Protofibrils

As a result, leather is made up of interlaced bundles of coUagen fibers (Fig. 1). A schematic model of coUagen bundles in leather is shown in Figure 2 (4). A coUagen bundle (about 80 )Tm in diameter) is made up of coUagen fibers (1—4 pm), composed of microfibrils (0.08—0.1 pm). Furthermore, a microfibril consists of many protofibrils (about 1.5 nm), which consist of several bundles of polypeptide chains. 

Filament (four right-hand twisted protofibrils)... 

Alzheimer s Disease. Figure 1 A(3 monomers can self-associate to form dimers, trimers and higher oligomers. Globular structures of synthetic A(342 are known as A(3-derived diffusible ligands (ADDLs) (3-12-mers of A(3). These structures are similar to the smallest protofibrils and represent the earliest macromolecular assembly of synthetic A(3. The characteristic amyloid fiber exhibits a high beta-sheet content and is derived in vitro by a nucleation-dependent self-association and an associated conformational transition from random to beta-sheet conformation of the A(3 molecule. Intermediate protofibrils in turn self-associate to form mature fibers. 

Harper JD, Wong SS, Lieber CM, Lansbury PT Jr. Assembly of A amyloid protofibrils an in vitro model for a possible early event in Alzheimer s disease. Biochemistry 1999 38 8972-8980. 

Lindgren M, Sorgjerd K, Hammarstrom P (2005) Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. Biophys J 88(6) 4200-4212... 

Caughey, B. and Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 26 267-298, 2003. 

Figure 17.5. The precursor molecule APP and the three different proteases a, (i, y secretase that are involved in the processing of APPto fS-amyloid peptide. The aberrant processing of the amyloid precursor protein (APP) leads to accumulation of beta-amyloid fragments, first as protofibrils and then as fibers that aggregate in the senile plaque structures. (See color insert.)...

Lazo, N. D., and Downing, D. T. (1998). Amyloid fibrils may be assembled from beta-helical protofibrils. Biochemistry 37, 1731-1735. 

DeMarco, M. L., and Daggett, V. (2004). From conversion to aggregation Protofibril formation of the prion protein. Proc. Natl. Acad. Sci. USA 101, 2293-2298. Diaz-Avalos, R., Long, C., Fontano, E., Balbirnie, M., Grothe, R., Eisenberg, D., and Caspar, D. L. D. (2003). Cross-beta structure of an amyloid-forming peptide studied by electron nano-crystallography. Fibre Diffract. Rev. 11, 79-86. 

Fig. 1. Structure of amyloid fibrils formed by the human amylin peptide. Negatively stained (A) and metal shadowed (B) fibrils formed by human amylin (adapted from Goldsbury et al., 2000a). (C) A human amylin fibril model formed by three protofibrils having a superpleated /i-structure (adapted from Kajava et al., 2005). Only Ca traces of the polypeptide chains are shown. (D) Atomic model of the cross-/ motif formed by the human amylin peptide (adapted from Kajava et al, 2005). Scale bar, 100 nm (A and B).

Fig. 2. Electron micrographs highlighting the polymorphism of amyloid fibrils. (A) A single human calcitonin protofibril with a diameter of 4 nm (adapted from Bauer et al., 1995). (B) Different morphologies present in a transthyretin fibril preparation. Black arrowheads show oligomers of different sizes, the black arrow points to a 9- to 10-nm-wide fibril, and the white arrowhead marks an 4-nm-wide fibril (adapted from Cardoso et al., 2002). (C-F) Human amylin fibril ribbons (adapted from Goldsbury et al., 1997). (C) A single 5-nm-wide protofibril. (D-F) Ribbons containing two (D), three (E), or five (F) 5-nm-wide protofibrils. (G) A twisted ribbon made of four 5-nm-wide protofibril subunits of Api-40 (adapted from Goldsbury et al., 2000b). Scale bar, 50 nm (A-G).

For calcitonin, the thinnest single fibril, the protofibril, had a diameter of 4-5 nm and was observed at low (0.1-1 mM) calcitonin concentration (Fig. 2A Bauer et al, 1995). For transthyretin, short and flexible protofibrils 4—5 nm in diameter were observed, but the prominent species was an 8-nm diameter and up to 300-nm-long fibril (Fig. 2B Cardoso et al., 2002). For human amylin, 5-nm-wide protofibrils were rarely depicted by themselves (Fig. 2C), but they could be readily identified as a distinct building block of wider fibrils (Fig. 2D-F Goldsbury et al., 1997). The wider fibrils... 

