title: Mutagenic analysis in a pure molecular system shows that thioredoxin-interacting protein residue Cys247 is necessary and sufficient for a mixed disulfide formation with thioredoxin.
abstract: The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²⁴⁷) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.
authors: Biotechnologies & Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
authors: Benjamin
authors: B
authors: Fould
authors: Véronique
authors: V
authors: Lamamy
authors: Sophie-Penelope
authors: SP
authors: Guenin
authors: Christine
authors: C
authors: Ouvry
authors: Francis
authors: F
authors: Cogé
authors: Jean A
authors: JA
authors: Boutin
authors: Gilles
authors: G
authors: Ferry
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010084:Oxidation-Reduction; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D004220:Disulfides; D011994:Recombinant Proteins; C491203:TXN protein, human; C486715:TXNIP protein, human; D013879:Thioredoxins; D003545:Cysteine