A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.
A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.
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DOI：
10.1371/journal.pgen.1010430
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发表时间：
2022-09
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
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糖基化途径的部分功能丧失突变是一组称为先天性糖基化疾病（CDGs）的罕见疾病的基础。特别是，DPAGT1- cdg是由编码n -糖基化第一步DPAGT1的基因突变引起的，这种疾病目前缺乏有效的治疗方法。为了确定DPAGT1- cdg的潜在治疗靶点，我们在果蝇细胞中进行了CRISPR敲除筛选，以寻找DPAGT1抑制下与更好的存活率和糖蛋白水平相关的基因。我们确定了数百个可能具有治疗益处的候选基因。有趣的是，抑制甘醇糖基转移酶Dpm1或其下游糖基化途径，可以挽救DPAGT1抑制和内质网应激的两种体内模型，尽管仅这些途径的损伤通常会导致CDGs。虽然这两种体内模型表面上都引起细胞应激（通过DPAGT1抑制或错误折叠的蛋白质），但我们发现果糖代谢的新差异可能表明糖酵解是DPAGT1- cdg的调节剂。我们的研究结果为DPAGT1- cdg提供了新的治疗靶点，包括DPAGT1相关通路的独特发现，并揭示了果糖代谢和内质网应激之间的新相互作用。糖修饰对蛋白质的正常折叠和功能至关重要。控制这些糖修饰的生物学途径被称为糖基化途径。当这些途径被破坏时，比如通过基因突变，它会导致罕见的发育障碍，称为先天性糖基化障碍（CDGs）。大多数cdg缺乏任何治疗选择，并且对可能改善这些疾病的相互作用基因或途径知之甚少。在这里，我们研究DPAGT1- cdg，涉及酶编码基因DPAGT1突变，通过果蝇模式生物，黑腹果蝇。使用抑制药物，我们使用CRISPR技术进行了两个基于细胞的筛选，以确定哪些基因在DPAGT1抑制下是重要的。然后，我们用一只活的苍蝇来模拟眼睛中的DPAGT1-CDG，以确定哪一个被筛选的基因可以改善受损的眼睛发育。令人惊讶的是，我们发现抑制其他cdg引起基因，如我们的顶击Dpm1，可以改善我们的DPAGT1-CDG细胞和苍蝇模型。此外，某些代谢基因的缺失和饮食的改变也改善了DPAGT1-CDG模型。本研究发现了与DPAGT1-CDG相关的多个新基因和途径，为新的CDG治疗奠定了基础。
Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress. Sugar modifications are important to proper protein folding and function. The biological pathways that control these sugar modifications are called glycosylation pathways. When these pathways are disrupted, such as through genetic mutation, it can cause rare developmental disorders called Congenital Disorders of Glycosylation (CDGs). Most CDGs lack any treatment options, and little is known about interacting genes or pathways that might improve these disorders. Here, we study DPAGT1-CDG which involves mutations in the enzyme-encoding gene, DPAGT1, by using the fruit fly model organism, Drosophila melanogaster. Using inhibitory drugs, we perform two cell-based screens with CRISPR technology to determine which genes are important under DPAGT1 inhibition. We then used a living fly, that models DPAGT1-CDG in the eye, in order to determine which of the top screened genes can improve its impaired eye development. Surprisingly, we found that inhibiting other CDG-causing genes, such as our top hit Dpm1, could improve our DPAGT1-CDG cell and fly models. In addition, loss of certain metabolism genes, and changes in diet, also improved the DPAGT1-CDG model. This study finds multiple new genes and pathways implicated in DPAGT1-CDG and lays the groundwork for new CDG therapies.
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