Regulation of interferon receptor by Leishmania kinase

描述(申请人提供)：由利什曼原虫引起的利什曼病是一个重要的公共卫生问题，目前影响着全球超过3.5亿人，而II型干扰素(干扰素？)在对利什曼原虫的抵抗力方面，根据目前的范例，I型干扰素(如IFNA种)并不被认为是主要的。这一思路源于这样的数据，即与野生型动物相比，IFNA受体亚单位1(IFNAR1)缺失的小鼠对利什曼原虫感染并不过于敏感。然而，利什曼原虫可能简单地灭活IFNAR1的另一种可能性尚未被探索。在这里，我们提出了一个范式转换假说，利什曼原虫前鞭毛体能够通过表达寄生虫酪蛋白激酶1(L-CK1)下调IFNAR1的水平，导致人IFNAR1(小鼠中的Ser526)Ser535磷酸化增加，从而触发-Trcp E3泛素连接酶的招募，随后IFNAR1泛素化和降解。这种L-CK1介导的IFNAR1的下调对于削弱IFNA信号(包括IFNA诱导的诱导型一氧化氮合酶的表达)以降低对利什曼原虫感染的抵抗力具有重要意义。这一假说基于我们的初步数据，这些数据表明：(I)通过依赖Ser535/526的磷酸化的泛素化和降解而发生的IFNAR1基因下调限制了细胞对IFNA的反应程度；(Ii)L-CK1的表达导致Ser535/526的磷酸化增加和IFNAR1的降解；(Iii)L.主要鞭毛虫蛋白激酶(以前被鉴定为L-CK1)能够使IFNAR1磷酸化；(Iv)L.重大感染或L-CK1的表达均抑制细胞对IFNA的反应程度。为了验证我们的假说，我们提出：(1)确定主要乳杆菌感染和L-CK1的表达/活性对IFNAR1下调的影响。我们将利用转基因L-CK1单倍体缺失和突变株以及表达稳定的IFNAR1突变体的宿主细胞，研究在表达L-CK1或主要乳杆菌感染(在允许或不允许L-CK1活性和IFNAR1磷酸化的条件下)是否增加IFNAR1Ser535/526的磷酸化，-Trcp E3泛素连接酶的募集，IFNAR1泛素化和降解；(2)确定L-CK1诱导的IFNAR1降解在改变细胞对IFNA的反应和对主要乳杆菌感染的抗性中的作用。我们将确定主要利什曼原虫感染或L-CK1的表达是否影响IFNA信号的大小和持续时间，以及在抗利什曼原虫防御中发挥重要作用的干扰素刺激基因(如诱导型一氧化氮合酶)的表达。我们将通过产生携带IFNAR1点突变(S526A)的敲入小鼠，使IFNAR1对L-CK1介导的磷酸化产生抗性，并检测这些小鼠对主要乳杆菌感染的敏感性，进一步研究IFNAR1磷酸化在这些反应中的作用。利什曼原虫属的单细胞寄生虫导致一种危险的疾病(利什曼病)，目前影响着世界各地超过3.5亿人，包括驻中东的美军。利什曼病的治疗受到药物储备有限、毒性高以及寄生虫对这些药物产生抗药性的阻碍。因此，必须确定新的治疗靶点，特别是那些影响细胞对利什曼原虫前鞭毛体的先天抵抗力的靶点。我们的研究(旨在鉴定和表征利什曼原虫酪蛋白激酶1作为细胞对I型干扰素等先天耐药调节剂的主要调节因子)应该能够深入了解利什曼原虫逃避哺乳动物细胞防御机制的机制，并潜在地确定治疗利什曼病的新靶点。

DESCRIPTION (provided by applicant): Leishmaniasis caused by Leishmania parasites represents an important public health problem that currently affects more than 350 million people around the world, Whereas a key role of Type II interferon (IFN?) in resistance to Leishmania is well established, according to the current paradigm, Type I interferons (such as IFNa species) are not considered of major importance. This line of thinking stems from the data that IFNa receptor subunit 1 (IFNAR1)-null mice are not overly sensitive to Leishmania infection compared to wild type animals. However, an alternative possibility that Leishmania may simply inactivate IFNAR1 has not been explored. Here we propose a paradigm-shifting hypothesis that Leishmania promastigotes are capable of down regulating IFNAR1 levels via expression of parasite casein kinase 1 (L-CK1) that leads to increased Ser535 phosphorylation of human IFNAR1 (Ser526 in mice), which triggers the recruitment of ¿-Trcp E3 ubiquitin ligase followed by IFNAR1 ubiquitination and degradation. This L-CK1-mediated down-regulation of IFNAR1 is important for impairing IFNa signaling (including IFNa-induced expression of inducible nitric oxide synthase, iNOS) to decrease the resistance against Leishmania infection. This hypothesis is based on our preliminary data indicating that: (i) down regulation of IFNAR1 that occurs via its Ser535/526 phosphorylation- dependent ubiquitination and degradation limits the extent of cellular responses to IFNa, (ii) expression of L- CK1 leads to increased Ser535/526 phosphorylation and degradation of IFNAR1, (iii) shed L. major promastigote kinase (previously identified as L-CK1) is capable of phosphorylating IFNAR1, (iv) L. major infection down regulates IFNAR1 levels in human dendritic cells; and (v) either L. major infection or expression of L-CK1 inhibits the extent of cellular responses to IFNa. To test our hypothesis we propose: (1) To determine the effect of L. major infection and expression/activity of L-CK1 on the down regulation of IFNAR1. We will investigate whether Ser535/526 phosphorylation of IFNAR1, recruitment of ¿-Trcp E3 ubiquitin ligase, IFNAR1 ubiquitination and degradation are increased in response to expression of L-CK1 or L. major infection (under the conditions that permit or disallow L-CK1 activity and IFNAR1 phosphorylation) using transgenic L. major L-CK1 haploinsufficient and mutant knock-in strains as well as host cells expressing a stabilized IFNAR1 mutant; (2) To determine the role of L-CK1-induced IFNAR1 degradation in modifying the cellular responses to IFNa and in resistance to L. major infection. We will determine whether L. major infection or L-CK1 expression affects the magnitude and duration of IFNa signaling and expression of IFN-stimulated genes (ISG) that play an important role in anti-Leishmania defense (e.g., iNOS). We will further examine the role of IFNAR1 phosphorylation in these responses via generating the knock-in mice that harbor an IFNAR1 point mutant (S526A) that should render IFNAR1 resistant to L-CK1-mediated phosphorylation and will examine the sensitivity of these mice to L. major infection. Single celled parasites of the genus Leishmania cause a dangerous disease (leishmaniasis) that currently affects more than 350 million people around the world, including the US troops in the Middle East. Treatment of leishmaniasis is impeded by a limited arsenal of drugs, their high toxicity, and emerging resistance of parasites to these agents. Hence, it is imperative to identify novel treatment targets, in particular, those that affect the innate resistance of cells to Leishmania promastigotes. Our studies (aimed at identification and characterization of Leishmania casein kinase 1 as a major regulator of cellular responses to such innate resistance regulators as Type I interferons) should gain the insight on the mechanisms by which Leishmania may evade the defensive mechanisms of mammalian cells and, potentially, identify the novel targets for the therapy against leishmaniasis.