jack L. Strominger
Peter A. Rubenstein的其他文献
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{{ truncateString('Peter A. Rubenstein', 18)}}的其他基金
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
7850295 - 财政年份:2009
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical Consequences of Deafness-causing Actin Mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
7476109 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
7384548 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
8009461 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
7738925 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
7534363 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
Biochemical consequences of Deafness-causing actin mutations
导致耳聋的肌动蛋白突变的生化后果
- 批准号:
8197273 - 财政年份:2007
- 资助金额:
$ 16.51万 - 项目类别:
MUTAGENIC STUDY OF YEAST ACTIN CONFORMATIONAL CHANGES
酵母肌动蛋白构象变化的诱变研究
- 批准号:
6519149 - 财政年份:1984
- 资助金额:
$ 16.51万 - 项目类别:
A Mutagenic Study of Yeast Actin Conformational Changes
酵母肌动蛋白构象变化的诱变研究
- 批准号:
7786244 - 财政年份:1984
- 资助金额:
$ 16.51万 - 项目类别:
A Mutagenic Study of Yeast Actin Conformational Changes
酵母肌动蛋白构象变化的诱变研究
- 批准号:
7037479 - 财政年份:1984
- 资助金额:
$ 16.51万 - 项目类别:
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