Otein radiolabeled by incubation with [3Hmethyl] AdoMet and HsCaM KMT in panel (A) as CaM by MS/MS analysis. Peptides identified after tryptic digestion are shown in bold, approximately 60 of the entire CaM sequence was identified. The arrow indicates the position of HsCaM KMT. doi:10.1371/journal.pone.0052425.gCaM KMT Interacts with Hsp90 Molecular ChaperonTo search for cellular proteins that specifically interact with CaM KMT, lysates of HEK293 cells expressing FLAG-CaM KMT were immunoprecipitated with Peptide M supplier anti-FLAG antibody. The immunoprecipitates were Coomassie stained and one predominant protein band of about 90 kDa that Tubastatin A appeared to specifically co-purify with FLAG-CaM KMT could be distinguished. The other less intensive bands of ,70 kDa were revealed as nonspecific in additional experiments (Fig. 4A). The 90 kDa band was excised from the Coomassie stained gel, subjected to mass spectrometry analysis and identified as the alpha and beta isoforms of the molecular chaperon Hsp90. The sequenced peptides represent 26 coverage of the amino acid sequences and allow differentiating between the a and b isoforms of Hsp90 (Fig. 4B) suggesting that both of them interact with CaM KMT. Human Hsp90a and Hsp90b homologs show approximately 85 identity to each other with molecular masses of 84 and 83 kDa, respectively. These homologs exhibit similar participation in multi-chaperon complexes and interact with the same substrates under normalconditions [17]. To ascertain the association between CaM KMT and Hsp90 we transiently transfected HEK293 cells with Myc-CaM KMT and performed immunoprecipitation with a monoclonal anti-Myc antibody. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with antiHsp90 a/b antibody. In agreement with the mass spectrometry results CaM KMT was found to bind 18325633 Hsp90 (Fig. 4 C-left). Conversely, the transfected cell lysates were precipitated with antiHsp90 antibody and then probed with anti-Myc (Fig. 4 C-right). Thus, CaM KMT and Hsp90 proteins are suggested to be in a protein complex.CaM KMT Binds to the Middle Domain of HspSequence alignments and proteolytic digests of Hsp90 have shown a modular structure of three domains: the N-terminal is an ATP binding domain; the C-terminal domain mediates the dimerization of the chaperons and the middle domain acts as a discriminator between different types of client and co-chaperon proteins [18,19]. Therefore, we next asked whether the interactionCharacterization of CaM KMTFigure 3. Subcellular localization of the CaM KMT-GFP fusion proteins in transiently transfected cells and expression in mouse tissues. (A) GFP- CaM KMT is localized in the cytoplasm and the nucleus. Confocal images of HeLa cells expressing CaM KMT-GFP (green), nuclear staining by DAPI (blue) and the merged image. (B) The expression of the GFP only. Confocal images of HeLa cells expressing GFP (green), staining ofCharacterization of CaM KMTnuclei by DAPI (blue), and the merged image. (C) Cell lysates (100 mg of protein/lane) from mouse muscle, heart, liver, kidney, brain and spleen were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with an affinity purified polyclonal anti-CaM KMT antibody (1) immune and (2) pre-immune serum. Anti-HSP90 antibody served for protein loading control, 100 mg protein/lane were analyzed. Positions of CaM KMT and HSP-90 are indicated by the arrows. (D) GFP- CaM KMTsh is localized to the Golgi. COS-7 cells were transfected with the G.Otein radiolabeled by incubation with [3Hmethyl] AdoMet and HsCaM KMT in panel (A) as CaM by MS/MS analysis. Peptides identified after tryptic digestion are shown in bold, approximately 60 of the entire CaM sequence was identified. The arrow indicates the position of HsCaM KMT. doi:10.1371/journal.pone.0052425.gCaM KMT Interacts with Hsp90 Molecular ChaperonTo search for cellular proteins that specifically interact with CaM KMT, lysates of HEK293 cells expressing FLAG-CaM KMT were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were Coomassie stained and one predominant protein band of about 90 kDa that appeared to specifically co-purify with FLAG-CaM KMT could be distinguished. The other less intensive bands of ,70 kDa were revealed as nonspecific in additional experiments (Fig. 4A). The 90 kDa band was excised from the Coomassie stained gel, subjected to mass spectrometry analysis and identified as the alpha and beta isoforms of the molecular chaperon Hsp90. The sequenced peptides represent 26 coverage of the amino acid sequences and allow differentiating between the a and b isoforms of Hsp90 (Fig. 4B) suggesting that both of them interact with CaM KMT. Human Hsp90a and Hsp90b homologs show approximately 85 identity to each other with molecular masses of 84 and 83 kDa, respectively. These homologs exhibit similar participation in multi-chaperon complexes and interact with the same substrates under normalconditions [17]. To ascertain the association between CaM KMT and Hsp90 we transiently transfected HEK293 cells with Myc-CaM KMT and performed immunoprecipitation with a monoclonal anti-Myc antibody. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with antiHsp90 a/b antibody. In agreement with the mass spectrometry results CaM KMT was found to bind 18325633 Hsp90 (Fig. 4 C-left). Conversely, the transfected cell lysates were precipitated with antiHsp90 antibody and then probed with anti-Myc (Fig. 4 C-right). Thus, CaM KMT and Hsp90 proteins are suggested to be in a protein complex.CaM KMT Binds to the Middle Domain of HspSequence alignments and proteolytic digests of Hsp90 have shown a modular structure of three domains: the N-terminal is an ATP binding domain; the C-terminal domain mediates the dimerization of the chaperons and the middle domain acts as a discriminator between different types of client and co-chaperon proteins [18,19]. Therefore, we next asked whether the interactionCharacterization of CaM KMTFigure 3. Subcellular localization of the CaM KMT-GFP fusion proteins in transiently transfected cells and expression in mouse tissues. (A) GFP- CaM KMT is localized in the cytoplasm and the nucleus. Confocal images of HeLa cells expressing CaM KMT-GFP (green), nuclear staining by DAPI (blue) and the merged image. (B) The expression of the GFP only. Confocal images of HeLa cells expressing GFP (green), staining ofCharacterization of CaM KMTnuclei by DAPI (blue), and the merged image. (C) Cell lysates (100 mg of protein/lane) from mouse muscle, heart, liver, kidney, brain and spleen were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with an affinity purified polyclonal anti-CaM KMT antibody (1) immune and (2) pre-immune serum. Anti-HSP90 antibody served for protein loading control, 100 mg protein/lane were analyzed. Positions of CaM KMT and HSP-90 are indicated by the arrows. (D) GFP- CaM KMTsh is localized to the Golgi. COS-7 cells were transfected with the G.
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