Tang Meng, Xia Ying, Xiao Taoran, Cao Ruiyu, Cao Yu, Ouyang Bo
State Key Laboratory of Molecular Biology, Centre for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Polymers (Basel). 2022 Jul 26;14(15):3013. doi: 10.3390/polym14153013.
DHHC3 belongs to a family of DHHC palmitoyltransferase, which catalyzes the S-palmitoylation of target proteins by attaching a fatty acyl group to a cysteine. Recently, DHHC3 has been demonstrated to be a promising antitumor target in cancer therapeutics. However, the detailed structure and catalysis mechanism of DHHC3 remain elusive, considering its sequence diversity from the DHHC homologues with known crystal structures. Here, we described the expression and purification of human DHHC3 (hDHHC3) and truncated hDHHC3 with the flexible N-terminal domain (NTD) removed. Purified hDHHC3 proteins were used under various conditions for protein crystallization. LAMTOR1, one of the interacting proteins of hDHHC3 to facilitate the crystallization, was further identified by mass spectrometry and co-immunoprecipitation assay. The structural exploration using cryogenic electronic microscopy (cryo-EM) on the inactive hDHHS3 mutant showed a typical sideview of membrane proteins. These results provide a preliminary guidance for the structural determination of DHHC3.
DHHC3属于DHHC棕榈酰转移酶家族，该家族通过将脂肪酰基连接到半胱氨酸上来催化靶蛋白的S-棕榈酰化。最近，DHHC3已被证明是癌症治疗中一个有前景的抗肿瘤靶点。然而，考虑到其与已知晶体结构的DHHC同源物的序列差异，DHHC3的详细结构和催化机制仍然不清楚。在这里，我们描述了人DHHC3（hDHHC3）以及去除了柔性N端结构域（NTD）的截短型hDHHC3的表达和纯化。纯化后的hDHHC3蛋白在各种条件下用于蛋白质结晶。通过质谱和免疫共沉淀分析进一步鉴定了LAMTOR1，它是hDHHC3的相互作用蛋白之一，有助于结晶。使用低温电子显微镜（cryo-EM）对无活性的hDHHS3突变体进行的结构探索显示出膜蛋白的典型侧视图。这些结果为DHHC3的结构测定提供了初步指导。
