Evolutionary and functional relationships between Lgr proteins and ubiquitin-specific proteases

Lgr 蛋白与泛素特异性蛋白酶之间的进化和功能关系

• 批准号: RGPIN-2021-02566
• 负责人: Gray, Douglas
• 金额: $ 2.33万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744320
• 关键词: Evolutionary; functional; relationships; between; Lgr

We have previously explored the evolutionary history of the deubiquitinating enzymes. We determined that the genes encoding USP4 and USP15 were generated by an ancient whole genome duplication event (and are therefore ohnologs), whereas USP11 arose from a small segmental duplication of USP4 and then rapidly diverged in sequence. To determine the level of functional redundancy we crossed mice with inactivating mutations of Usp4 and Usp15 and found that while mice were viable when null for either gene no progeny could be generated that were null for both. This suggests that there are essential protein substrates that can be deubiquitinated by either, but in the absence of both such substrates are ubiquitinated and degraded by the proteasome. We found that the Wnt/beta catenin pathway is deficient in compound null cells, and have established that LGR5 (a modulator of Wnt signaling) is absent in the liver of midgestation embryos of that genotype. This would explain the failure of hematopoiesis that we believe is the cause of embryonic lethality. To accelerate the research we established a collaboration to generate zebrafish in which the orthologs of Usp4 and Usp15 have been deleted and are currently performing genetic crosses to determine if our mouse findings are recapitulated in this species. A failure of hematopoiesis would be much easier to study in the transparent zebrafish embryo. Single cell RNAseq will be performed on blood cells from fish of various genotypes to determine which cell types and molecular pathways are deficient. Our next objective will be an analysis of lgr genes in teleost fish and their potential role in hematopoiesis. Given that Lgr5 is an essential gene in mouse we were surprised to discover that lgr5 does not exist in zebrafish, but that the ohnolog genes lgr4 and lgr6 were present in the zebrafish genome. A graduate student will be recruited to establish the evolutionary history of the teleost lgr genes and determine temporal relationships to evolution of the deubiquitinating enzyme genes. I propose to use in situ hybridization to study gene expression in the zebrafish to determine if lgr4 and/or lgr6 play any role in hematopoiesis. In parallel lgr genes will be studied in medaka, a fish species with all three lgr ohnologs. The lgr genes of both zebrafish and medaka will be deleted using CRISPR/Cas9 methodology and phenotypes will be analyzed with particular attention to hematopoiesis. Collectively these experiments will provide insight into the evolution and subfunctionalization of duplicated genes, the understanding of which is our long term objective.

我们先前已经探索了去泛素化酶的进化历史。我们确定编码USP 4和USP 15的基因是由一个古老的全基因组复制事件产生的（因此是ohnologs），而USP 11是由USP 4的一个小片段复制产生的，然后在序列上迅速分化。为了确定功能冗余的水平，我们将具有Usp 4和Usp 15失活突变的小鼠杂交，并发现当两种基因都无效时，小鼠是存活的，但不能产生两种基因都无效的后代。这表明，有必要的蛋白质底物，可以去泛素化的任何一个，但在缺乏这两种底物的泛素化和降解的蛋白酶体。我们发现Wnt/β连环蛋白途径在化合物无效细胞中缺乏，并且已经确定LGR 5（Wnt信号传导的调节剂）在该基因型的妊娠中期胚胎的肝脏中不存在。这就解释了造血功能的失败，我们认为这是胚胎死亡的原因。为了加速研究，我们建立了一个合作，以产生斑马鱼，其中Usp 4和Usp 15的直系同源物已被删除，目前正在进行遗传杂交，以确定我们的小鼠研究结果是否在该物种中重现。在透明的斑马鱼胚胎中研究造血失败要容易得多。将对各种基因型鱼类的血细胞进行单细胞RNA测序，以确定哪些细胞类型和分子途径存在缺陷。我们的下一个目标将是分析硬骨鱼lgr基因及其在造血中的潜在作用。考虑到Lgr 5是小鼠的必需基因，我们惊讶地发现Lgr 5不存在于斑马鱼中，但ohnolog基因Lgr 4和Lgr 6存在于斑马鱼基因组中。一名研究生将被招募来建立硬骨鱼lgr基因的进化历史，并确定去泛素化酶基因进化的时间关系。我建议使用原位杂交研究基因表达的斑马鱼，以确定是否lgr 4和/或lgr 6发挥任何作用，在造血。与此同时，我们将在青鳉鱼中研究lgr基因，这是一种具有所有三种lgr基因的鱼类。将使用CRISPR/Cas9方法删除斑马鱼和青鳉的lgr基因，并将分析表型，特别关注造血。总的来说，这些实验将提供深入了解重复基因的进化和亚功能化，这是我们的长期目标。