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1.  Ubiquitin-Binding Site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23 
Nature communications  2014;5:4638.
Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by Ubiquitin-Binding Site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.
doi:10.1038/ncomms5638
PMCID: PMC4237202  PMID: 25144244
Deubiquitinase; Drosophila; Neurodegeneration; Neuroprotection; Polyglutamine; Proteasome; hHR23; Spinocerebellar Ataxia; Ubiquitin
2.  Ubiquitin-Specific Protease 25 Functions in Endoplasmic Reticulum-Associated Degradation 
PLoS ONE  2012;7(5):e36542.
Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome.
doi:10.1371/journal.pone.0036542
PMCID: PMC3348923  PMID: 22590560
3.  An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein 
AoB Plants  2012;2012:pls003.
Filter cubes made with machine-vision dichroic filters and illuminated with a royal blue light emitting diode can be used to produce an epifluorescent digital camera attachment that improves whole organism green fluorescent protein (GFP) photography. Mean pixel intensity responds linearly to purified GFP titration.
Background and aims
Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants.
Methodology
Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically.
Principal findings
Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29).
Conclusions
Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets.
doi:10.1093/aobpla/pls003
PMCID: PMC3296078  PMID: 22479674

Results 1-3 (3)