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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gerratana, Barbara
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Medicine
New Haven
United States
Zip Code
Hickey, Christopher M; Xie, Yang; Hochstrasser, Mark (2018) DNA binding by the MAT?2 transcription factor controls its access to alternative ubiquitin-modification pathways. Mol Biol Cell 29:542-556
Budenholzer, Lauren; Cheng, Chin Leng; Li, Yanjie et al. (2017) Proteasome Structure and Assembly. J Mol Biol 429:3500-3524
Ronau, Judith A; Hochstrasser, Mark (2017) The DUB blade goes snicker-snack: Novel ubiquitin cleavage by a Legionella effector protein. Cell Res 27:845-846
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
Berk, Jason M; Hochstrasser, Mark (2016) Protein Modification: Bacterial Effectors Rewrite the Rules of Ubiquitylation. Curr Biol 26:R539-R542
Hochstrasser, Mark (2016) Gyre and gimble in the proteasome. Proc Natl Acad Sci U S A 113:12896-12898
Hu, Ronggui; Hochstrasser, Mark (2016) Recent progress in ubiquitin and ubiquitin-like protein (Ubl) signaling. Cell Res 26:389-90
Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark (2016) Substrate specificity of the ubiquitin and Ubl proteases. Cell Res 26:441-56
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
Li, Xia; Li, Yanjie; Arendt, Cassandra S et al. (2016) Distinct Elements in the Proteasomal ?5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly. J Biol Chem 291:1991-2003

Showing the most recent 10 out of 28 publications