GFP reporter mouse models of UPS proteolytic function.
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Animals↗
Biomedical subjects
Victoria Menéndez-Benito
Publications and source records attributed to Victoria Menéndez-Benito.
Endoplasmic reticulum stress compromises the ubiquitin-proteasome system.
The presence of endoplasmic reticulum (ER) stress and impaired ubiquitin-proteasome system (UPS) activity has been independently implicated in the pathophysiology of conformational diseases. Here, we reveal a link between ER stress and the functionality of the UPS. Treatment of cells with different ER stressors delayed the degradation of an ER reporter substrate and caused a subtle but consistent accumulation of three independent nuclear/cytosolic UPS reporter substrates. A similar signature increase was observed upon induction of ER stress in transgenic mice expressing a reporter substrate. Cells undergoing ER stress failed to clear efficiently UBB+1, an aberrant ubiquitin found in conformational diseases, which in turn caused general impairment of the UPS. We conclude that ER stress has a general inhibitory effect on the UPS. The compromised UPS during ER stress may explain the long-term gradual accumulation of misfolded proteins as well as the selective vulnerability of particular cell populations in conformational diseases.
Animals↗
Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.
A reliable and robust means of evaluating the functional status of ubiquitin-dependent proteolysis in living cells is to follow the turnover of readily detectable reporter substrates. During the past few years, several reporter substrates have been generated by use of the green fluorescent protein (GFP), which is converted for this purpose from a normally very stable protein into a short-lived substrate of the ubiquitin/proteasome system. These short-lived substrates are valuable tools providing researchers with unique information about the absence or presence of blockades in this system in living cells. We have recently generated the first transgenic mouse model for monitoring the ubiquitin/proteasome system based on the ubiquitous expression of a GFP-based proteasome substrate. Together these models can be used to study ubiquitin-dependent degradation in health and disease and for the identification of small synthetic compounds or proteins capable of modifying the activity of the system. In this chapter, we describe the basic principles of GFP-based reporter substrates, their strengths and weaknesses, and a number of protocols that can be used to study the ubiquitin/proteasome system in yeast, cell lines, and transgenic mice.
Animals↗
A transgenic mouse model of the ubiquitin/proteasome system.
Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.
Alzheimer Disease↗