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Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination
RSC Chemical Biology Pub Date : 2021-07-29 , DOI: 10.1039/d1cb00088h
Mengyun Zhang 1, 2 , Joseph M McEwen 3 , Nicole M Sjoblom 3 , Kristin M Kotewicz 1, 2 , Ralph R Isberg 1 , Rebecca A Scheck 3
Affiliation  

Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family.

中文翻译:

Legionella pneumophila Sde 家族的成员靶向酪氨酸残基进行磷酸核糖基连接的泛素化

嗜肺军团菌通过 IV 型分泌系统 (T4SS) 转运数百​​个蛋白质效应器来建立复制液泡。在这些易位效应器中,有 Sde 家族的成员,它们催化宿主靶标的磷酸核糖基连接的泛素化 (pR-Ub)。先前的工作已经假定 Sde 蛋白仅针对受体蛋白底物中的丝氨酸 (Ser) 残基。我们在这里展示了 SdeC 介导的 pR-Ub 修饰是由逐步反应产生的,该反应也修饰了酪氨酸 (Tyr) 残基。出乎意料的是,Ub 上 HA 标签的存在导致了多聚 pR 泛素化,这与充当受体目标的 HA 标签一致。基于对交联产物的 LC-MS/MS 分析,对磷酸核糖基连接的 HA-Ub 的询问显示 Tyr4 是首选的目标残基。使用合成 HA 变体的进一步分析揭示了 Tyr 的混杂修饰,因为仅通过构建三重突变体来阻止交联,其中 HA 序列中的所有三个 Tyr 都被 Phe 取代。尽管以前的工作表明 Ser 是唯一的受体残基,但我们没有发现使用 Tyr → Ser 替换突变体的 Ser 偏好于 Tyr 的证据。这项工作表明 Sde 家族的 pR 泛素化不限于先前提出的 Ser 修饰,并扩大了该家族的潜在目标位点。我们没有发现使用 Tyr → Ser 替换突变体的 Ser 优先于 Tyr 的证据。这项工作表明 Sde 家族的 pR 泛素化不限于先前提出的 Ser 修饰,并扩大了该家族的潜在目标位点。我们没有发现使用 Tyr → Ser 替换突变体的 Ser 优先于 Tyr 的证据。这项工作表明 Sde 家族的 pR 泛素化不限于先前提出的 Ser 修饰,并扩大了该家族的潜在目标位点。
更新日期:2021-08-04
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