Direct cellular delivery of human proteasomes to delay tau aggregation

Abstract

The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins.

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

“…Native gel analysis using purified proteasomes or whole cell lysate analysis was performed largely as previously described [15]. Samples were resolved by 3.5 % nondenaturing PAGE for 850 volt-hours and proteasomes were visualized using the fluorogenic substrate suc-LLVY-AMC (Bachem).…”

“…A genetic knockdown of Ubp6, the ortholog of mammalian USP14, accelerates degradation of various target substrates [11], suggesting that USP14 may be a potential therapeutic target in diseases involving accumulation of toxic proteins, such as Alzheimer's and Parkinson's diseases [14, 15]. The noncatalytic inhibition of the proteasome by USP14 is mediated by the direct interaction with the ATPase ring of the proteasome; this process results in strong interference with RPN11 function and with a conformational change of the proteasome for proper substrate translocation [16].…”

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

“…63 MAPT-BiFC cells were seeded in a 96-well plate at a density of 10 5 cells/well and treated with 30 nM okadaic acid (LC Laboratories, O-5857) for 24 h to accelerate MAPT oligomerization. The cells were then treated with PCA (50 mM) for 4 h. Fluorescence images were quantified with ImageJ software (ver.…”

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

“…Large-pore MSNs were surface-functionalized with NTA-Ni complexes (MSN-Ni) through a series of modifications (Figure 2(a)) to accommodate pH dependent binding and release of the His-tagged FRB-VC[10,11,19]. STEM and SEM images (Figure 2(b)) indicated that the resulting nitrilotriacetic acid-modified MSNs (MSN-NTAs) exhibit uniform particle size and coral surface morphology with irregular pore shape (pore size ranging from 10 – 40 nm) consistent with the structure of the un-functionalized form (Figure S1).…”