Ube2g2 binding domain in the ubiquitin ligase gp78 and methods of use thereof
Inventors
Weissman, Allan • Mariano, Jennifer • Chen, Bo • Tsai, Yien Che
Assignees
DEPARTMENT OF HEALTH AND HUMAN SERVICES GOVERNMENT OF United States, Secretary of • US Department of Health and Human Services
Publication Number
US-8420776-B2
Publication Date
2013-04-16
Expiration Date
2025-04-27
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Abstract
The present invention features isolated nucleic acid molecules, designated G2BD nucleic acid molecules, which encode the binding site from the gp78 ubiquitin ligase that binds to the Ube2G2 ubiquitin conjugating enzyme. The invention further provides isolated G2BD proteins and fusion proteins. Still further provided are diagnostic and therapeutic methods, as well as screening assays utilizing compositions of the invention.
Core Innovation
The invention features isolated nucleic acid molecules, designated G2BD nucleic acid molecules, encoding the binding site from the gp78 ubiquitin ligase that binds specifically to the Ube2G2 ubiquitin conjugating enzyme. It further provides isolated G2BD proteins and fusion proteins, and methods of use including diagnostic and therapeutic methods, as well as screening assays utilizing these compositions. The G2BD serves as a modulating agent in regulating cellular processes such as endoplasmic reticulum-associated degradation (ERAD).
The problem addressed arises from the need to modulate the activity of gp78, a ubiquitin ligase critically involved in ERAD and human disease. Detailed understanding of how gp78 binds to its cognate E2 enzyme, Ube2G2, was lacking, including the specific binding domain and the capacity to regulate this interaction. Given gp78's role in ERAD, cell migration, and metastasis, controlling its activity could impact conditions like cystic fibrosis, cancer invasiveness, and other ERAD associated disorders.
Claims Coverage
The patent provides two independent claims covering isolated polypeptides for binding Ube2G2 and methods of detecting Ube2G2 using these polypeptides.
Isolated polypeptides comprising specific sequences for Ube2G2 binding
The invention covers isolated polypeptides consisting of the amino acid sequence Q-R-M-L-V-Q-R-K-D-E-L-L-Q-Q-A-R-K-R-F-L-N-K (SEQ ID NO:34), polypeptides with at least 80% or 90% sequence identity to SEQ ID NO:34, or polypeptides of 22-30 amino acids containing SEQ ID NO:34 or closely related sequences including conservative variations and consensus sequences defined by sequences such as A-A-A-A-G-Q-R-M-L-V-Q-Xn-R-F-L-N-K (SEQ ID NO:59) or Q-R-M-L-V-Q-Xn-R-F-L-N-K (SEQ ID NO:48), where X is any amino acid and n is from 0 to 50.
Method of detecting Ube2G2 using G2BD polypeptides
Providing a method of detecting Ube2G2 in a sample by contacting the sample with the aforementioned isolated polypeptides and determining the binding of the polypeptide to Ube2G2 to thereby detect its presence.
The claims focus on isolated polypeptides containing the specific G2BD sequences for Ube2G2 binding with defined identities and lengths, and methods of using these polypeptides to detect Ube2G2 in biological samples, emphasizing the specific interaction and utility in detection.
Stated Advantages
The G2BD nucleic acid and protein molecules serve as novel diagnostic targets and therapeutic agents for ERAD-associated disorders, including cystic fibrosis and various cancers.
Modulation of G2BD activity enables regulation of ERAD processes, potentially increasing cell surface expression of proteins such as CFTR and MHC class I molecules.
Use of the G2BD molecules can suppress cancer invasiveness and metastasis by blocking E2 binding and gp78 function.
Expression of the isolated Ube2G2 binding domain can limit ERAD with little toxicity, offering therapeutic and bioproduction advantages.
Documented Applications
Treatment of ERAD-associated disorders such as cystic fibrosis by modulating ERAD.
Increasing expression of cell surface receptors and proteins that traverse the secretory pathway, including MHC class I molecules.
Use in cancer treatment to suppress tumor invasiveness and metastasis.
Screening assays to identify reagents that block G2BD on endogenous gp78 or modulate ERAD.
Diagnostic methods for detecting Ube2G2 levels using labeled G2BD polypeptides.
Enhancement of recombinant antibody production and biotechnology applications via modulation of ERAD.
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