Human stefin B readily forms amyloid fibrils in vitro.
Human stefin B (cystatin B) is an intracellular cysteine proteinase inhibitor broadly distributed in different tissues. Here, we show that recombinant human stefin B readily forms amyloid fibrils in vitro. It dimerises and further oligomerises, starting from the native-like acid intermediate, I(N), populated at pH 5. On standing at room temperature it produces regular (over 4 microm long) fibrils over a period of several months. These have been visualised by transmission electron microscopy and atomic force microscopy. Their cross-sectional diameter is about 14 nm and blocks of 27 nm repeat longitudinally. The fibrils are smooth, of unbranched surface, consistent with findings of other amyloid fibrils. Thioflavin T fluorescence spectra as a function of time were recorded and Congo red dye binding to the fibrils was demonstrated. Adding 10% (v/v) trifluoroethanol resulted in an increased rate of fibrillation with a typical lag phase. The finding that human stefin B, in contrast to the homologue stefin A, forms amyloid fibrils rather easily should promote further studies of the protein's behaviour in vivo, and/or as a model system for fibrillogenesis.
Zerovnik
Eva
E
Department of Biochemistry and Molecular Biology, Institute Jozef Stefan, Jamova 39, 1000 Ljubljana, Slovenia. eva.zerovnik@ijs.si
Pompe-Novak
Marusa
M
Skarabot
Miha
M
Ravnikar
Maja
M
Musevic
Igor
I
Turk
Vito
V
D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000737:chemistry; D000818:Animals; D052160:Benzothiazoles; D002021:Buffers; D004396:Coloring Agents; D003224:Congo Red; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013844:Thiazoles; D014961:X-Ray Diffraction
D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D002021:Buffers; C524994:CSTB protein, human; D004396:Coloring Agents; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D055313:Cystatin B