CHARACTERIZATION OF THE PRELAMIN A ENDOPROTEASE

前层蛋白A内切酶在哺乳动物细胞中催化层蛋白A成熟的终末反应。这是从层蛋白A前体前层素A的羧基末端通过内切酶去除15个氨基酸残基的过程。该酶需要一个法尼化、羧基末端、甲基化的半胱氨酸肽作为底物。它还识别一个六肽，RSY产生LLG，在指定的位置切割这个序列。一般认为法尼化作用是蛋白质与膜脂双层之间的锚定。然而，这些关于Prelamin A内切酶的结果与另一个假说是一致的：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。为了进一步了解另一种蛋白质识别法尼化蛋白质的机制，我们建议更详细地表征前层蛋白A和内切酶之间的这些相互作用的机制，特别是法尼基结合结构域(特定目标2)。这样的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)。我们还提议进一步深入了解这一不寻常的蛋白质加工途径的生物学意义。RSYLLG裂解序列在瑞士PROT数据库中的哺乳动物蛋白质中是唯一的。我们将确定内蛋白降解裂解位点的底物特异性，以检验该反应是Prelamin A加工所特有的假设(特定目标3)。我们已经证明，该酶既受Prelamin A底物的表达调控，也受培养细胞的增殖状态调节。我们建议进一步确定这些调节反应的机制，这项工作需要对酶进行分子克隆和重组表达(特定目标4)。为了达到这些目的，我们提出了以下具体目标：1)扩大Prelamin A内切酶制备的规模，可能通过使用亲和层析；2)通过亲和标记、肽图谱和测序鉴定Prelamin A内切酶的法尼基结合结构域和活性部位；3)通过建立一种更简单的方法和筛选多肽底物来表征该酶的氨基酸底物专一性；4)Prelamin A内切酶的分子克隆和重组表达。

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.