Regulation of the histone acetyltransferase p300 by TRIM25

由于其乙酰转移酶活性、E3连接酶活性以及作为蛋白质-蛋白质相互作用的支架，p300对许多细胞过程至关重要，其失调与几种疾病有关。然而，尽管它很重要，我们对其监管的了解仍然非常有限。当我们研究TRIM25对p53的调控时（Zhang et al., 2015），我们观察到在TRIM25的存在下，p300的丰度和p300依赖性的p53活性都明显降低。TRIM25是TRIM蛋白家族的成员，该蛋白家族的特征是在TRIM蛋白的n端存在一个三方基元，由一个ring结构域、一个或两个b -box和一个卷曲-线圈区域组成。尽管TRIM25具有环结构域和E3连接酶活性，例如指向14-3-3 sigma，但它无法靶向p300以使其被26S蛋白酶体降解。相反，我们可以通过溶酶体抑制剂抑制p300的降解。TRIM25如何靶向p300使其溶酶体降解尚不清楚。我们假设TRIM25以某种方式介导p300在细胞聚集体中的聚集，然后融入自噬途径。在这种情况下，TRIM25可能参与p300在聚合体中的聚集或p300向自噬体的传递。通过使用各种降解途径的抑制剂和显性负突变体，通过下调可能参与p300与RNAi降解的蛋白质，通过研究是否以及哪些翻译后修饰参与，通过研究蛋白质-蛋白质相互作用和微观研究，我们将挑战我们的假设，并确定(i) p300是否通过自噬途径被降解（ii） p300的降解是否涉及聚合体的形成以及（iii） TRTIM25如何介导p300的降解。由于p300是转录控制的中心节点，我们进一步假设，通过调节p300， TRIM25对基因转录的影响尚未被低估。我们建议在全基因组范围内研究具有和不具有TRIM25的细胞和组织的转录组。在一些细胞中，RNAi对p300的下调将使我们能够区分p300依赖性和p300非依赖性过程。总之，所提出的实验将极大地增加我们对p300调控和TRIM25活性的认识，此外，它们可能允许对TRIM蛋白作为自噬-re/adaptors的功能提供额外的和新颖的见解，这一功能直到最近才被阐明。

Due to its acetyltransferase-activity, its E3 ligase activity and by acting as a scaffold for protein-protein interactions, p300 is essential for a multitude of cellular processes and its misregulation is involved in several diseases. Yet, despite its importance, our knowledge about its regulation is still very limited. While we were investigating the regulation of p53 by TRIM25 (Zhang et al., 2015), we observed that in the presence of TRIM25 p300 abundance and p300-dependent p53 activity were strongly decreased. TRIM25 is a member of the TRIM-protein family, a protein family that is characterized by the presence of a tripartite motif at the N-terminus of TRIM proteins consisting of a RING-domain, one or two B-boxes and a coiled-coil region. Despite having a RING-domain and E3 ligase activity e.g. towards 14-3-3 sigma, TRIM25 was unable to target p300 for degradation by 26S proteasomes. Instead, we could inhibit p300 degradation by inhibitors of lysosomes. How TRIM25 targets p300 to lysosomal degradation is not known. We hypothesize that TRIM25 somehow mediates that p300 aggregates in cellular aggresomes that are then fitted into the autophagy pathway. In this context, TRIM25 could participate in the aggregation of p300 in aggresomes or in the delivery of p300 to autophagosomes. By using inhibitors of various degradation pathways and dominant negative mutants, by downregulating proteins that may be involved in p300 degradation with RNAi, by investigating whether and which post-translational modifications are involved, by investigating protein-protein-interactions and by microscopic studies, we will challenge our hypothesis and determine whether (i) p300 is degraded by the authophagy pathway (ii) whether degradation of p300 involves the formation of aggresomes and (iii) how TRTIM25 mediates the degradation of p300. Since p300 is a central node of transcriptional control, we further hypothesize that via the regulation of p300, TRIM25 has an as yet undervalued impact on gene transcription. We propose to investigate the transcriptome of cells and tissues with and without TRIM25 in a genome wide manner. Downregulation of p300 by RNAi in some of the cells will allow us to distinguish between p300-dependent and p300-independent processes. In summary, the proposed experiments will strongly increase our knowledge about the regulation of p300 and the activity of TRIM25, In addition, they may allow additional and novel insights into the function of TRIM proteins as autophagy-re/adaptors, a function that only recently has been elucidated.