Lange Sven M, McFarland Matthew R, Lamoliatte Frederic, Carroll Thomas, Krshnan Logesvaran, Pérez-Ràfols Anna, Kwasna Dominika, Shen Linnan, Wallace Iona, Cole Isobel, Armstrong Lee A, Knebel Axel, Johnson Clare, De Cesare Virginia, Kulathu Yogesh
MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Nat Struct Mol Biol. 2024 Dec;31(12):1872-1887. doi: 10.1038/s41594-024-01354-y. Epub 2024 Jul 8.
Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48-K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48-K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48-K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48-K63-linked Ub, these results suggest a function for K48-K63-branched chains in VCP/p97-related processes.
分支泛素（Ub）链在人类细胞的泛素聚合物中占相当大的比例。尽管它们数量众多，但由于缺乏可及的方法和工具，我们对细胞信号传导中分支泛素功能的理解一直受到阻碍。在这里，我们鉴定了细胞内分支链特异性结合蛋白，并设计了探究K48-K63分支泛素功能的方法。我们建立了一种监测复杂泛素链内连接键切割的方法，并揭示了ATXN3和MINDY作为去分支酶。我们设计了一种K48-K63分支特异性纳米抗体，并在纳米抗体-分支泛素链复合物的晶体结构中揭示了其特异性的分子基础。使用这种纳米抗体，我们检测到在含缬酪肽蛋白（VCP）/p97受到抑制后以及DNA损伤后，K48-K63-Ub分支增加。连同我们发现多个与VCP/p97相关的蛋白与K48-K63连接的泛素结合或使其去分支，这些结果表明K48-K63分支链在VCP/p97相关过程中具有功能。
