Eukaryotes have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins, and often these proteins are then targeted for degradation by the proteasome. Substrates include naturally short-lived regulatory factors and aberrant "protein quality control" (PQC) substrates. Many human disorders, including neurodegenerative diseases such as Alzheimer's and Parkinson's disease, diabetes, cystic fibrosis, and different forms of cancer, are associated with abnormalities in Ub-dependent proteolysis. The Ub-proteasome system presents promising drug targets for treating these diseases. In this renewal, the PI proposes to extend studies on Ub-dependent proteolysis, focusing on endoplasmic reticulum (ER)- associated degradation (ERAD) and basic features of membrane and nuclear protein ubiquitination and degradation. The proposed research will concentrate on the yeast Saccharomyces cerevisiae because of its experimental advantages and the fact that the Ub system in general, and the ERAD machinery in particular, is highly conserved. Recent work has identified a yeast Ub-ligase complex embedded in the ER and nuclear envelope membranes that is capable of recognizing a wide array of regulatory and PQC substrates. This unusual complex includes a large integral membrane Ub ligase (E3) called Doa10 and two Ub-conjugating enzymes (E2s), Ubc6 and Ubc7. Doa10 is the prototype for a broadly conserved class of viral and eukaryotic Ub ligases. It was discovered from an analysis of a soluble nuclear substrate, the Mat2 transcription factor, but it also has membrane substrates. The latter proteins need additional factors that are not required for the degradation of soluble targets. The overarching goal of the proposal is to define the key biochemical, structural and cell biological determinants of specificity for this highly conserved Ub-ligation system. The following Aims are proposed: (1) Determine how Doa10 interacts with its cofactors and recognizes its substrates by point mutagenesis coupled with genetic and biochemical assays;(2) Investigate how Doa10-dependent degradation of integral membrane proteins differs from that of soluble ones using matched soluble and membrane substrates bearing identical degradation signals (degrons);and (3) Compare different Doa10-dependent degrons to determine if common features characterize both naturally short-lived and PQC substrates and examine Doa10- dependent ubiquitination in the context of the native Mat2 protein.

Public Health Relevance

A small protein called ubiquitin regulates the growth and behavior of all human cells;ubiquitin is attached to specific cell proteins, and this directs these proteins to a large protein complex that then degrades them. Defects in the enzymes controlling these processes are known to cause neurodegenerative disorders, developmental abnormalities, and various cancers. This project aims to deepen our understanding of the enzymes that attach ubiquitin to damaged proteins as well as to important regulatory proteins, with the long-term goal of developing therapies to treat patients suffering from Alzheimer's disease, diabetes, cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM046904-22
Application #
8213489
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gerratana, Barbara
Project Start
1992-02-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
22
Fiscal Year
2012
Total Cost
$356,079
Indirect Cost
$140,457
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Hochstrasser, Mark (2016) Gyre and gimble in the proteasome. Proc Natl Acad Sci U S A 113:12896-12898
Berk, Jason M; Hochstrasser, Mark (2016) Protein Modification: Bacterial Effectors Rewrite the Rules of Ubiquitylation. Curr Biol 26:R539-42
Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark (2016) Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells. Cell Rep 14:2962-74
Li, Xia; Li, Yanjie; Arendt, Cassandra S et al. (2016) Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly. J Biol Chem 291:1991-2003
Hu, Ronggui; Hochstrasser, Mark (2016) Recent progress in ubiquitin and ubiquitin-like protein (Ubl) signaling. Cell Res 26:389-90
Zattas, Dimitrios; Berk, Jason M; Kreft, Stefan G et al. (2016) A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation. J Biol Chem 291:12105-18
Huber, Eva M; Heinemeyer, Wolfgang; Li, Xia et al. (2016) A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat Commun 7:10900
Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark (2016) Substrate specificity of the ubiquitin and Ubl proteases. Cell Res 26:441-56
Zattas, Dimitrios; Hochstrasser, Mark (2015) Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope. Crit Rev Biochem Mol Biol 50:1-17
Hickey, Christopher M; Hochstrasser, Mark (2015) STUbL-mediated degradation of the transcription factor MATα2 requires degradation elements that coincide with corepressor binding sites. Mol Biol Cell 26:3401-12

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