CHARACTERIZATION OF THE PRELAMIN A ENDOPROTEASE

前核纤层蛋白A内切蛋白酶催化哺乳动物细胞中核纤层蛋白A成熟的终末反应。 这是从核纤层蛋白A前体（prelamin A）的羧基末端内切蛋白水解去除15个氨基酸残基。 该酶需要法尼基化的羧基末端甲基化的半胱氨酰肽作为底物。 它还识别六肽，RSY产生LLG，在指示处切割该序列。一般认为法尼基化作为蛋白质与膜脂双层的锚。 然而，这些结果的prelamin A内切蛋白酶是一致的替代假设：法尼基化的行为，结合特定的氨基酸序列，介导蛋白质异源二聚体的形成。 为了进一步深入了解法尼基化蛋白质识别另一种蛋白质的机制，我们建议更详细地描述这些相互作用的机制，这些相互作用的前层蛋白A和内切蛋白酶，特别是法尼基结合结构域（具体目标2）。 这些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。 RSYLLG切割序列在Swiss-Prot数据库中的哺乳动物蛋白中是独特的。 我们将确定内切蛋白水解切割位点的底物特异性，以检验该反应对于前层蛋白A加工是独特的假设（特异性目的3）。 我们已经表明，这种酶是由表达的前层蛋白A基板和培养细胞的增殖状态。 我们建议进一步表征这些调节反应的机制，这需要酶的分子克隆和重组表达（具体目标4）。 为了实现这些目标，我们提出了以下具体目标：1）可能通过使用亲和色谱法来放大前层蛋白A内切蛋白酶的制备; 2）通过亲和标记、肽图谱和测序来表征前层蛋白A内切蛋白酶的法尼基结合结构域和活性位点; 3）通过开发更简单的测定和筛选多种肽底物来表征酶的氨基酸底物特异性; 4）前层蛋白A内切蛋白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.