Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization.

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.

Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis.

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps-) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps- groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps- samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection.