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SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation.

Lee JA, Yerbury JJ, Farrawell N, Shearer RF, Constantinescu P, Hatters DM, Schroder WA, Suhrbier A, Wilson MR, Saunders DN, Ranson M - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

SerpinB2 attenuates Htt42q fibril formation in vitro.A. Soluble Htt remaining in Httex146Q-Cerulean aggregation reactions in the absence or presence of SerpinB2 (equimolar ratio); B. TEM images of fibril formation at 72 h; C. Representative ligand blot (n = 3) showing differences in binding of SerpinB2 to soluble and aggregated Httex146Q. Binding was determined by immunoassay. SerpinB14 or clusterin binding to soluble and aggregated Httex146Q are also shown.
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pone.0130136.g002: SerpinB2 attenuates Htt42q fibril formation in vitro.A. Soluble Htt remaining in Httex146Q-Cerulean aggregation reactions in the absence or presence of SerpinB2 (equimolar ratio); B. TEM images of fibril formation at 72 h; C. Representative ligand blot (n = 3) showing differences in binding of SerpinB2 to soluble and aggregated Httex146Q. Binding was determined by immunoassay. SerpinB14 or clusterin binding to soluble and aggregated Httex146Q are also shown.

Mentions: Given that SerpinB2 expression can drive changes in protein inclusion formation and cell viability, we tested the ability of recombinant SerpinB2 to suppress in vitro fibril formation by Httex146Q fused to the monomeric cyan fluorescent protein Cerulean. To prevent aggregation during recombinant protein production, Httex146Q-Cerulean was expressed as a fusion protein with a cleavable MBP tag. As previously reported [20], the Httex146Q-Cerulean formed insoluble, fibrillar aggregates over 72 h following cleavage with TEV protease to remove the MBP tag (Fig 2A). The presence of recombinant SerpinB2 modestly attenuated the amount of insoluble Httex146Q-Cerulean but the morphology of Httex146Q-Cerulean insoluble fibrils was indistinguishable in the presence or absence of SerpinB2 (Fig 2B). To examine if the modest attenuation of fibril formation was due to interaction of SerpinB2 with Httex146Q-Cerulean, a solid phase ligand-blotting assay was used [17]. SerpinB2 bound to non-aggregated—but not aggregated—Httex146Q-Cerulean (Fig 2C). Furthermore, the closely-related SerpinB14 displayed only limited or no binding to non-aggregated Httex146Q-Cerulean, or aggregated Httex146Q-Cerulean, respectively (Fig 2C). The established extracellular chaperone clusterin [17] bound to both insoluble and soluble Httex146Q-Cerulean (Fig 2C).


SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation.

Lee JA, Yerbury JJ, Farrawell N, Shearer RF, Constantinescu P, Hatters DM, Schroder WA, Suhrbier A, Wilson MR, Saunders DN, Ranson M - PLoS ONE (2015)

SerpinB2 attenuates Htt42q fibril formation in vitro.A. Soluble Htt remaining in Httex146Q-Cerulean aggregation reactions in the absence or presence of SerpinB2 (equimolar ratio); B. TEM images of fibril formation at 72 h; C. Representative ligand blot (n = 3) showing differences in binding of SerpinB2 to soluble and aggregated Httex146Q. Binding was determined by immunoassay. SerpinB14 or clusterin binding to soluble and aggregated Httex146Q are also shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470917&req=5

pone.0130136.g002: SerpinB2 attenuates Htt42q fibril formation in vitro.A. Soluble Htt remaining in Httex146Q-Cerulean aggregation reactions in the absence or presence of SerpinB2 (equimolar ratio); B. TEM images of fibril formation at 72 h; C. Representative ligand blot (n = 3) showing differences in binding of SerpinB2 to soluble and aggregated Httex146Q. Binding was determined by immunoassay. SerpinB14 or clusterin binding to soluble and aggregated Httex146Q are also shown.
Mentions: Given that SerpinB2 expression can drive changes in protein inclusion formation and cell viability, we tested the ability of recombinant SerpinB2 to suppress in vitro fibril formation by Httex146Q fused to the monomeric cyan fluorescent protein Cerulean. To prevent aggregation during recombinant protein production, Httex146Q-Cerulean was expressed as a fusion protein with a cleavable MBP tag. As previously reported [20], the Httex146Q-Cerulean formed insoluble, fibrillar aggregates over 72 h following cleavage with TEV protease to remove the MBP tag (Fig 2A). The presence of recombinant SerpinB2 modestly attenuated the amount of insoluble Httex146Q-Cerulean but the morphology of Httex146Q-Cerulean insoluble fibrils was indistinguishable in the presence or absence of SerpinB2 (Fig 2B). To examine if the modest attenuation of fibril formation was due to interaction of SerpinB2 with Httex146Q-Cerulean, a solid phase ligand-blotting assay was used [17]. SerpinB2 bound to non-aggregated—but not aggregated—Httex146Q-Cerulean (Fig 2C). Furthermore, the closely-related SerpinB14 displayed only limited or no binding to non-aggregated Httex146Q-Cerulean, or aggregated Httex146Q-Cerulean, respectively (Fig 2C). The established extracellular chaperone clusterin [17] bound to both insoluble and soluble Httex146Q-Cerulean (Fig 2C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.

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.

No MeSH data available.


Related in: MedlinePlus