Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY 40536, USA; Department of Neuroscience, University of Kentucky, Lexington, KY 40536, USA.
Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY 40536, USA.
Biochim Biophys Acta Mol Cell Res. 2022 Sep;1869(9):119298. doi: 10.1016/j.bbamcr.2022.119298. Epub 2022 May 26.
Calpain-5 (CAPN5) is a member of the calpain family of calcium-activated neutral thiol proteases. CAPN5 is partly membrane associated, despite its lack of a transmembrane domain. Unlike classical calpains, CAPN5 contains a C-terminal C2 domain. C2 domains often have affinity to lipids, mediating membrane association. We recently reported that the C2 domain of CAPN5 was essential for its membrane association and the activation of its autolytic activity. However, despite the removal of the C2 domain by autolysis, the N-terminal fragment of CAPN5 remained membrane associated. S-acylation, also referred to as S-palmitoylation, is a reversible post-translational lipid modification of cysteine residues that promotes membrane association of soluble proteins. In the present study several S-acylated cysteine residues were identified in CAPN5 with the acyl-PEG exchange method. Data reported here demonstrate that CAPN5 is S-acylated on up to three cysteine residues including Cys-4 and Cys-512, and likely Cys-507. The D589N mutation in a potential calcium binding loop within the C2 domain interfered with the S-acylation of CAPN5, likely preventing initial membrane association. Mutating specific cysteine residues of CAPN5 interfered with both its membrane association and the activation of CAPN5 autolysis. Taken together, our results suggest that the S-acylation of CAPN5 is critical for its membrane localization which appears to favor its enzymatic activity.
钙蛋白酶-5（CAPN5）是钙激活中性丝氨酸蛋白酶家族的成员。尽管 CAPN5 缺乏跨膜结构域，但它部分与膜结合。与经典的钙蛋白酶不同，CAPN5 含有一个 C 末端 C2 结构域。C2 结构域通常与脂质具有亲和力，介导膜结合。我们最近报道，CAPN5 的 C2 结构域对于其与膜的结合以及其自身水解活性的激活是必不可少的。然而，尽管 C2 结构域通过自身水解被去除，CAPN5 的 N 末端片段仍与膜结合。S-酰化，也称为 S-棕榈酰化，是半胱氨酸残基的一种可逆的翻译后脂质修饰，可促进可溶性蛋白与膜的结合。在本研究中，我们使用酰基-PEG 交换法在 CAPN5 中鉴定了几个 S-酰化的半胱氨酸残基。这里报道的数据表明，CAPN5 可以在多达三个半胱氨酸残基（包括 Cys-4 和 Cys-512，可能还有 Cys-507）上发生 S-酰化。C2 结构域中一个潜在钙结合环中的 D589N 突变干扰了 CAPN5 的 S-酰化，可能阻止了初始的膜结合。CAPN5 特定半胱氨酸残基的突变干扰了其与膜的结合及其自身水解活性的激活。总之，我们的结果表明，CAPN5 的 S-酰化对于其膜定位至关重要，这似乎有利于其酶活性。
