title: SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation.
abstract: SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS.
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Jodi A
authors.initials: JA
authors.lastname: Lee
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Yerbury
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Natalie
authors.initials: N
authors.lastname: Farrawell
authors.affiliation: Kinghorn Cancer Centre and Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia; St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Shearer
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Patrick
authors.initials: P
authors.lastname: Constantinescu
authors.affiliation: Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Vic., Australia.
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Qld, Australia.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Schroder
authors.affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Qld, Australia.
authors.forename: Andreas
authors.initials: A
authors.lastname: Suhrbier
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wilson
authors.affiliation: Kinghorn Cancer Centre and Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia; St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Darren N
authors.initials: DN
authors.lastname: Saunders
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Marie
authors.initials: M
authors.lastname: Ranson
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry; D000818:Animals; D019610:Cytoprotection / Q000187:drug effects*; D005091:Exons / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects*; D006801:Humans; D002479:Inclusion Bodies / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D010455:Peptides / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000378:metabolism* / Q000494:pharmacology*; D011247:Pregnancy; D066329:Protein Aggregates; D011485:Protein Binding; D017510:Protein Folding / Q000187:drug effects*; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D010455:Peptides; D017396:Plasminogen Activator Inhibitor 2; D066329:Protein Aggregates; D050486:Serotonin Plasma Membrane Transport Proteins; C493231:Slc6a4 protein, mouse; D025801:Ubiquitin; C097188:polyglutamine