Psoriasis Upregulated Phorbolin-1 Shares Structural but not Functional Similarity to the mRNA-Editing Protein Apobec-1 Peder Madsen, Julio E. Celis,

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Psoriasis Upregulated Phorbolin-1 Shares Structural but not Functional Similarity to the mRNA-Editing Protein Apobec-1 Peder Madsen, Julio E. Celis, Hanne H. Rasmussen, Henrik Vorum Journal of Investigative Dermatology Volume 113, Issue 2, Pages 162-169 (August 1999) DOI: 10.1046/j.1523-1747.1999.00682.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nucleotide and deduced amino acid sequence of the phorbolin-1 cDNA. Nucleotide and corresponding amino acid sequence of the cDNA clone encoding for isoelectric focusing SSP 2116 (Rasmussen & Celis 1993). The cDNA, which contains 1348 bp, codes for a protein having 199 amino acids starting at nucleotide position 76 and ending with a termination codon at position 675. Potential AUG start codons are marked with asterisks, and partial peptide sequences obtained by microsequencing are underlined. Accession number U03891. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of clone 4359. (A) 2D PAGE autoradiogram of [35S]methionine-labeled polypeptides from COS-1 cells transfected with clone 4359, inserted into the pMT21 eukaryotic expression vector. Overexpressed proteins are indicated by arrows, these proteins are not detected in non-transfected COS-1 cells (not shown). (B) 2D PAGE autoradiogram of [35S]methionine-labeled polypeptides from primary human keratinocytes treated for 20 h with PMA (100 ng per ml), 5 d after plating. PMA-induced proteins are indicated by name. (C) 2D immunoblot of COS-1 cells transfected with clone 4359, using a rabbit anti-serum directed against the C-terminal of clone 4359. (D) 2D PAGE autoradiogram of clone 4359 assayed in the in vitro transcription/translation system. (E) 2D PAGE autoradiogram of the phorbolin-1-related cDNA expressed by the in vitro transcription/translation system. Overexpressed proteins in C–E are indicated by arrows, acidic variants are indicated by arrowheads. Asterisks in D and E indicates a common translational product that is not related to the expressed cDNA, as this protein also is detected when expressing unrelated cDNA (Madsen et al. 1995). Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Alignment of the phorbolins and apobec-1. Identical residues are highlighted in a black background whereas similar residues are in gray. The region containing the active site and zinc coordinating spans residues 61–96 in the apobec-1 amino acid sequence. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Nucleotide and deduced amino acid sequence of the phorbolin-1-related cDNA. Nucleotide and corresponding amino acid sequence of the phorbolin-1-related cDNA clone. The cDNA, which contains 1526 bp, codes for a protein having 235 amino acids starting at nucleotide position 486 and ending with a termination codon at position 1191. The in-frame upstream stop codon at position 372 is underlined and in italics. ORF start codons are numbered with their corresponding nucleotide positions and are marked with asterisks. uORF AUG codons are underlined. Accession number U61084. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Phylogenetic analysis of the active sites of cytidine deaminases and the Phorbolin-1. The active site domain containing the zinc coordinating and proton donor residues were aligned as previously described based on the crystal structure of the E. coli cytidine deaminase (Betts et al. 1994;Anant et al. 1997). The distance matrix between the various cytidine deaminases was calculated using the PROTDIST program and used to generate a tree by the KITSCH program. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 In vitro RNA editing assay. A 470 nucleotide apoB RNA was incubated with 10 μg of chicken enterocyte S-100 extract and in vitro transcribed/translated proteins, followed by primer extension using a radiolabeled primer (Anant et al. 1995). Lane 1, in vitro transcription/translation extract without plasmid DNA; lane 2, in vitro synthesized apobec-1; lane 3, in vitro synthesized phorbolin-1; lane 4, in vitro synthesized phorbolin-1 plus apobec-1; lane 5, in vitro synthesized PA-FABP (Madsen et al. 1992) plus apobec-1; lane 6, bacterially expressed and purified GST/apobec-1; lane 7, negative control, apoB RNA alone. This is a representative of two independent assays. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Phorbolin-1-related does not bind to an apoB RNA template. Ultraviolet cross-linking was performed using a radiolabeled 105 nucleotide rat apoB RNA template in the presence of 0.25 or 1 μg of recombinant GST/APOBEC-1 or GST/phorbolin-1. After digestion with RNAse T1, the samples were size fractionated in to a 10% SDS–PAGE gel, dried, and autoradiographed. Molecular weight markers are shown to the left. This is a representative of two independent assays. Journal of Investigative Dermatology 1999 113, 162-169DOI: (10.1046/j.1523-1747.1999.00682.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions