In eukaryotic cells, proteins are often targeted to specfic compartments. This targeting ususally depends on signals within the proteins themselves that are recognized by specific intracellular receptors. One compartment of interest are peroxisomes. Most proteins are targeted to peroxisomes by virtue of sequences found at their carboxyl-termini that approximate the sequence -SerLysLeu. These are recognized by the protein Pex5p. Sequence variants in the proteins that participate in this protein targeting process have been associated with developmental disorders termed peroxisome biogenesis disorders. Through studies of the binding of a series of peptides to wild-type human Pex5p as well as two disease-associated sequence variants, we have developed a model that rationalizes the peroxisome targeting properties of many proteins within the human proteome. This model provides an explanation of the variation in the severity of disease with Pex5p sequence variation. We have now tested this model by synthesizing peptides that correspond to essentially all proteins encoded by the human genome that are targeted to the inside of the peroxisome by -SerLysLeu-like sequences. The results of binding studies confirm almost all aspects of the model. Furthermore, the affinities of the peptides for Pex5p have been compared with expression levels for the corresponding genes in a wide range of tissues suggesting co-evolution of targeting signals and expression levels with the avoidance of high-affinity targeting signals on highly expressed proteins. Processes such as inflammation involve cytokines that initiate often complex sigmaling pathways inside cells. Cytokines such as tumor necrosis factor alpha bind to receptors expressed on the surfaces of responsive cells. This binding triggers a signal transduction cascade that eventually leads to changes in gene expression including, in many cases, the expression of additional cytokines. These signal transduction cascades involve many protein-protein interactions. In addition, they also involve chemical modification of specific proteins including the addition and removal of phosphoryl groups and the addition and removal of small proteins such as ubiquitin or chains of ubiquitin. In the case of the tumor necrosis factor alpha-initiated signal tranduction pathway, many of the participating proteins include small structural domains organized around bound zinc ions. We have synthesized peptides that correpond to these zinc-binding domains and found that these protein fragments can often participate in specific protein-protein interactions and in protein modification reactions. In particular, we have found that zinc-binding domains from the protein A20 can both affect ubiquitation reactions involving other proteins and can, themselves, be modified by the addition of ubiquitin. These observations set the groundwork for more detailed studies directed toward determining the structural features that are required for participation in these reactions. We have now completed metal-binding studies on peptides corresponding to all seven zinc-binding domains in A20 and are testing a variety of assays for examining peptide-ubiquitin interactions.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$517,352
Indirect Cost
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State
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Wang, Renjing; Ilangovan, Udayar; Leal, Belinda Z et al. (2011) Identification of nucleic acid binding residues in the FCS domain of the polycomb group protein polyhomeotic. Biochemistry 50:4998-5007
Ghosh, Debdip; Berg, Jeremy M (2010) A proteome-wide perspective on peroxisome targeting signal 1(PTS1)-Pex5p affinities. J Am Chem Soc 132:3973-9
Berg, J M; Rogers, M E; Lyster, P M (2010) Systems biology and pharmacology. Clin Pharmacol Ther 88:17-9
Guerrerio, Anthony L; Berg, Jeremy M (2010) Design of single-stranded nucleic acid binding peptides based on nucleocapsid CCHC-box zinc-binding domains. J Am Chem Soc 132:9638-43
Jantz, Derek; Berg, Jeremy M (2010) Probing the DNA-binding affinity and specificity of designed zinc finger proteins. Biophys J 98:852-60
Davis, Alisa M; Berg, Jeremy M (2009) Homodimerization and heterodimerization of minimal zinc(II)-binding-domain peptides of T-cell proteins CD4, CD8alpha, and Lck. J Am Chem Soc 131:11492-7