Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

The cholera toxin B pentamer (CtxB5), which belongs to the AB5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB5 and the related heat labile enterotoxin LTB5 have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them

Metal chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence of NNN' N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn2+ By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu2+ Or Zn2+ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress