PDF(Pubmed)
Abstract：
During autophagy, potentially toxic cargo is enveloped by a newly formed autophagosome and trafficked to the lysosome for degradation. Ubiquitinated protein aggregates, a key target for autophagy, are identified by multiple autophagy receptors. NBR1 is an archetypal autophagy receptor and an excellent model for deciphering the role of the multivalent, heterotypic interactions made by cargo-bound receptors. Using NBR1 as a model, we find that three critical binding partners - ATG8-family proteins, FIP200, and TAX1BP1 - each bind to a short linear interaction motif (SLiM) within NBR1. Mutational peptide arrays indicate that these binding events are mediated by distinct overlapping determinants, rather than a single, convergent, SLiM. AlphaFold modeling underlines the need for conformational flexibility within the NBR1 SLiM, as distinct conformations mediate each binding event. To test the extent to which overlapping SLiMs exist beyond NBR1, we performed peptide binding arrays on >100 established LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Comparative analysis of phosphomimetic peptides highlights that while FIP200 and Atg8-family binding are generally augmented by phosphorylation, TAX1BP1 binding is nonresponsive, suggesting differential regulation of these binding events. In vivo studies confirm that LIR-mediated interactions with TAX1BP1 enhance NBR1 activity, increasing autophagosomal delivery by leveraging an additional LIR from TAX1BP1. In sum, these results reveal a one-to-many binding modality in NBR1, providing key insights into the cooperative mechanisms among autophagy receptors. Furthermore, these findings underscore the pervasive role of multifunctional SLiMs in autophagy, offering substantial avenues for further exploration into their regulatory functions.
摘要：
在自噬过程中,潜在有毒的货物被新形成的自噬体包裹，并被运输到溶酶体进行降解。泛素化蛋白质聚集体，自噬的关键靶点,由多个自噬受体鉴定。NBR1是一种原型自噬受体，是解读多价细胞作用的极好模型，货物结合受体产生的异型相互作用。使用NBR1作为模型，我们发现三个关键的结合伙伴-ATG8家族蛋白，FIP200和TAX1BP1-各自与NBR1内的短线性相互作用基序(SLiM)结合。突变肽阵列表明这些结合事件是由不同的重叠决定簇介导的。而不是一个人，convergent,SLiM。AlphaFold建模强调了NBR1SLiM内构象灵活性的需要，不同的构象介导每个结合事件。为了测试超出NBR1的重叠SLiM存在的程度，我们在>100个已建立的LC3相互作用区（LIR）上进行了肽结合阵列，揭示FIP200和/或TAX1BP1与LIR结合是一种常见现象，并提示LIR是蛋白质相互作用热点。磷酸模拟肽的比较分析强调，虽然FIP200和Atg8家族结合通常通过磷酸化增强，TAX1BP1结合是无反应的，提示这些结合事件的差异调节。体内研究证实，LIR介导的与TAX1BP1的相互作用增强NBR1活性，通过利用来自TAX1BP1的额外LIR来增加自噬体递送。总之,这些结果揭示了NBR1中一对多的结合模式,为自噬受体之间的协同机制提供了关键见解.此外，这些发现强调了多功能SLiMs在自噬中的普遍作用，为进一步探索其监管职能提供了实质性途径。
