The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have previously reported UBR1 and UBR2 as its functionally overlapping E3s and elucidated their in vivo roles using mouse knockout approach. Unexpectedly, although both UBR1 and UBR2 strongly bound to N-degrons, UBR1-/-UBR2-/- cells still retained the N-end rule E3 activities, indicating the presence of yet unidentified N-recognins (N-degron-Recognizing E3s). The goal of this study is to identify and characterize N-recognins and their substrates and to elucidate the physiological meaning of their E3-substrate interaction. To identify mammalian N-recognins, we developed a novel affinity-based proteomic approach using synthetic peptides bearing N-degron, yielding a novel 570 kDa-protein named UBR4 and a 300 kDa-E3 ligase EDO (termed UBR5). UBR1, -2, -4, and -5 shared a zinc finger-like domain named the UBR box. Mammalian genome appears to encode seven UBR proteins, named UBR1 through UBR7. Further, by using a functional proteomic approach, we have obtained -35 candidate N-end rule substrates from -14,000 different proteins expressed in the rabbit reticulocyte lysates. Preliminary characterization of candidate substrates unveiled several novel in vivo N-end rule substrates (RGS4, RGS5, RGS16, and CDC6), the first to be identified in mammals. To further extend our current understanding of the N-end rule pathway, we propose the following Aims.
Aim 1. To characterize UBR box proteins as candidate N-recoqnins. We will examine: 1) proteolysis of model N-end rule substrates in UBR mutant cells, 2) the interaction and specificity of UBR box proteins with N-degrons, and 3) in vitro ubiquitylation of model substrates by UBR box proteins.
Aim 2. To determine whether UBR box motif is the recognition domain for N-degron. We will determine whether the UBR boxes of N-recognins are required and sufficient for direct binding to N-degrons and identify essential residues for recognition of N-degron.
Aim 3. To identify physiological N-end rule substrates. We will dissect proteolysis of candidate substrates in reticulocyte lysates and UBR mutant cells, determine the N-recognin-substrate interaction, and test whether N-recognins support substrate ubiquitylation in vitro.
Aim 4. To dissect physiological processes underlying identified N-end rule substrates. We will examine the physiological significance underlying the N-end rule dependent proteolysis of RGS4, -5, and -16, emerging in vivo N-end rule substrates. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074000-02
Application #
7174858
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Shapiro, Bert I
Project Start
2006-02-01
Project End
2010-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
2
Fiscal Year
2007
Total Cost
$265,603
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Meisenberg, Cornelia; Tait, Phillip S; Dianova, Irina I et al. (2012) Ubiquitin ligase UBR3 regulates cellular levels of the essential DNA repair protein APE1 and is required for genome stability. Nucleic Acids Res 40:701-11
Yang, Fang; Cheng, Yong; An, Jee Young et al. (2010) The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis. PLoS One 5:e14017
Tasaki, Takafumi; Zakrzewska, Adriana; Dudgeon, Drew D et al. (2009) The substrate recognition domains of the N-end rule pathway. J Biol Chem 284:1884-95
Sriram, Shashikanth M; Banerjee, Rajkumar; Kane, Ravi S et al. (2009) Multivalency-assisted control of intracellular signaling pathways: application for ubiquitin- dependent N-end rule pathway. Chem Biol 16:121-31
Lee, Min Jae; Pal, Krishnendu; Tasaki, Takafumi et al. (2008) Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway. Proc Natl Acad Sci U S A 105:100-5
Tasaki, Takafumi; Kwon, Yong Tae (2007) The mammalian N-end rule pathway: new insights into its components and physiological roles. Trends Biochem Sci 32:520-8
Tasaki, Takafumi; Sohr, Reinhard; Xia, Zanxian et al. (2007) Biochemical and genetic studies of UBR3, a ubiquitin ligase with a function in olfactory and other sensory systems. J Biol Chem 282:18510-20
Hu, Rong-Gui; Brower, Christopher S; Wang, Haiqing et al. (2006) Arginyltransferase, its specificity, putative substrates, bidirectional promoter, and splicing-derived isoforms. J Biol Chem 281:32559-73
An, Jee Young; Seo, Jai Wha; Tasaki, Takafumi et al. (2006) Impaired neurogenesis and cardiovascular development in mice lacking the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway. Proc Natl Acad Sci U S A 103:6212-7