{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T03:48:31Z","timestamp":1790999311135,"version":"4.1.0"},"reference-count":67,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2012,1,3]],"date-time":"2012-01-03T00:00:00Z","timestamp":1325548800000},"content-version":"vor","delay-in-days":581,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology of the Cell"],"published-print":{"date-parts":[[2010,6]]},"abstract":"<jats:p><jats:italic>Background<\/jats:italic>. Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the <jats:italic>CSTB<\/jats:italic> (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation <jats:italic>in vitro<\/jats:italic> and, as we show here, to study protein aggregation in cells.<\/jats:p><jats:p><jats:italic>Results<\/jats:italic>. Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin\u2013proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T\u2010Sapphire\u2010tagged StB on overexpression in mammalian cells. Compared with wild\u2010type StB, the EPM1 missense mutant (G4R), the aggregate\u2010prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non\u2010homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB\u2010positive cytoplasmic aggregates were partially co\u2010localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co\u2010localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability.<\/jats:p><jats:p><jats:italic>Conclusions<\/jats:italic>. We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.<\/jats:p>","DOI":"10.1042\/bc20090163","type":"journal-article","created":{"date-parts":[[2010,1,18]],"date-time":"2010-01-18T12:55:46Z","timestamp":1263819346000},"page":"319-334","source":"Crossref","is-referenced-by-count":26,"title":["Intracellular aggregation of human stefin B: confocal and electron microscopy 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