CHARACTERIZATION OF THE PRELAMIN A ENDOPROTEASE

在哺乳动物细胞中，纤层蛋白A内蛋白酶催化纤层蛋白A成熟的终末反应。这是从层状蛋白A前体（前层状蛋白A）的羧基端去除15个氨基酸残基的蛋白内水解过程。酶需要一个法酰化的羧基端甲基化的半胱氨酸肽作为底物。它也识别一个六肽，RSY产生LLG，在指示的地方切割这个序列。一般认为，法尼基化作用作为一个锚蛋白到膜脂双分子层。然而，这些关于前纤层蛋白A内源性蛋白酶的结果与另一种假设是一致的：法尼化作用，与特定的氨基酸序列一起，介导蛋白质异二聚体的形成。为了进一步了解法尼基化蛋白被另一种蛋白质识别的机制，我们建议更详细地描述前纤层蛋白A与内源性蛋白酶之间相互作用的机制，特别是法尼基结合域（specific aim 2）。此类研究将需要开发更有效和更高容量的酶纯化方法（具体目标1）。我们还建议进一步深入了解这种不寻常的蛋白质加工途径的生物学意义。rsylg切割序列在Swiss-Prot数据库中的哺乳动物蛋白中是独一无二的。我们将确定内源性蛋白水解裂解位点的底物特异性，以验证该反应是pre - lamin A加工所特有的假设（特异性目的3）。我们已经证明，这种酶受前纤层蛋白A底物的表达和培养细胞的增殖状态的调节。我们建议进一步表征这些调控反应的机制，这需要对酶进行分子克隆和重组表达（specific aim 4）。为了实现这些目标，我们提出以下具体目标：1)扩大pre - lamin A内源性蛋白酶制备的规模，可能通过使用亲和层析；2)通过亲和标记、多肽作图和测序等方法，鉴定prelamin A内源性蛋白酶的法尼基结合域和活性位点；3)通过开发一种更简单的试验和筛选多种肽底物来表征酶的氨基酸底物特异性；4) prelamin A内源性蛋白酶的分子克隆与重组表达。

The prelamin A endoprotease catalyzes the terminal reaction of lamin A maturation in mammalian cells. This is an endoproteolytic removal of 15 amino acid residues from the carboxy terminus of the lamin A precursor, prelamin A. The enzyme requires a farnesylated, carboxy terminal, methylated cysteinyl peptide as a substrate. It also recognizes a hexapeptide, RSY yields LLG, cleaving this sequence where indicated. It has generally been assumed that farnesylation acts as an anchor for proteins to membrane lipid bilayers. However, these results on the prelamin A endoprotease are consistent with an alternative hypothesis: that farnesylation acts, in conjunction with specific amino acid sequences, to mediate protein heterodimer formation. To gain further insight into the mechanism of farnesylated protein recognition by another protein, we are proposing to characterize, in more detail, the mechanism of these interactions between prelamin A and the endoprotease, particularly the farnesyl binding domain(specific aim 2). Such studies will require the development of more efficient and higher capacity purification methods for the enzyme(specific aim 1). We are also proposing to gain further insight into the biological significance of this unusual protein processing pathway. The RSYLLG cleavage sequence is unique among mammalian proteins in the Swiss-Prot data base. We will determine the substrate specificity of the endoproteolytic cleavage site, to test the hypothesis that this reaction is unique to prelamin A processing (specific aim 3). We have shown that this enzyme is regulated both by expression of the prelamin A substrate and the proliferation status of cultured cells. We propose to further characterize the mechanisms of these regulatory responses, an undertaking which requires molecular cloning and recombinant expression of the enzyme(specific aim 4). To achieve these ends, we are proposing the following specific aims: 1) Scale-up of prelamin A endoprotease preparation, possibly through the use of affinity chromatography; 2) Characterization of the farnesyl binding domain and active site of the prelamin A endoprotease through affinity labeling, peptide mapping and sequencing; 3) Characterization of the amino acid substrate specificity of the enzyme through the development of a simpler assay and screening of multiple peptide substrates;4) Molecular cloning and recombinant expression of the prelamin A endoprotease.