MilliporeSigma
  • Home
  • Search Results
  • Human stefin B role in cell's response to misfolded proteins and autophagy.

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

PloS one (2014-07-23)
Mira Polajnar, Tina Zavašnik-Bergant, Katja Škerget, Matej Vizovišek, Robert Vidmar, Marko Fonović, Nataša Kopitar-Jerala, Uroš Petrovič, Susanna Navarro, Salvador Ventura, Eva Žerovnik
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Fast Garnet GBC sulfate salt, diazonium dye
Supelco
Sucrose, analytical standard, for enzymatic assay kit SCA20
Sigma-Aldrich
Sucrose, Grade I, suitable for plant cell culture
Sigma-Aldrich
Sucrose, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, ≥99.5% (GC)
Sigma-Aldrich
DAPI, for nucleic acid staining
Millipore
Sucrose, ACS reagent, suitable for microbiology, ≥99.0%
Sigma-Aldrich
Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)
Sigma-Aldrich
Sucrose, ≥99.5%
Sigma-Aldrich
Sucrose, Grade II, suitable for plant cell culture
Sigma-Aldrich
Sucrose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, ACS reagent
Sigma-Aldrich
Sucrose, meets USP testing specifications
USP
Sucrose, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Sucrose, puriss., meets analytical specification of Ph. Eur., BP, NF
Supelco
Sucrose, Pharmaceutical Secondary Standard; Certified Reference Material
Sucrose, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Paraformaldehyde, reagent grade, crystalline
Sigma-Aldrich
Nα-Benzoyl-DL-arginine β-naphthylamide hydrochloride, trypsin substrate
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Methyl cellulose, viscosity: 15 cP
Sigma-Aldrich
Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Anti-p62 (Sequestosome-1) Antibody, clone 11C9.2, clone 11C9.2, from mouse
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution