Mycobacterium tuberculosisChaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?

Abstract

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a ␤-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N-and C-truncated forms of the protein, the C-and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately ␤-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.

Crystal Structure of the 65-Kilodalton Heat Shock Protein, Chaperonin 60.2, ofMycobacterium tuberculosis

“…Levels of sequence identity with GroEL of more than 50% for both the proteins suggest that these proteins might also function as molecular chaperones in M. tuberculosis. Interestingly, the mycobacterial chaperonins have previously been shown to be secreted into the extracellular environment, although their role as molecular chaperones is limited to the cytosol (10,14). The existence of chaperonins in the extracellular environment thus suggests a possible alternate functional role.…”

“…However, translation searches with this orientation and with different models did not yield a satisfactory solution. The best solution for the translation function was obtained by using the E. coli 14 complex structure (Protein Data Bank code 1KP8) as the model. The 1KP8 model was oriented according to the common orientation at the start of translation searches.…”

Heat shock fusion protein induces both specific and nonspecific anti‐tumor immunity

“…LPS was removed by passing the protein through a polymyxin B-agarose column (Detoxigel column; Pierce, Rockford, IL). The fusion protein of HSP65 and PSA peptide (HSP65-PSA) [32] and the fusion protein of Chap10 and MUC1 peptide (Chap10-MUC1) [33] were prepared similarly as described above. The MUC1-derived peptide (NH 2 -SAPDTRPAP) and the PSA-derived peptide (NH 2 -FLTPKKLQCV) were synthesized using standard F-moc chemistry on a peptide synthesizer (model 432A; PE Applied Biosystems).…”

Heat shock protein 10 of Chlamydophila pneumoniae induces proinflammatory cytokines through Toll-like receptor (TLR) 2 and TLR4 in human monocytes THP-1

“…HSP10 (GroES), a cochaperonin to HSP60 (GroEL) shared the same GroE operons and co-expressed the GroE complex with HSP60. It is located in cytosol, cell membrane, intercellular space, and periphery with mounting evidence demonstration (Fossati et al 2003;Jia et al 2011) and may interact with various surrounding environment. The association between the presence of anti-HSP10 antibodies and adult onset asthma or Chlamydia trachomatis genital tract infection (Betsou et al 1999;LaVerda et al 2000;Betsou et al 2003) indicated that CHSP10 had been exposed to the host components and triggered the immune system to produce the specific anti- Figure 7.…”