Alzheimer's associated variant ubiquitin causes inhibition of the 26S proteasome and chaperone expression

Intracellular protein inclusions in Alzheimer's disease and progressive supranuclear palsy contain UBB+1, a variant ubiquitin. UBB+1 is able block the 26S proteasome in cell lines. Proteasome inhibition by drug action has previously been shown to induce a heat-shock response and render protection against stress. We investigated UBB+1 by developing a stable, conditional expression model in SH-SY5Y human neuroblastoma cells. Induction of UBB+1 expression caused proteasome inhibition as was confirmed by reduced ability to process misfolded canavanyl proteins, accumulation of GFPu, a proteasome substrate, and reduced cleavage of a fluorogenic substrate. We show that expression of UBB+1 induces expression of heat-shock proteins. This priming of the chaperone system in these cells promotes a subsequent resistance to tert-butyl hydroperoxide-mediated oxidative stress. We conclude that although UBB+1-expressing cells have a compromised ubiquitin-proteasome system, they are protected against oxidative stress conditions.     

The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy—the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼ 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.     

Structural Basis of E2–25K/UBB+1 Interaction Leading to Proteasome Inhibition and Neurotoxicity

E2–25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB⁺¹) and has been identified as a crucial factor regulating amyloid-β neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2–25K-UBB⁺¹ interaction, we determined the three-dimensional structures of UBB⁺¹, E2–25K and the E2–25K/ubiquitin, and E2–25K/UBB⁺¹ complex. The structures revealed that ubiquitin or UBB⁺¹ is bound to E2–25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2–25K mutants showed that disrupting UBB⁺¹ binding markedly diminishes synthesis of neurotoxic UBB⁺¹-anchored polyubiquitin. These results suggest that the interaction between E2–25K and UBB⁺¹ is critical for the synthesis and accumulation of UBB⁺¹-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.