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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.Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs.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 protects cells from Htt Exon1 polyQ expansion-induced toxicity.A, B. Viability of wild-type (A) or SerpinB2-/- (B) MEFs following transient transfection with Httex125Q-mCherry, Httex146Q-mCherry, or mCherry expression alone (control) vectors. Data represent mean percentage of viable cells (as measured by SytoxRed exclusion and flow cytometry) normalised to mCherry only controls (n = 3 ± SEM). * Htt25q and Htt46q values significantly different from mCherry at 48 h post transfection, P < 0.01; C. Inclusion formation in wild-type versus SerpinB2-/- MEFs 48 h post transfection with either Httex125Q-mCherry or Httex146Q-mCherry. Two types of Htt foci were observed, small (< 2um; white arrow heads) and large (> 2 um; white arrows); D. Distribution of Htt in MEFs was quantified as diffuse, small foci, or inclusion in each cell; E, F, G. Viability of wild-type (WT) (E) or SerpinB2-/- (F) MEFS transduced with pMIG control empty vector (vector), or SerpinB2-/- MEFS transduced with pMIG-SerpinB2 vector (WT rescue) (G) following transfection with Httex125Q-mCherry, Httex146Q-mCherry or mCherry expression vectors. Data represent mean percentage of viable cells normalised to mCherry only controls (n = 3 ± SEM). * Htt46Q value significantly different from mCherry at 48 h post transfection, P = 0.011.
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pone.0130136.g001: SerpinB2 protects cells from Htt Exon1 polyQ expansion-induced toxicity.A, B. Viability of wild-type (A) or SerpinB2-/- (B) MEFs following transient transfection with Httex125Q-mCherry, Httex146Q-mCherry, or mCherry expression alone (control) vectors. Data represent mean percentage of viable cells (as measured by SytoxRed exclusion and flow cytometry) normalised to mCherry only controls (n = 3 ± SEM). * Htt25q and Htt46q values significantly different from mCherry at 48 h post transfection, P < 0.01; C. Inclusion formation in wild-type versus SerpinB2-/- MEFs 48 h post transfection with either Httex125Q-mCherry or Httex146Q-mCherry. Two types of Htt foci were observed, small (< 2um; white arrow heads) and large (> 2 um; white arrows); D. Distribution of Htt in MEFs was quantified as diffuse, small foci, or inclusion in each cell; E, F, G. Viability of wild-type (WT) (E) or SerpinB2-/- (F) MEFS transduced with pMIG control empty vector (vector), or SerpinB2-/- MEFS transduced with pMIG-SerpinB2 vector (WT rescue) (G) following transfection with Httex125Q-mCherry, Httex146Q-mCherry or mCherry expression vectors. Data represent mean percentage of viable cells normalised to mCherry only controls (n = 3 ± SEM). * Htt46Q value significantly different from mCherry at 48 h post transfection, P = 0.011.

Mentions: Htt polyQ expansion is characterized by the aggregation of Htt and decreased cell survival in a polyQ-expansion dependent manner in neuronal models [30]. Expression of either wild-type Httex125Q-mCherry or pathogenic Httex146Q-mCherry induced a similar and significant loss (approximately 25%) in viability of SerpinB2-/- MEFs 48 h after transfection, compared to the mCherry alone vector, with little effect on wild-type MEFs observed (Fig 1A and 1B). This is consistent with previous work using wild-type MEFs that showed no difference in toxicity responses between cells expressing Httex125Q and Httex197Q [31]. The sensitization of cells to overexpression of both wild-type and pathogenic Httex1polyQ variants in the absence of SerpinB2 suggests a cytoprotective role for SerpinB2.


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 protects cells from Htt Exon1 polyQ expansion-induced toxicity.A, B. Viability of wild-type (A) or SerpinB2-/- (B) MEFs following transient transfection with Httex125Q-mCherry, Httex146Q-mCherry, or mCherry expression alone (control) vectors. Data represent mean percentage of viable cells (as measured by SytoxRed exclusion and flow cytometry) normalised to mCherry only controls (n = 3 ± SEM). * Htt25q and Htt46q values significantly different from mCherry at 48 h post transfection, P < 0.01; C. Inclusion formation in wild-type versus SerpinB2-/- MEFs 48 h post transfection with either Httex125Q-mCherry or Httex146Q-mCherry. Two types of Htt foci were observed, small (< 2um; white arrow heads) and large (> 2 um; white arrows); D. Distribution of Htt in MEFs was quantified as diffuse, small foci, or inclusion in each cell; E, F, G. Viability of wild-type (WT) (E) or SerpinB2-/- (F) MEFS transduced with pMIG control empty vector (vector), or SerpinB2-/- MEFS transduced with pMIG-SerpinB2 vector (WT rescue) (G) following transfection with Httex125Q-mCherry, Httex146Q-mCherry or mCherry expression vectors. Data represent mean percentage of viable cells normalised to mCherry only controls (n = 3 ± SEM). * Htt46Q value significantly different from mCherry at 48 h post transfection, P = 0.011.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4470917&req=5

pone.0130136.g001: SerpinB2 protects cells from Htt Exon1 polyQ expansion-induced toxicity.A, B. Viability of wild-type (A) or SerpinB2-/- (B) MEFs following transient transfection with Httex125Q-mCherry, Httex146Q-mCherry, or mCherry expression alone (control) vectors. Data represent mean percentage of viable cells (as measured by SytoxRed exclusion and flow cytometry) normalised to mCherry only controls (n = 3 ± SEM). * Htt25q and Htt46q values significantly different from mCherry at 48 h post transfection, P < 0.01; C. Inclusion formation in wild-type versus SerpinB2-/- MEFs 48 h post transfection with either Httex125Q-mCherry or Httex146Q-mCherry. Two types of Htt foci were observed, small (< 2um; white arrow heads) and large (> 2 um; white arrows); D. Distribution of Htt in MEFs was quantified as diffuse, small foci, or inclusion in each cell; E, F, G. Viability of wild-type (WT) (E) or SerpinB2-/- (F) MEFS transduced with pMIG control empty vector (vector), or SerpinB2-/- MEFS transduced with pMIG-SerpinB2 vector (WT rescue) (G) following transfection with Httex125Q-mCherry, Httex146Q-mCherry or mCherry expression vectors. Data represent mean percentage of viable cells normalised to mCherry only controls (n = 3 ± SEM). * Htt46Q value significantly different from mCherry at 48 h post transfection, P = 0.011.
Mentions: Htt polyQ expansion is characterized by the aggregation of Htt and decreased cell survival in a polyQ-expansion dependent manner in neuronal models [30]. Expression of either wild-type Httex125Q-mCherry or pathogenic Httex146Q-mCherry induced a similar and significant loss (approximately 25%) in viability of SerpinB2-/- MEFs 48 h after transfection, compared to the mCherry alone vector, with little effect on wild-type MEFs observed (Fig 1A and 1B). This is consistent with previous work using wild-type MEFs that showed no difference in toxicity responses between cells expressing Httex125Q and Httex197Q [31]. The sensitization of cells to overexpression of both wild-type and pathogenic Httex1polyQ variants in the absence of SerpinB2 suggests a cytoprotective role for SerpinB2.

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.Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs.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