真核细胞中的蛋白降解过程主要由泛素-蛋白酶体通路和自噬-溶酶体通路两大途径主导。其中，泛素-蛋白酶体通路负责细胞内绝大多数蛋白质的降解。实验室前期研究发现自噬关键蛋白LC3-II前体LC3-I通过以PA28γ为激活因子的蛋白酶体降解，提示自噬与蛋白酶体降解途径存在相互调控。p62，也被称为A170/SQSTM1，起初被发现是一种自噬底物受体蛋白，其特有的LIR结构域能够与LC3和GABARAP相互作用，从而协助底物与自噬小体实现对接。然而，p62/SQSTM1在泛素-蛋白酶体通路底物降解中的作用尚不清楚。

实验室前期尚未发表的蛋白酶体质谱数据显示，p62与蛋白酶体有着密切的关联。本研究从小鼠组织中成功纯化出蛋白酶体，通过免疫印迹实验证明，体内的蛋白酶体上有p62的结合。体外实验发现，蛋白酶体与纯化的p62蛋白孵育后在非变性胶中的迁移变慢，证实p62能与蛋白酶体结合。进一步发现，p62的缺失能够促进外源蛋白酶体底物Ub-R-GFP和内源蛋白酶体底物p21的降解。回补p62后，这两种蛋白酶体底物的降解恢复正常。这些结果表明，p62通常抑制蛋白酶体底物经泛素-蛋白酶体途径的降解。

蛋白酶体在特定条件下会发生相分离从而产生点状聚集。本研究发现，细胞中敲除p62导致蛋白酶体泛素受体Rpn10与Rpn13形成的点状聚集数量显著增多，且这些聚集大多分布在细胞核中。p62回补能够抑制敲除引起的细胞核内Rpn10与Rpn13点状聚集的形成。反过来，细胞中敲除Rpn13会引起p62形成点状聚集，其数量及面积均增加，进一步说明Rpn13与p62在点状聚集的形成过程中存在着相互影响。最后，在蛋白酶体泛素受体Rpn13缺失的情况下，过表达p62可以维持底物p21的正常降解，说明p62可以补偿Rpn13招募底物的功能。我们猜测，由于p62具有结合泛素的UBA结构域，在Rpn13缺失的情况下，p62可能会通过结合泛素化底物、调控蛋白酶体相分离并补偿Rpn13的作用。而在Rpn13存在的情况下，p62则可能通过结合泛素化底物而与Rpn13进行竞争性抑制，以使自噬途径和泛素-蛋白酶体途径达到相对平衡。

综上所述，虽然自噬底物受体p62通常抑制底物经泛素-蛋白酶体途径的降解以使自噬途径和泛素-蛋白酶体途径达到相对平衡，但可能通过调控蛋白酶体相分离补偿蛋白酶体泛素受体亚基Rpn13的作用而维持底物通过蛋白酶体的正常降解。这些结果为深入理解自噬与泛素-蛋白酶体通路间的交互调控机制提供了重要的数据。

The degradation of proteins within eukaryotic cells is primarily mediated by two principal pathways: the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. Among these, the ubiquitin-proteasome pathway holds primary responsibility for the degradation of the majority of intracellular proteins. Prior research conducted in our laboratory has revealed that the autophagy-associated protein LC3-I, the precursor of LC3-II, undergoes degradation via the ubiquitin-proteasome pathway, facilitated by the activating factor PA28γ. This finding suggests a complex interplay between autophagy and proteasome degradation mechanisms. p62, also known as A170/SQSTM1, was initially identified as an autophagy receptor protein. Its LIR domain enables interactions with LC3 and GABARAP, thereby facilitating the docking of substrates with autophagosomes. However, the specific role of p62 in the substrates degradation of ubiquitin-proteasome pathway remains elusive.

The laboratory's unpublished proteasome mass spectrometry data indicates a close association between p62 and the proteasome. In this study, we successfully purified the proteasome from mouse tissues and confirmed by immunoblotting experiments the presence of p62 binding to endogenous proteasomes. Furthermore, in vitro assays demonstrated that the migration of the proteasome in native gels slows significantly upon incubation with purified p62 protein, validating the interaction between p62 and proteasome. Remarkably, the absence of p62 was observed to accelerate the degradation of both exogenous proteasome substrate Ub-R-GFP and endogenous proteasome substrate p21. Conversely, the replenishment of p62 restored the normal degradation rates of these substrates. Upon complementing p62, the degradation of these two proteasome substrates returned to normal levels. These results indicate that p62 typically inhibits the degradation of proteasome substrates via the ubiquitin-proteasome pathway.

Remarkably, the proteasome undergoes phase separation under certain conditions, resulting in the formation of puncta. Our study revealed that knockout of p62 in cells leads to a significant increase in the number of puncta formed by proteasome ubiquitin receptors Rpn10 and Rpn13, predominantly localized within the nucleus. The replenishment of p62 effectively suppresses the formation of these nuclear puncta. Conversely, the knockout of Rpn13 triggers the formation of p62 puncta, exhibiting an increase in both number and size. These observations suggest a mutual regulatory interplay between Rpn13 and p62 in the formation of punctate aggregates. Notably, in the absence of the proteasome ubiquitin receptor Rpn13, overexpression of p62 maintains the normal degradation of p21. It indicates that p62 can functionally compensate for the loss of Rpn13 in recruiting and targeting substrates for degradation. We speculate that since p62 has a ubiquitin-binding UBA domain, it may bind to ubiquitinated substrates in the absence of Rpn13, regulate proteasome phase separation, and functionally compensate for Rpn13. However, in the presence of Rpn13, p62 may engage in competitive inhibition with Rpn13 by binding to ubiquitinated substrates, thereby maintaining a delicate balance between autophagy and the ubiquitin-proteasome pathway.

In conclusion, while the autophagy receptor p62 typically serves to inhibit the degradation of substrates via the ubiquitin-proteasome pathway, promoting a homeostatic balance between autophagy and the ubiquitin-proteasome pathway, it may also maintain normal substrate degradation through the proteasome by regulating proteasome phase separation and compensating for the function of the proteasome ubiquitin receptor subunit Rpn13. These findings provide crucial insights into the intricate crosstalk mechanisms underlying the interaction between autophagy and the ubiquitin-proteasome pathway.