Human stefin B role in cell's response to misfolded proteins and autophagy.

Mira Polajnar,Salvador Ventura,Matej Vizovišek,Eva Žerovnik,Susanna Navarro,Nataša Kopitar-Jerala,Marko Fonović,Tina Zavašnik-Bergant,Uroš Petrovič,Robert Vidmar,Katja Škerget,Edathara Abraham

doi:10.1371/journal.pone.0102500

Abstract

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

Highlights

Stefin B belongs to a superfamily of cystatins, cysteine protease inhibitors
As aggregates are considered to be mostly cleared via autophagy [16], we presumed that their levels will be reduced after treating KO astrocytes with an autophagy inducer
To assess whether wt stefin B monomers, dimers and tetramers could have a function in aggregate clearance, the KO astrocytes were incubated with isolated stefin B monomers, dimers, tetramers and higher oligomers [10] for 24 hours

Summary

Introduction

Stefin B belongs to a superfamily of cystatins, cysteine protease inhibitors. It is localized both in the nucleus and in the cytoplasm where it inhibits activity of cathepsins B, H, K, L, and S [1]. We proposed in 2005 [7] that the prefibrillar oligomers of stefin B (and its aggregation-prone EPM1 mutants) might be toxic to cells, as it is the case for other amyloidogenic proteins. Stefin B forms dimers, tetramers and higher-order oligomers, these latter assemblies being the cytotoxic species [10]. We have shown that wt stefin B and especially the missense and truncated EPM1 mutants form intracellular aggregates [16]. The endogenous protein formed small oligomers whereas wt stefin B and its EPM1 mutants all aggregated upon over expression. Wt aggregated to a lower extent than disease-linked mutants

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