title: Human stefin B role in cell's response to misfolded proteins and autophagy.
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.
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Ljubljana, Slovenia.
authors.forename: Mira
authors.initials: M
authors.lastname: Polajnar
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Tina
authors.initials: T
authors.lastname: Zavašnik-Bergant
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Department of Molecular and Biomedical Science, Jožef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Škerget
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Ljubljana, Slovenia.
authors.forename: Matej
authors.initials: M
authors.lastname: Vizovišek
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Ljubljana, Slovenia.
authors.forename: Robert
authors.initials: R
authors.lastname: Vidmar
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Fonović
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Nataša
authors.initials: N
authors.lastname: Kopitar-Jerala
authors.affiliation: Department of Molecular and Biomedical Science, Jožef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Uroš
authors.initials: U
authors.lastname: Petrovič
authors.affiliation: Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Spain.
authors.forename: Susanna
authors.initials: S
authors.lastname: Navarro
authors.affiliation: Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Ventura
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; Jožef Stefan International Postgraduate School, Ljubljana, Slovenia; CipKeBip - Center of Excellence for integrated approaches in chemistry and biology of proteins, Ljubljana, Slovenia.
authors.forename: Eva
authors.initials: E
authors.lastname: Žerovnik
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D001343:Autophagy; D002478:Cells, Cultured; D003001:Cloning, Molecular; D055313:Cystatin B / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D018384:Oxidative Stress; D066329:Protein Aggregates; D017510:Protein Folding; D055503:Protein Multimerization
chemical_list: D066329:Protein Aggregates; D055313:Cystatin B