Parsons Victoria A, Vadlamudi Swarooparani, Voos Kayleigh M, Rohy Abigail E, Moxley Anne H, Cannon Maren E, Rosen Jonathan D, Mills Christine A, Herring Laura E, Broadaway K Alaine, Lorenzo Damaris N, Mohlke Karen L
Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Diabetologia. 2025 Jun;68(6):1169-1183. doi: 10.1007/s00125-025-06391-w. Epub 2025 Mar 10.
AIMS/HYPOTHESIS: Components of the insulin processing and secretion pathways remain incompletely understood. Here, we examined a genome-wide association study (GWAS) signal for plasma proinsulin levels. Lead GWAS variant rs150781447-T encodes an Arg279Cys substitution in TBC1 domain family member 30 (TBC1D30), but no role for this protein in insulin processing or secretion has been established previously. This study aimed to evaluate whether TBC1D30 drives the GWAS association signal by determining whether TBC1D30 is involved in proinsulin secretion and, if so, to examine the effects of variant alleles and potential mechanisms.
Using CRISPR/Cas9 genome editing to create double-strand breaks and prime editing to install substitutions in INS1 832/13 insulinoma cells, we generated clonal cell lines with altered TBC1D30, as well as homozygous and heterozygous lines carrying the lead GWAS variant. We characterised lines by Sanger sequencing, quantitative PCR and ELISAs to measure glucose-stimulated proinsulin and insulin secretion. We also tested the effects of TBC1D30 knockdown on proinsulin and insulin secretion in human islets. We further assessed TBC1D30's contribution to secretory pathways by examining the effects of altered gene function on intracellular proinsulin and insulin content and insulin localisation, and by identifying potential proteins that interact with TBC1D30 using affinity purification mass spectrometry.
Compared with mock-edited cells, cell lines with reduced TBC1D30 expression or altered Rab GTPase-activating protein (RabGAP) domain had significantly more secreted proinsulin, 1.8- and 2.6-fold more than controls, respectively. Similarly, cells expressing the variant substitution demonstrated increased proinsulin secretion. Cell lines with a partial deletion of a critical functional domain showed 1.8-fold higher expression of Tbc1d30 and at least 2.0-fold less secreted proinsulin. Cells with altered RabGAP domain sequence also demonstrated, to a lesser extent, changes in secreted insulin levels. TBC1D30 knockdown in human islets resulted in increased insulin secretion with no significant effect on proinsulin secretion. The effects of altered TBC1D30 on mislocalisation of insulin, intracellular proinsulin and insulin content and the identities of interacting proteins are consistent with a role for TBC1D30 in proinsulin and insulin secretion.
CONCLUSIONS/INTERPRETATION: These findings suggest that effects on TBC1D30 are responsible for the GWAS signal and that TBC1D30 plays a critical role in the secretion of mature insulin.
目的/假设：胰岛素加工和分泌途径的组成部分仍未完全明确。在此，我们研究了一项全基因组关联研究（GWAS）中血浆胰岛素原水平的信号。GWAS的主要变异体rs150781447 - T在TBC1结构域家族成员30（TBC1D30）中编码一个Arg279Cys替换，但此前尚未确定该蛋白在胰岛素加工或分泌中的作用。本研究旨在通过确定TBC1D30是否参与胰岛素原分泌来评估其是否驱动GWAS关联信号，如果是，则研究变异等位基因的影响及潜在机制。
利用CRISPR/Cas9基因组编辑技术在INS1 832/13胰岛素瘤细胞中产生双链断裂，并通过碱基编辑技术进行替换，我们构建了TBC1D30表达改变的克隆细胞系，以及携带GWAS主要变异体的纯合子和杂合子细胞系。我们通过桑格测序、定量PCR和酶联免疫吸附测定（ELISA）来表征这些细胞系，以测量葡萄糖刺激的胰岛素原和胰岛素分泌。我们还测试了TBC1D30敲低对人胰岛中胰岛素原和胰岛素分泌的影响。我们通过检查基因功能改变对细胞内胰岛素原、胰岛素含量及胰岛素定位的影响，以及使用亲和纯化质谱法鉴定与TBC1D30相互作用的潜在蛋白质，进一步评估TBC1D30对分泌途径的贡献。
与模拟编辑的细胞相比，TBC1D30表达降低或Rab GTP酶激活蛋白（RabGAP）结构域改变的细胞系分泌的胰岛素原显著增多，分别比对照多1.8倍和2.6倍。同样，表达变异替换的细胞胰岛素原分泌增加。关键功能结构域部分缺失的细胞系Tbc1d30表达高1.8倍，分泌的胰岛素原至少少2.0倍。RabGAP结构域序列改变的细胞在分泌胰岛素水平上也有较小程度的变化。人胰岛中TBC1D30敲低导致胰岛素分泌增加，而对胰岛素原分泌无显著影响。TBC1D30改变对胰岛素错误定位、细胞内胰岛素原和胰岛素含量以及相互作用蛋白身份的影响与TBC1D30在胰岛素原和胰岛素分泌中的作用一致。
结论/解读：这些发现表明对TBC1D30的影响是导致GWAS信号的原因，并且TBC1D30在成熟胰岛素的分泌中起关键作用。
