Ubiquitin is directly linked via an ester to protein-conjugated mono-ADP-ribose

The prevailing view on post-translational modifications (PTMs) is that a single amino acid is modified with a single PTM at any given time. However, recent work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation. For example, DELTEX E3 ligases were reported to ubiquitylate a hydroxyl group on free NAD+ and ADP-ribose in vitro, generating a noncanonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester. We call this process mono-ADP-ribosyl ubiquitylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that multiple PARPs are MARUbylated and extended with K11-linked polyubiquitin chains when exogenously expressed. Finally, we show that in response to type I interferon stimulation, MARUbylation can occur endogenously on PARP targets. Thus, MARUbylation represents a new dual PTM that broadens our understanding of the function of PARP-mediated ADP-ribosylation in cells.

目前学界关于翻译后修饰（post-translational modifications, PTMs）的主流认知认为，任一时刻下单个氨基酸残基仅会被一种翻译后修饰所修饰。但近期研究证实，不同翻译后修饰之间存在串扰现象，部分修饰甚至可发生于同一氨基酸残基上。ADP核糖基化（ADP-ribosylation）与泛素化（ubiquitylation）之间便存在这类相互作用。例如，有研究报道DELTEX E3泛素连接酶（DELTEX E3 ligases）可在体外对游离烟酰胺腺嘌呤二核苷酸（free NAD+）及ADP核糖（ADP-ribose）上的羟基基团进行泛素化修饰，生成非经典泛素酯连接型产物。本研究首次证实，这类双翻译后修饰可在细胞内发生于单ADP核糖基化（mono-ADP-ribosylated, MARylated）的多聚ADP核糖聚合酶10（PARP10）的谷氨酸/天冬氨酸位点，形成单ADP核糖基泛素酯修饰。我们将该修饰过程命名为单ADP核糖基泛素化（mono-ADP-ribosyl ubiquitylation），简称MARUbylation。通过化学与酶学实验手段，包括新鉴定的具有酯酶特异性活性的细菌去泛素化酶（deubiquitinase），我们发现当外源表达时，多种多聚ADP核糖聚合酶家族（PARPs）蛋白会发生MARUbylation修饰，并被连接上K11连接型多泛素链（K11-linked polyubiquitin chains）。最后我们证实，在I型干扰素（type I interferon）刺激条件下，MARUbylation修饰可内源性地发生于PARP蛋白的靶标上。综上，MARUbylation作为一种新型双翻译后修饰，拓展了我们对细胞内多聚ADP核糖聚合酶介导的ADP核糖基化功能的认知。