Characterisation of serpin polymers in vitro and in vivo.
Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.
Dept. of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
Didier
D
Belorgey
James A
JA
Irving
Ugo I
UI
Ekeowa
Joanna
J
Freeke
Benoit D
BD
Roussel
Elena
E
Miranda
Juan
J
Pérez
Carol V
CV
Robinson
Stefan J
SJ
Marciniak
Damian C
DC
Crowther
Claire H
CH
Michel
David A
DA
Lomas
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular / Q000379:methods; D004195:Disease Models, Animal; D004331:Drosophila melanogaster / Q000235:genetics; D004831:Epilepsies, Myoclonic / Q000473:pathology; D020271:Heredodegenerative Disorders, Nervous System / Q000473:pathology; D006801:Humans; D007106:Immune Sera; D002479:Inclusion Bodies / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods; D020125:Mutation, Missense; D055503:Protein Multimerization; D058849:Protein Refolding; D057165:Proteostasis Deficiencies / Q000235:genetics / Q000473:pathology*; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D007106:Immune Sera; D015843:Serpins