Abstract
Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation.
Highlights
Serpins (SERine Protease INhibitor) are highly conserved proteins that control serine protease activities in different extracellular environments [1]
Several mutations identified in humans are at the origin of a large class of pathologies, known as serpinopathies, which share a common molecular mechanism: the polymerization of a mutant serpin and its accumulation within the endoplasmic reticulum (ER) [2]
Serpins are composed of three b-sheets (A–C) and an exposed mobile reactive centre loop (RCL), acting as a target for the serine protease [3]
Summary
Serpins (SERine Protease INhibitor) are highly conserved proteins that control serine protease activities in different extracellular environments [1]. The RCL may be inserted into the sheet of a neighbor serpin molecule forming dimers and eventually polymers [9] For these characteristics, serpin polymers are ordered structures, which do not require extensive protein unfolding and, in general, do not activate the unfolded protein response or other stress signaling pathways from the ER [10]. Contrary to other serpins, NS is a transient inhibitor of tPA and the acyl-enzyme NS-tPA complexes are short-lived, [23,38] The fragility of both polymers and complexes may be related to the relative stability of the different neuroserpin conformers, and may be relevant both for the pathology and for the physiological role of NS
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