A functional analysis of PCNA-binding peptides derived from protein sequence, interaction screening and rational design.
Proliferating cell nuclear antigen (PCNA) has no intrinsic enzymatic function, but functions as a sliding platform to mediate protein interactions with the DNA strand. Many proteins interact with PCNA through a small conserved motif with consensus QxxLxxFF. This work uses Schizosaccharomyces pombe and human cells to analyse the function of PCNA-binding peptides. Interacting peptides were identified using two-hybrid screening; one (pep102) binds directly to a physiologically relevant site on PCNA. The EGFP-pep102 overexpression phenotype is consistent with competitive blocking of PCNA-protein interactions. Various PCNA-binding peptides were all shown to inhibit PCNA function by competitive binding in both human and S. pombe cells as EGFP fusion proteins. The action of a p21(WAF1/Cip1)-derived peptide was complicated by the presence of additional functional domains and possible post-translational modification. The activity of pep102 was hampered by low expression in both model systems. The peptide derived from rational design (con1) was stable, highly active in inhibiting PCNA function both S. pombe and human cells and showed a high affinity for PCNA both in vitro and in vivo. These results validate the use of functional screening in yeast to identify peptide aptamers that are functional in mammalian cells; such aptamers provide excellent leads for small molecule antiproliferative therapies.
Warbrick
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Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, UK. c.warbrick@dundee.ac.uk
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003114:Colony-Forming Units Assay; D015195:Drug Design; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins