Interleukin-1β drives NEDD8 nuclear-to-cytoplasmic translocation, fostering parkin activation via NEDD8 binding to the P-ubiquitin activating site

Abstract

BackgroundNeuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and β, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1β on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8.MethodsImmunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1β effects on PINK1, P-Ub, parkin, P-parkin, and GSK3β—as well as phosphorylation of parkin by GSK3β—were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3β. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1β, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates.ResultsIL-1α, IL-1β, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1β-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3β-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser65 for parkin activation.ConclusionsThe promotion of parkin- and NEDD8-mediated ubiquitination by IL-1β is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1β suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1β elevation, easily envisioned considering its early induction in Down’s syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

Structural modeling of GSK3β implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs

“…This was followed by another equilibration phase before the actual Molecular Dynamics (MD) run using the NPT protocol. The actual MD run followed a metadynamic simulation protocol as described previously 11 , 39 . Briefly, for the metadynamics run, collective variables (CVs) play crucial roles 40 .…”

Structural and functional consequences of NEDD8 phosphorylation

“…While this suggests that phosphorylation of NEDD8 contributes to the mitochondrial stress response, NEDD8 has been associated with several other stress pathways and, thus, it seems likely that NEDD8 phosphorylation plays a more general role in the cellular stress response. For instance, it was reported that upon neuronal stress, IL-1β fosters NEDD8-induced Parkin activation by inducing translocation of NEDD8 from the nucleus to the cytoplasm 31 and that NEDD8 and Parkin colocalize in the cytoplasm resulting in Parkin neddylation and activation 18 . However, neither study investigated the phosphorylation status of NEDD8.…”

“…However, neither study investigated the phosphorylation status of NEDD8. Furthermore, in silico simulations predicted that NEDD8 binds to the same region of Parkin as pUb and induces the release of Parkin’s Ub-like domain 31 , which is critical for subsequent phosphorylation and complete activation of Parkin. We now confirm and refine this prediction by showing that pNEDD8 but not unmodified NEDD8 activates Parkin and propose that upon neuronal stress, NEDD8 is phosphorylated which leads to activation of Parkin.…”

The Many Potential Fates of Non-Canonical Protein Substrates Subject to NEDDylation

“…NEDD8 was shown to be a "molecular switch" whereby NEDDylation of VHL prevents its incorporation into a CRL and promotes its ability to interact with fibronectin [94]. Several reports have also demonstrated that Parkin activity was enhanced following NEDDylation [95][96][97]. Interestingly, VHL, Parkin, and Mdm2 (discussed previously) are all E3 ligases which contain a RING domain; however, NEDD8-enhancement of activity is not limited to RING ligases.…”