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Background: Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes., Principal Findings: To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates., Conclusions: Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.

Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration. [ABSTRACT FROM AUTHOR]

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with ‘rasosomes’, randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function. [ABSTRACT FROM AUTHOR]