Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species
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DOI：
10.3791/4132
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发表时间：
2012-08-01
影响因子：
1.2
通讯作者：
Korth, Carsten
中科院分区：
文献类型：
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作者：
Bader, Verian;Ottis, Philipp;Korth, Carsten
蛋白质聚集被视为慢性、变性脑病症的一般标志，例如在神经变性疾病阿尔茨海默病（A β，tau）、帕金森病（α-突触核蛋白）、亨廷顿病（聚谷氨酰胺，亨廷顿蛋白）等中。蛋白质聚集被认为是由于蛋白质稳态失调而发生的，即错误折叠蛋白质的产生和降解之间的不平衡。值得注意的是，在这些疾病的散发形式中发现了相同的蛋白质聚集，而这些疾病在家族形式的罕见变体中是突变的。精神分裂症是一种慢性进行性脑部疾病，在许多情况下会沿着永久性和不可逆的认知缺陷。在候选基因方法中，我们研究了在苏格兰家族中克隆的与慢性精神疾病相关的基因DISC 1（DISC 1）是否可以在精神分裂症散发病例的大脑中发现不溶性聚集体（3）。使用SMRI CC，我们在大约20%的CMD病例中鉴定了肌氨酰不溶性DISC 1免疫反应性，但在生化分离后未鉴定出正常对照或神经退行性疾病患者。随后的体外研究表明，DISC 1的聚集倾向受疾病相关多态性S704 C的影响（4），体外产生的DISC 1侵袭体具有细胞侵袭性（5），与A β（6）、tau（7-9）、α-突触核蛋白（10）、聚谷氨酰胺（11）或SOD 1聚集体（12）的表现相似。这些发现促使我们提出，至少有一个子集的情况下，CMD，那些与聚集DISC 1可能是蛋白质构象disorders.Here，我们描述了我们如何产生DISC 1侵略在哺乳动物细胞，纯化他们的蔗糖梯度，并使用它们的细胞侵袭性研究。同样，我们描述了我们如何产生一个专门的多聚体C-末端DISC 1片段，标签和纯化细胞侵袭力的研究。使用DISC 1的重组多聚体，我们实现了类似的细胞侵袭性作为一个类似的标记的合成asynuclein片段。我们还表明，该片段在体内立体定向注射到受体动物的大脑中时被吸收。
Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A beta, tau), Parkinson's Disease (alpha-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease(1,2), could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia(3). Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C(4), and that DISC1 aggresomes generated in vitro were cell-invasive(5), similar to what had been shown for A beta(6), tau(7-9), alpha-synuclein(10), polyglutamine(11), or SOD1 aggregates(12). These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic asynuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.