Serpin polymerization in vitro

Abstract

Serpin polymerization is an event which generally occurs within living tissue as a consequence of a folding defect caused by point mutations. Major advances in cell biology and imaging have allowed detailed studies into subcellular localization, processing, and clearance of serpin polymers, but to understand the molecular basis of the misfolded state and polymeric linkage, it has been and continues to be necessary to generate polymers in vitro. The goal of this chapter is to outline the principal techniques that have been developed over the past 20 years to produce and characterize serpin polymerization in vitro. For the majority of this time, all data were interpreted in accordance with the so-called "loop-sheet" hypothesis, where polymers form through the intermolecular incorporation of the reactive center loop (RCL) of one serpin monomer into the β-sheet A of another. This hypothesis is supported by the ability of serpins to incorporate exogenous peptides into sheet A in an identical manner to the insertion of its own RCL upon cleavage by protease or conversion to the latent state. However, a recent crystal structure of an intact serpin dimer showed that much larger "domain swaps" are possible that would also lead to hyperstable linkage between serpin monomers. This chapter is therefore not limited to a description of experimental technique, but discusses the findings in light of the two current models of serpin polymerization. We would encourage readers to reevaluate the literature on serpin polymerization and to expand on the experiments outlined here in order to differentiate between possible domain-swapping mechanisms.