The Slow Wallerian Degeneration Protein, Wld^S, Binds Directly to VCP/p97 and Partially Redistributes It within the Nucleus

Abstract

Slow Wallerian degeneration (Wld S ) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD؉ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld S targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld S lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD ؉ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld S protein influence the intranuclear location of both ubiquitin proteasome and NAD ؉ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP. INTRODUCTIONThe E4 ubiquitination factor Ube4b (or Ufd2a) has a 123-amino acid N-terminal region that is essential for ubiquitination activity (Mahoney et al., 2002). It is unclear why this region is essential because it does not contain the U box, and it appears to be absent in invertebrate orthologues that ubiquitinate effectively (Koegl et al., 1999;Hatakeyama et al., 2001;Mahoney et al., 2002;Hoppe et al., 2004;Richly et al., 2005). It is important to understand the molecular mechanism of Ube4b because it has a key role in the ubiquitin proteasome system (UPS; Hoppe, 2005), it is neuroprotective in polyglutamine disorders and an important candidate gene for neuroblastoma (Krona et al., 2003). Information on the substrates of Ube4b is beginning to emerge (Hoppe et al., 2004;Okumura et al., 2004;Spinette et al., 2004;Richly et al., 2005) but there is much still to learn about its regulation.In the slow Wallerian degeneration mutant mouse (Wld S ), 70 amino acids of this essential domain of Ube4b form the N-terminus of a chimeric protein that delays Wallerian degeneration of injured axons in mice and rats by 10-fold (see Figure 1A; Lunn et al., 1989;Mack et al., 2001;Adalbert et al., 2005). The chimeric protein is absent in wild-type mice. This sequence (N70) is fused in Wld S protein to the full coding sequence of nicotinamide mononucleotide adenylyltransferase (Nmnat1; Conforti et al., 2000;Emanuelli et al., 2001;Mack et al., 2001) in regulating axon degeneration. Wld S also delays axon degeneration in a wide range of neurodegenerative disorders and acute retrograde axonal degeneration after spinal injury, indicating that axon degeneration mechanisms are more closely related th...

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

“…First, valosin-containing protein (VCP/p97) binds to the N-terminal 16 amino acids of Wld S and Ube4b. 30 Consequently, Wld S partially redistributes VCP into punctate intranuclear foci in some neuronal subtypes. Removing the N-terminal 16 amino acids of Wld S abolishes the targeting of VCP to intranuclear foci, and we confirm here that overexpressed Nmnat1 also fails to target VCP to intranuclear foci (Supplementary Figure 5).…”

Non-Nuclear WldSDetermines Its Neuroprotective Efficacy for Axons and SynapsesIn Vivo

“…Other data regarding the molecular mechanism of Wld S mediated axon protection suggest involvement of valosincontaining protein (VCP) that binds the N-terminal 16 aa of Wld S through a VCP binding motif (Laser et al, 2006). This region is necessary but not sufficient for axon protection in mice (Conforti et al, 2007) (Conforti, Wilbrey, Morreale, Janeckova, Beirowski, Adalbert, Mazzola, Di Stefano, Hartley, Babetto, Smith, Gilley, Billington, Genazzani, Ribchester, Magni, and Coleman, unpublished work) and is necessary for full strength phenotype in Drosophila (Avery, Sheehan, Kerr, Wang, and Freeman, unpublished work).…”

“…Thus, the previously identified Nmnat1 NLS (Sasaki et al, 2006) is also the main determinant of nuclear localization of full-length Wld S protein. We additionally identified a weak predicted NLS (PSORTII and NucPred predictions) within the N-terminal 16 aa of Wld S but chose not to mutate this region because its influence on Wld S biochemistry (Laser et al, 2006) could affect phenotype in other ways.…”

NAD and axon degeneration: From the Wlds gene to neurochemistry

“…Interestingly, this study also showed that UFD2 interacted with calosin-containing protein (VCP), a member of the large family of AAA-type ATPases that are thought to possess chaperone activity, indicating that the mammalian UFD2a/VCP complex might function as an E3/chaperon complex in the response of neurons to stress. Recently, it has been reported the N70 of the Wlds protein bind to VCP directly, 87 indicating that the N70 might increase the expression level and protein stability of Wlds especially under stress conditions such as axonal injury. On the other hand, virus mediated in vitro overexpression may generate at high concentrations (1 to 20 mM) does not require any pretreatment, arguing against the need for nuclear effects.…”