Genetic suppression of loss of TPP1

TPP1 缺失的基因抑制

基本信息

批准号：
9477794
负责人：
Richard H Gomer
金额：
$ 17.36万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9477794
关键词：
Ammonium Chloride Amoeba genus Animal Model Autophagocytosis Biological Models CLN2 gene Caenorhabditis elegans Cell Nucleus Cells Child Childhood Chloroquine Cholesterol Cleaved cell Code Complementary DNA DNA cassette Defect Deposition Development Dictyostelium Dictyostelium discoideum Drosophila genus Drug Targeting Enzymes Exhibits Fibroblasts Future Genes Genetic Genetic Screening Genetic Suppression Genetic Transcription Growth Human Hybrids In Vitro Insertional Mutagenesis Jansky-Bielschowsky Disease Knock-out Lysosomes Mediating Messenger RNA Methods Modeling Movement Mus Mutagenesis Mutate Mutation Names Neurodegenerative Disorders Neuronal Ceroid-Lipofuscinosis Neurons Nomenclature Orthologous Gene Patients Peptide Hydrolases Pharmacology Phenotype Proteins Psyche structure RNA Saccharomyces cerevisiae Shotguns Site Spielmeyer-Vogt Disease System Technology Testing Therapeutic Vertebrates Vision Work Yeasts cDNA Library cell transformation cell type disease-causing mutation effective therapy enzyme activity genetic approach high reward high risk homologous recombination knockout gene oxysterol binding protein restriction enzyme screening social tool tripeptidyl aminopeptidase vector

项目摘要

Neuronal ceroid lipofuscinosis (NCL) is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is no effective therapy. Children with NCL show a normal early growth but then exhibit a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. A subtype of NCL called Late-Infantile NCL (LINCL) is caused by mutations in the protease tripeptidyl peptidase 1 (TPP1; encoded by the CLN2 gene). Little is known about the normal function of TPP1, and an intriguing possibility is that an identification of genetic suppressors of a loss of TPP1 might identify pharmacological targets to ameliorate the effects of TPP1 loss. Although TPP1 is highly conserved among vertebrates, TPP1 orthologs have not been detected in Drosophila, C. elegans, or S. cerevisiae. In the genetically tractable social amoeba Dictyostelium discoideum, DdTpp1 is a TPP1 ortholog, and there are several similarities between Dictyostelium tpp1¯ cells and cells from children with LINCL. In a preliminary genetic screen for suppressors of the tpp1¯ phenotype, and screening for a reversion of just one of the phenotypes of tpp1¯ cells, we found that disruption of a protein with similarity to mammalian oxysterol-binding proteins suppresses some but not all of the tpp1¯ phenotypes. Preliminary work then indicated that fibroblasts from some children with LINCL have abnormally high levels of cholesterol. The existence of a partial genetic suppressor of tpp1¯, and the usefulness of this approach to guide work on cells from LINCL patients, suggests the exciting possibility that targeting specific proteins could be a viable way to suppress some of the effects of loss of TPP1 function. In this high risk/ high reward R21 proposal, we propose to use the power of Dictyostelium genetic screens to identify the genes, which, when disrupted, suppress tpp1¯ phenotypes. In Aim 1 we will use random insertional mutagenesis to complete the partial genetic screen for suppressors, and screen for a reversion of multiple phenotypes. In Aim 2 we will use a complementary genetic approach, shotgun antisense, to similarly screen for revertants. The sustained impact of the proposed studies will be the identification, in a genetically tractable system, of the key downstream effectors of TPP1. This work will impact our understanding of TPP1 in a model system, and will serve as a necessary basis for future work to test the hypothesis that, in a mammalian system, blocking the function of one or more proteins identified in the Dictyostelium genetic screen could be useful as a therapeutic for LINCL.

神经元的脂肪促脂肪促（NCL）是最常见的童年神经退行性疾病。 NCL不可避免地致命，并且没有有效的疗法。 NCL儿童表现出正常的早期增长，但然后暴露了运动，视力和心理能力的逐步下降，并积累了神经元和其他细胞类型中的自动荧光沉积物。 NCL的子类型，称为晚期NCL（LINCL）是由蛋白酶三肽基肽酶1（TPP1；由CLN2基因编码）引起的。几乎没有知道TPP1的正常功能以及有趣的可能性是通用的识别损失TPP1的倡导者可能会鉴定出药物靶标，以改善TPP1损失的影响。尽管TPP1在脊椎动物中是高度保守的，但在果蝇中尚未检测到TPP1直系同源物秀丽隐杆线虫或酿酒酵母。在遗传性的社交变形虫dictyostelium discoideum中，DDTPP1是一个 TPP1直系同源 Lincl。在TPP1表型补充的初步遗传筛选中，并筛选反向在TPP1细胞的表型之一中，我们发现与哺乳动物相似的蛋白质破坏氧蛋白酶结合蛋白抑制了一些但不是全部TPP1表型。然后，初步工作表明一些LINCL儿童的成纤维细胞具有绝对高的胆固醇。这存在TPP1的部分遗传抑制剂，以及这种指导细胞工作的方法的实用性从Lincl患者中，提出靶向特定蛋白可能是一种可行的方式的令人兴奋的可能性抑制TPP1功能丧失的某些影响。在这个高风险/高奖励R21提案中，我们提出了使用dictyostelium遗传筛选的力量来识别这些基因，当被破坏时，它会抑制TPP1 表型。在AIM 1中，我们将使用随机插入诱变来完成部分遗传筛选补充和筛选多种表型的反向。在AIM 2中，我们将使用完整的通用 shot弹枪反义的方法类似地筛选了恢复物。拟议研究的持续影响将是TPP1的关键下游效应的一般可牵引系统中的识别。这项工作将影响我们在模型系统中对TPP1的理解，并将作为未来工作的必要基础为了检验以下假设：在哺乳动物系统中，阻止了在 Dictyostelium遗传筛选可作为LINCL的治疗方法。