Sending proteins to the lysosome for degradation is one of the chief mechanisms cells use to control the activity of cell-surface proteins. Ubiquitination of membrane proteins serves as a major signal for their sorting to lysosomes. The post-translational ligation of ubiquitin (Ub) to target proteins initiates their internalization from the cell surface and their transport into lumenal vesicles of multivesicular endosomes/multivesicular bodies (MVBs). A series of ESCRTs (Endosomal Sorting Complex Required for Transport: e.g. ESCRT-0,I,II,III)) are known to coordinate the sorting of ubiquitinated membrane protein cargo (Ub-cargo) with the formation of MVB lumenal vesicles and thus play critical roles in the process of lysosomal degradation. Yet, how ESCRT proteins recognize and move Ub-cargo as well as how they might participate in coordinating the activity of Ub ligases and Ub peptidases to modify and thus regulate the fate of Ub-cargo are unclear. The work proposed here has two main objectives: The first is to establish which proteins serve as endosomal Ub sorting receptors, and how they move Ub-cargo in coordination with their other functions. The second objective centers on the emerging concept that since ubiquitination is dynamic, there are many instances in which Ub ligases and peptidases can compete for the final disposition of cargo. We now propose that other protein complexes that work in parallel as "ESCRT-0- like" proteins, as well as other endosomal sorting complexes, associate with DUbs and ligases to acutely influence the sorting fate of specific Ub-cargo.
The Specific Aims are to: Investigate the roles of Ub-binding by ESCRT-I and -II. Test the function of alternate "ESCRT-0-like" complexes. Test the role of Deubqiutinating enzymes in recycling proteins from endosomes

Public Health Relevance

Failure to target particular membrane proteins to lysosomes leads to inappropriately hyperactive channels, transporters, or signaling receptors, which in turn contribute to a number of diseases including cancer, metabolic disorders, developmental abnormalities, heart disease, and hypertension. The work proposed here will investigate the biochemical and cell biological processes that ensure proper recognition and sorting of membrane proteins that are marked by ubiquitin for degradation in lysosomes. We believe that such understanding will open new avenues for therapeutic interventions that manipulate the process of lysosomal degradation to target a range of biological processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058202-16
Application #
8511684
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Ainsztein, Alexandra M
Project Start
1998-08-05
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
16
Fiscal Year
2013
Total Cost
$328,044
Indirect Cost
$110,796
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Piper, Robert C; Dikic, Ivan; Lukacs, Gergely L (2014) Ubiquitin-dependent sorting in endocytosis. Cold Spring Harb Perspect Biol 6:
Ver Heul, Aaron M; Fowler, C Andrew; Ramaswamy, S et al. (2013) Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2. J Biol Chem 288:6890-902
Pashkova, Natasha; Gakhar, Lokesh; Winistorfer, Stanley C et al. (2013) The yeast Alix homolog Bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes. Dev Cell 25:520-33
Kamadurai, Hari B; Qiu, Yu; Deng, Alan et al. (2013) Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. Elife 2:e00828
MacDonald, Chris; Buchkovich, Nicholas J; Stringer, Daniel K et al. (2012) Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation. EMBO Rep 13:331-8
Pashkova, Natasha; Piper, Robert C (2012) UBAP1: a new ESCRT member joins the cl_Ub. Structure 20:383-5
Stringer, Daniel K; Piper, Robert C (2011) A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination. J Cell Biol 192:229-42
Piper, Robert C; Lehner, Paul J (2011) Endosomal transport via ubiquitination. Trends Cell Biol 21:647-55
Shields, S Brookhart; Piper, Robert C (2011) How ubiquitin functions with ESCRTs. Traffic 12:1306-17
Pashkova, Natasha; Gakhar, Lokesh; Winistorfer, Stanley C et al. (2010) WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins. Mol Cell 40:433-43

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