Refolding and polymerization pathways of neuroserpin

Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate I(IN) and then underwent further refolding to the native form through a late refolding intermediate, I(R). The late-onset mutant S49P was also able to refold to the native form through I(IN) and I(R), but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed I(R) through the same pathway as S49P, but the protein could not attain the native state and remained as I(R). The I(R)s of the mutants had a long lifespan at 4 °C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, I(R) formed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.