Application and Analysis of the GFPu Family of Ubiquitin‐Proteasome System Reporters

Bence, Neil; Bennett, Eric J.; Kopito, Ron R.

doi:10.1016/s0076-6879(05)99033-2

Cited by 77 publications

(93 citation statements)

References 11 publications

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“…For chase analysis, HeLa cells were treated with 75 mg ml À 1 cycloheximide and samples were isolated at chase times 0, 0.5, 1 and 1.5 h. For overexpression of GFPu 30 and GFP-cODC 31 , a fusion of the CL1 and ornithine decarboxylase (ODC) degrons on the carboxyl terminus of EGFP, respectively, HeLa cells were transfected with 2-4 mg of plasmid DNA in a six-well plate for 4 h using Lipofectamine 3000 (Invitrogen). Cells were lysed 36-48 h post transfection in RIPA buffer after treated with 50 mg ml À 1 of MSNPN or proteasome-MSNPN complexes for 4 h. Total cell lysates were analysed by immunoblotting with anti-GFP antibody (Enogene).…”

Section: Discussionmentioning

confidence: 99%

Direct cellular delivery of human proteasomes to delay tau aggregation

Han

Choi

et al. 2014

Nat Commun

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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.

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Section: Discussionmentioning

confidence: 99%

Direct cellular delivery of human proteasomes to delay tau aggregation

Han

Choi

et al. 2014

Nat Commun

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“…GFPu (in EGFP-C1 plasmid backbone; Clontech, Palo Alto, CA), a fusion of the CL1 degron (degradation signal) on the carboxyl terminus of GFP, was kindly provided by Ron Kopito (Stanford University, Palo Alto, CA). GFPu is ubiquitinated and specifically degraded by the proteasome (27)(28)(29). GFP-odc (in EGFP-C1 plasmid backbone; Clontech), a fusion of the ornithine decarboxylase (odc) degron on the carboxyl terminus of GFP, was also kindly provided by Ron Kopito.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Djakovic

Schwarz

Baryłko

et al. 2009

Journal of Biological Chemistry

209

213

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Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulindependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

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“…The aggregate fraction on the membrane was detected with an anti-CryAB antibody. The activity of the ubiquitin-proteosome system (UPS) was estimated using a modified GFP construct, which was kindly provided by Dr. Kopito (Stanford University) (20). The modified GFP construct was used to generate adenoviral constructs as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…The UPS controls many cellular processes, and the disturbance of this system is thought to be one of the causal mechanisms of the amyloid degenerative disease (20). Recent findings indicate that in situ UPS activity using modified GFP signals correlates much more with cellular conditions than does enzyme activity in the lysate (25).…”

Section: Inhibitory Effects Of Hsp22 and Hsp25 On Amyloid Oligomer Fomentioning

confidence: 99%

Interruption of CryAB-Amyloid Oligomer Formation by HSP22

Sanbe

Yamauchi

Miyamoto

et al. 2007

Journal of Biological Chemistry

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An R120G missense mutation in ␣-B-crystallin (CryAB), a small heat-shock protein (HSP), causes a desmin-related cardiomyopathy (DRM) that is characterized by the formation of aggregates containing CryAB and desmin. The mutant CryAB protein leads to the formation of inclusion bodies, which contain amyloid oligomer intermediates (amyloid oligomer) in the cardiomyocytes. To further address the underlying mechanism(s) of amyloid oligomer formation in DRM linked to the CryAB R120G, a recombinant CryAB R120G protein was generated. The purified CryAB R120G protein can form a toxic amyloid oligomer, whereas little immunoreactivity was observed in the wild-type CryAB protein. A native PAGE showed that the oligomerized form was present in the CryAB R120G protein, whereas only a high molecular mass was detected in the wildtype CryAB. The oligomerized CryAB R120G of around 240 -480 kDa showed strong positive immunoreactivity against an anti-oligomer antibody. The CryAB R120G amyloid oligomer was unstable and easily lost its conformation by ␤-mercaptoethanol and SDS. Recombinant HSP25 or HSP22 proteins can directly interrupt oligomer formation by the CryAB R120G protein, whereas the amyloid oligomer is still present in the mixture of the wild-type CryAB and CryAB R120G proteins. This interruption by HSP25 and HSP22 was confirmed in a cardiomyocyte-based study using an adenoviral transfection system. Blockade of amyloid oligomer formation by HSP25 and HSP22 recovered the ubiquitin proteosomal activity and cellular viability. Blockade of oligomer formation by small HSP may be a new therapeutic strategy for treating DRM as well as other types of amyloid-based degenerative diseases.Many systemic and neurodegenerative disorders whose etiologies are linked to misfolded or unfolded proteins are characterized by the accumulation of intracellular or extracellular protein deposits or aggregates (1). The pathologies of these diseases are complex, and the accumulation of misfolded and unfolded proteins in the cells can contribute to or be causal for at least some of these neurodegenerative and systemic diseases (2). However, the direct relationship between the protein deposition and the disease pathology is still controversial. Recent reports suggest that amyloid ␤ (A␤) 2 and other amyloidogenic proteins exert their cellular toxicity as soluble amyloid oligomeric intermediates (amyloid oligomer) but not as insoluble aggregates or fibrils (3-6). These soluble amyloid oligomer proteins generally appear to have ␤-sheet structures, whose formation is correlated with the appearance of a hydrophobic environment (6, 7). Several reports indicate that the soluble amyloid oligomer may be more important in pathogenesis than the insoluble fibrillar amyloid deposits (8 -11). An antibody that specifically recognizes the structure of the amyloid oligomer reacts with oligomers generated from all types of amyloidogenic proteins and peptides, such as A␤-(1-42), ␣-synuclein, polyglutamine, and prions (6). This result implies that the amyloid oligomer has ...

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Application and Analysis of the GFPu Family of Ubiquitin‐Proteasome System Reporters

Cited by 77 publications

References 11 publications

Direct cellular delivery of human proteasomes to delay tau aggregation

Direct cellular delivery of human proteasomes to delay tau aggregation

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Interruption of CryAB-Amyloid Oligomer Formation by HSP22

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