State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, 510060, P. R. China.
Department of Oncology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, P. R. China.
Cancer Commun (Lond). 2021 Jul;41(7):615-630. doi: 10.1002/cac2.12163. Epub 2021 May 28.
Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of aberrant plasma cells within the bone marrow. The high frequent mutation of family with sequence similarity 46, member C (FAM46C) is closely related with the occurrence and progression of MM. Recently, FAM46C has been identified as a non-canonical poly(A) polymerase (PAP) that functions as a tumor suppressor in MM. This study aimed to elucidate the structural features of this novel non-canonical PAP and how MM-related mutations affect the structural and biochemical properties of FAM46C, eventually advancing our understandings towards FAM46C mutation-related MM occurrence.
We purified and crystallized a mammalian FAM46C construct, and solved its structure. Next, we characterized the property of FAM46C as a PAP through a combination of structural analysis, site-directed mutagenesis and biochemical assays, and by comparison with its homolog FAM46B. Finally, we structurally analyzed MM-related FAM46C mutations and tested the enzymatic activity of corresponding mutants.
We determined the crystal structure of a mammalian FAM46C protein at 2.35 Å, and confirmed that FAM46C preferentially consumed adenosine triphosphate (ATP) and extended A-rich RNA substrates. FAM46C showed a weaker PAP activity than its homolog FAM46B, and this difference was largely dependent on the residue variance at particular sites. Of them, residues at positions 77, 290, and 298 of mouse FAM46C were most important for the divergence in enzymatic activity. Among the MM-associated FAM46C mutants, those residing at the catalytic site (D90G and D90H) or putative RNA-binding site (I155L, S156F, D182Y, F184L, Y247V, and M270V) showed abolished or compromised PAP activity of FAM46C, while N72A and S248A did not severely affect the PAP activity. FAM46C mutants D90G, D90H, I155L, S156F, F184L, Y247V, and M270V had significantly lower inhibitory effect on apoptosis of RPMI-8226 cells as compared to wild-type FAM46C.
FAM46C is a prokaryotic-like PAP with preference for A-rich RNA substrates, and showed distinct enzymatic efficiency with its homolog FAM46B. The MM-related missense mutations of FAM46C lead to various structural and biochemical outcomes to the protein.
多发性骨髓瘤（MM）是一种血液恶性肿瘤，其特征是骨髓中异常浆细胞的积累。家族性 46 号成员 C（FAM46C）的高频突变与 MM 的发生和进展密切相关。最近，FAM46C 被鉴定为一种非典型多聚（A）聚合酶（PAP），在 MM 中作为肿瘤抑制因子发挥作用。本研究旨在阐明这种新型非典型 PAP 的结构特征，以及 MM 相关突变如何影响 FAM46C 的结构和生化特性，从而深入了解 FAM46C 突变相关 MM 的发生机制。
我们纯化并结晶了一种哺乳动物 FAM46C 构建体，并解析了其结构。接下来，我们通过结构分析、定点突变和生化测定，并与同源物 FAM46B 进行比较，鉴定了 FAM46C 作为 PAP 的特性。最后，我们对 MM 相关的 FAM46C 突变进行了结构分析，并测试了相应突变体的酶活性。
我们确定了 2.35 Å 哺乳动物 FAM46C 蛋白的晶体结构，并证实 FAM46C 优先消耗三磷酸腺苷（ATP）并延伸富含 A 的 RNA 底物。FAM46C 的 PAP 活性比其同源物 FAM46B 弱，这种差异在很大程度上取决于特定位置的残基变化。其中，小鼠 FAM46C 的位置 77、290 和 298 的残基对酶活性的差异最为重要。在与 MM 相关的 FAM46C 突变体中，位于催化位点（D90G 和 D90H）或假定的 RNA 结合位点（I155L、S156F、D182Y、F184L、Y247V 和 M270V）的突变体完全丧失或削弱了 FAM46C 的 PAP 活性，而 N72A 和 S248A 对 PAP 活性没有严重影响。与野生型 FAM46C 相比，突变体 D90G、D90H、I155L、S156F、F184L、Y247V 和 M270V 对 RPMI-8226 细胞凋亡的抑制作用明显降低。
FAM46C 是一种具有 A 偏好性的原核样 PAP，与同源物 FAM46B 相比，其具有明显不同的酶效率。MM 相关的 FAM46C 错义突变导致该蛋白发生各种结构和生化变化。
