Thorpe Lab

oxidative protein folding

Inhibitors of Oxidative Protein Folding

Studies aimed towards the generation of inhibitors directed towards QSOX, and other enzymes with CxxC motifs, have led to the unexpected observation that arsenic(III) species can interfere with protein folding pathways by sequestration of reduced unfolded proteins.  This binding is dependent on the arsenic species and on the concentration of competing reduced glutathione.  Binding is of sufficient avidity to warrant consideration as one contributor to the physiological and pharmacological effects of arsenicals.

Arsenical are now being used for the treatment of acute promyelocytic leukemia and are being tested with other cancers.  In addition to specific protein targets of arsenicals, we have shown that As(III) species can have a more general effect by binding to protein thiols during protein folding.  This effect is likely to be most pronounced for cysteine-rich proteins – for example those found in secreted proteins prior to disulfide bond generation.

In the figure above, "A" is the unfolded reduced protein could yield the correctly-folded disulfide-bridged protein B.  Our experiments show that three representative reduced proteins (schematically represented by "A") bind a range of arsenicals with stoichiometries reflecting the nature of the arsenic(III) species and the number of free thiols available (see below).  This binding is sufficiently tight to remain significant in the presence of 5 mM reduced glutathione.  We hypothesize that some proteins may exhibit particularly avid dithiol or trithiol site for arsenicals and that binding could persist as disulfide bonds are inserted around these loci (C).  We have also shown that the monomethyl metabolite of arsenite (MMA, pink square) generates amyloid-like filaments with reduced RNase (E).  We have also suggested that arsenicals could also compromise some fraction of protein folding in the absence of disulfide bond generation.

The titration of reduced riboflavin bindiing protein with three arsenicals is shown below.  Binding is also followed by fluorescence and by the suppression of thiol reactivity after complexation by As(III) species.

These initial experiments suggest new strategies for the design of reagents that might selectively inhibit cells that carry a heavy secretory load.

 

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