Abstract：
Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of \'neosubstrate\' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating \'neointeractors\' distinct from previously disclosed \'neosubstrates\'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.
摘要：
免疫调节酰亚胺药物（IMiDs）是多发性骨髓瘤（MM）治疗的核心组成部分。IMiD结合小脑（CRBN），CUL4-DDB1-RBX1E3连接酶的衔接子，以改变其底物特异性并诱导对MM细胞至关重要的“新底物”转录因子的降解。迄今为止，机理研究主要集中在治疗活性的介体上，对临床IMiD毒性的了解尚不完善。我们采用了依赖BioID2的邻近标记（BioID2-CRBN）来表征在存在和不存在各种IMiD和蛋白酶体抑制剂的情况下的CRBN相互作用组，硼替佐米.我们旨在利用这项技术来进一步绘制超出常规蛋白质组学技术所实现的CRBN相互作用。为了支持这种方法，IMiD处理后表达BioID2-CRBN的细胞的分析显示已知CRBN相互作用物和新底物的生物素化。我们观察到硼替佐米单独显著修饰CRBN相互作用组。邻近标记还表明IMiDs增强了CRBN和未降解的蛋白质之间的相互作用，因此指定“新相互作用者”与先前公开的“新底物”不同。在这里，我们将非肌肉肌球蛋白重链IIA（MYH9）确定为推定的CRBN新相互作用因子，可能有助于IMiDs的血液学毒性。这些研究为IMiD和相关E3连接酶调节药物的机理分析中的邻近标记技术提供了概念证明。
