Caspase recruitment domain-containing protein (CARD)9, CARD10, CARD11, and CARD14 all belong to the CARD-coiled coil (CC) protein family and originated from a single common ancestral protein early in vertebrate evolution. All four proteins form CARD-CC/BCL10/MALT1 (CBM) complexes leading to nuclear factor-kappa-B (NF-κB) activation after upstream phosphorylation by various protein kinase C (PKC) isoforms. CBM complex signaling is critical for innate and adaptive immunity, but aberrant activation can cause autoimmune or autoinflammatory diseases, or be oncogenic. CARD9 shows a superior auto-inhibition compared with other CARD-CC family proteins, with very low spontaneous activity when overexpressed in HEK293T cells. In contrast, the poor auto-inhibition of other CARD-CC family proteins, especially CARD10 (CARMA3) and CARD14 (CARMA2), is hampering characterization of upstream activators or activating mutations in overexpression studies. We grafted different domains from CARD10, 11, and 14 on CARD9 to generate chimeric CARD9 backbones for functional characterization of activating mutants using NF-κB reporter gene activation in HEK293T cells as readout. CARD11 (CARMA1) activity was not further reduced by grafting on CARD9 backbones. The chimeric CARD9 approach was subsequently validated by using several known disease-associated mutations in CARD10 and CARD14, and additional screening allowed us to identify several previously unknown activating natural variants in human CARD9 and CARD10. Using Genebass as a resource of exome-based disease association statistics, we found that activated alleles of CARD9 correlate with irritable bowel syndrome (IBS), constipation, osteoarthritis, fibromyalgia, insomnia, anxiety, and depression, which can occur as comorbidities.

中文翻译：

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嵌合和突变 CARD9 构​​建体能够分析 CARD-CC 蛋白家族中保守和差异的自抑制机制

含半胱天冬酶募集结构域的蛋白 (CARD)9、CARD10、CARD11 和 CARD14 均属于 CARD 卷曲线圈 (CC) 蛋白家族，起源于脊椎动物进化早期的单一共同祖先蛋白。所有四种蛋白形成 CARD-CC/BCL10/MALT1 (CBM) 复合物，在上游被各种蛋白激酶 C (PKC) 同工型磷酸化后，导致核因子-κ-B (NF-κB) 激活。CBM 复合体信号传导对于先天性和适应性免疫至关重要，但异常激活可能导致自身免疫或自身炎症性疾病，或者致癌。与其他 CARD-CC 家族蛋白相比，CARD9 显示出优异的自身抑制作用，在 HEK293T 细胞中过表达时自发活性非常低。相比之下，其他 CARD-CC 家族蛋白，尤其是 CARD10 (CARMA3) 和 CARD14 (CARMA2) 的自身抑制能力较差，阻碍了上游激活剂的表征或过表达研究中的激活突变。我们将 CARD10、11 和 14 的不同结构域移植到 CARD9 上，生成嵌合 CARD9 主链，用于使用 HEK293T 细胞中 NF-κB 报告基因激活作为读数来表征激活突变体的功能。CARD11 (CARMA1) 活性并未因嫁接至 CARD9 主链而进一步降低。随后，通过使用 CARD10 和 CARD14 中的几种已知的疾病相关突变来验证嵌合 CARD9 方法，并且额外的筛选使我们能够识别人类 CARD9 和 CARD10 中的几种先前未知的激活自然变异。使用 Genebass 作为基于外显子组的疾病关联统计数据的资源，我们发现CARD9的激活等位基因与肠易激综合征 (IBS)、便秘、骨关节炎、纤维肌痛、失眠、焦虑和抑郁相关，这些可能作为合并症发生。