This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ubiquitin signaling is an important mechanism is nearly every cellular process. E3 ubiquitin ligases specify the substrate and catalyze the transfer from an E2 ubiquitin conjugating enzyme to that protein. Though E3 enzymes are a large and well-studied class of proteins, little is known about how the E3 catalyzes this transfer. We plan to interrogate the functional consequences of mutation at every amino acid of an E3 ligase. To this end, we have chosen to study the U-box domain of UBE4B and constructed a library of 1 million UBE4B mutants displayed on the coat of T7 phage. We use the UBE4B phage in in vitro ubiquitination reactions to test their E3 activity. With the addition of E1 and E2 enzymes, the UBE4B catalyzes autoubiquitination using Flag-tagged ubiquitin. This procedure allows us to select for enzymatically active UBE4B-phage by incubation with anti-Flag beads. Nonspecifically bound phage are washed away and bound phage are eluted by competition with Flag peptide. The eluted phage are amplified and subjected to more rounds of selection. Using high throughput sequencing to determine genotypes in the input pool of phage versus selected phages, we can track how each mutation performs during the selection experiments. This experiment will give us insight into the function of every amino acid in the U-box domain of UBE4B and may reveal the functional elements of E3 catalysis.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CB-L (40))
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Washington
Schools of Medicine
United States
Zip Code
Shan, Chun-Min; Wang, Jiyong; Xu, Ke et al. (2016) A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading. Elife 5:
Kim, Tae Kwon; Tirloni, Lucas; Pinto, Antônio F M et al. (2016) Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding. PLoS Negl Trop Dis 10:e0004323
McBride, Ryan; Paulson, James C; de Vries, Robert P (2016) A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins. J Vis Exp :
McClatchy, D B; Savas, J N; Martínez-Bartolomé, S et al. (2016) Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex. Mol Psychiatry 21:205-15
Wang, Jiyong; Cohen, Allison L; Letian, Anudari et al. (2016) The proper connection between shelterin components is required for telomeric heterochromatin assembly. Genes Dev 30:827-39
Di Maggio, Lucía Sánchez; Tirloni, Lucas; Pinto, Antonio F M et al. (2016) Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study. Sci Rep 6:32796
Homer, Christina M; Summers, Diana K; Goranov, Alexi I et al. (2016) Intracellular Action of a Secreted Peptide Required for Fungal Virulence. Cell Host Microbe 19:849-64
Silva, Erica; Betleja, Ewelina; John, Emily et al. (2016) Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left-right asymmetry. Mol Biol Cell 27:48-63
Tang, Wen; Tu, Shikui; Lee, Heng-Chi et al. (2016) The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. elegans. Cell 164:974-84
Carvalho, Paulo C; Lima, Diogo B; Leprevost, Felipe V et al. (2016) Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0. Nat Protoc 11:102-17

Showing the most recent 10 out of 561 publications