Regulation of NEDD8/Rub1 and its substrates
NEDD8 is a small ubiquitin like modifier (UBL) essential for life in almost all known eukaryotes. The primary objective of this work is to determine NEDD8 specific regulatory mechanisms. Like ubiquitin, NEDD8 is first matured and then conjugated via a tripartite conjugation cascade (E1, E2, E3). Although Arg72 in ubiquitin prevents misactivation of ubiquitin by the NEDD8 E1, the ubiquitin E1 is not as selective (Souphron et al., 2008). Under conditions of stress or NEDD8 overexpression, the ubiquitin E1 can be charged with NEDD8 (Hjerpe et al.; Hjerpe et al., 2012b; Leidecker et al., 2012). Most ubiquitin E2s can accept the aberrantly activated NEDD8 which then ultimately becomes conjugated to substrates that are normally ubiquitylated (Hjerpe et al., 2012a). Consequently, maintaining the ratio of NEDD8 to ubiquitin is important for maintaining the integrity of the ubiquitin pathway. The mechanism by which the ubiquitin to NEDD8 ratio is regulated remains unclear. In order to insulate the ubiquitylation pathway from NEDD8, we postulated that the ratio of free NEDD8 to free ubiquitin would be tightly controlled. The rapid turnover reported to exist for NEDD8 (Hipp et al., 2004) could be one means of regulating that ratio. However, we have discovered that in yeast that tagging the NEDD8 homolog, related to ubiquitin 1 (Rub1) results in abnormally fast degradation through the unfolded protein response (UPR). Endogenous Rub1 is actually quite stable. The pathway by which endogenous NEDD8 is degraded remains unknown. Another way of distinguishing the two pathways and uncovering NEDD8 specific regulation is to identify genuine substrates of the neddylation pathway. We were unable to uncover novel substrates in yeast. We used several methods to enrich and identify NEDD8 substrates in mammalian cells including cultured human cancer cells and wild type mouse tissue. Mouse testes have an increased neddylation profile by Western Blot (WB). We therefore attempted to identify NEDD8 substrates in mouse testes. We examined both the NEDD8 associated proteome and NEDD8 conjugates by using native and denaturing immunoprecipitation experiments paired with mass spectrometric (MS) analysis. First, we are confident in these experiments because we identified NEDD8 itself. We also observed the cullin family of proteins which have been well characterized as the main substrates of neddylation. One surprising find in the NEDD8 associated proteome was cullin associated, NEDD8 dissociated 1 (CAND1) which binds to the cullins and precludes NEDD8 conjugation to the cullins. We show that CAND1 is unneddylated itself but comes down in association with neddylated proteins. Interestingly, we also found the ubiquitin E1associated with NEDD8, indicating that there may be endogenous charging of the ubiquitin E1 with NEDD8 at low levels. The only direct NEDD8 conjugates well established in this work were the cullins. Further work is needed to completely rule out the existence of non-cullin substrates of the neddylation machinery.
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