Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers

Abstract

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.

Lu TH y: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

“…Accordingly, the highest cBRET ratios were obtained when VCP was tagged at its N‐terminus and the UBX proteins at their C‐terminus, respectively (Fig D). Exceptions were UBXD1, which binds to the C‐terminus of VCP and UBXD9, which is known to remodel VCP into a heterotetrameric structure (Arumughan et al , ). This suggests that for BRET experiments, fusion tags should be added in close proximity to the interaction domain.…”

“…Protein–protein interactions (PPIs) play an essential role in the proper functioning of living cells (Perkins et al , ; Cafarelli et al , ). They transmit information between signaling proteins, regulate enzymatic activities of proteins, and control the cellular tasks of molecular machines (Couzens et al , ; Taipale et al , ; Arumughan et al , ). Mutation‐dependent perturbations of PPIs play a crucial role in the development of diseases (Wang et al , ; Sahni et al , ).…”

The AAA+ ATPase p97, a cellular multitool

“…The proteins UBXD7, UBXD8, FAF1, SAKS1 (SAPK substrate protein 1) and p47 show this domain arrangement. Using immunoprecipitation experiments with all five proteins, the UBX–UBA proteins were shown to be somewhat promiscuous, binding to K11-, K33-, K48- and K63-linked ubiquitin chains to varying degrees, with K11 and K48 chains the most prevalent [ 92 ].…”

Involvement of VCP/UFD1/Nucleolin in the viral entry of Enterovirus A species

“…Alveolar soft part sarcoma locus (ASPL), a cofactor of VCP with high-affinity, is reported to promote VCP hexamer disassembly and disrupt the D2 ATPase activity of VCP (Arumughan et al, 2016). Its extended UBX domain-containing fragment (residues 313-553, termed ASPL-C), is critical for the interaction with VCP, while its mutant (two Proline amino acids of residues 437-438 were replaced by two Alanine amino acids, termed ASPL-C PP 437-438 AA) loses the binding for VCP Fig.…”

“…*, p < 0.05; **, p < 0.01. (Arumughan et al, 2016). Therefore, we constructed the ASPL-C and ASPL-C PP 437-438 AA to further assess the necessity of VCP's enzymatic activity.…”