Multistranded cables and ribbons were also observed for calcitonin (Bauer et al, 1995). These appeared to contain laterally associated 8-nm-wide fibrils, whereas the diameter of the individual strands within the multistranded cables could not be measured directly from the images. To estimate this diameter, the authors plotted the helical crossover spacing of the cables as a function of their diameter. Using a linear regression fit, the data were extrapolated to zero, yielding a width of 4.1 nm, similar to the width of the single protofibrils (Fig. 2A Bauer et al., 1995). 

For A/i it is worth noting that while single 5-nm protofibrils were rarely imaged by EM or SFM, the thinnest single fibrils had a diameter around 8-9 nm and were termed protofibrils by many researchers in the field (Goldsbury et al., 2005 Harper et al., 1997, 1999 Lambert et al, 1998 Nielsen et al., 1999 Walsh et al., 1997, 1999). Flat and twisted ribbons formed from 5-nm-wide subunits, as seen with human amylin, were also depicted for AjSi 4o (Fig- 2G Goldsbury et al., 2000b, 2005). 

Even though for each peptide or protein the MPL value for the protofibril may be different, all the available data can be summarized in a fairly simple scheme. The basic subunit of all amyloid fibrils is a 4- to 5-nm-wide protofibril whose detailed molecular architecture is species dependent. All the other structures observed can be either described as ribbons, sheets, or multistranded cables of protofibrils (Fig. 3). 

Fig. 3. A generalized model of amyloid fibril polymorphism based on the formation of straight or coiled fibrils composed of several 4- to 5-nm-wide protofibril subunits. Notice that the flat ribbons containing several protofibril strands may twist (Fig. 2F and G) and may ultimately form tubes (Bauer et al, 1995).

Amylin fibrils growing on mica were rather straight and exhibited various heights. They were compatible with the protofibril hypothesis of amyloid fibril polymorphism (Fig. 3), but no multistranded cables were present (Goldsbury et al, 1999). In contrast, coiled fibrils were often observed by SFM for fibrils assembled in solution prior to being adsorbed to mica (Jansen et al, 2005 Kad et al, 2003 Relini et al, 2004). In the case of... 

Fig. 4. Time-lapse SFM experiments revealing the growth of single protofibrils and mature fibrils on mica. (A) Fluman amylin protofibrils (adapted from Goldsbury et al., 1999). (B) Bidirectional growth of a single A/ protofibril (adapted from Goldsbury et al., 2005). (C) Unidirectional growth of a mature A/ fibril (adapted from Goldsbury et al., 2005). Scale bar, 200 nm (A, B, and C).

In the case of human amylin and Afi our understanding of the diversity in amyloid fibril architecture is the result of a recursive process, since the early morphological observations were followed by assessment of the assembly pathway which in turn yielded a better understanding of fibril polymorphism. However, this structural knowledge is secondary compared to the discovery of small oligomers, globular oligomers, and early protofibrils that appear to be extremely cytotoxic (Hartley etal., 1999 Lambert et al, 1998 Walsh et al, 1999). 

Harper, J. D., Wong, S. S., Lieber, C. M., and Lansbury, P. T. (1997). Observation of metastable A beta amyloid protofibrils by atomic force microscopy. Chem. Biol. 4, 119-125. 

Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowak, R. J., Walz, T., and Lansbury, P. T., Jr. (2002). Alpha-synuclein, especially the Parkinson s disease-associated mutants, forms pore-like annular and tubular protofibrils./. Mol. Biol. 322,1089-1102. LeVine, H. (1993). Thioflavine T interaction with synthetic Alzheimer s disease beta-amyloid peptides Detection of amyloid aggregation in solution. Protein Sci. 2, 404—410. Lin, H., Bhatia, R., and Lai, R. (2001). Amyloid beta protein forms ion channels Implications for Alzheimer s disease pathophysiology. FASEB J. 15, 2433-2444. Lorenzo, A., and Yankner, B. A. (1994). Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc. Natl. Acad. Sci. USA 91, 12243-12247. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H., Schubert, D., and Riek, R. (2005). 3D structure of Alzheimer s amyl o id-( be la) (1—12) fibrils. Proc. Natl. Acad. Sci. USA 102, 17342-17347. 

Destabilization Mechanism of Alzheimer s Ap42 Protofibrils With a Small-Molecule Inhibitor A Molecular Dynamics Simulation Study... 

Increasing evidence indicates that accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin-proteasome system dysfunction, and the direct or indirect consequences of abnormal protein aggregation and accumulation represent deleterious events linked to neurodegeneration (255,256). Ubiquitination is an essential cellular process affected by a multienzyme cascade involving Els (ubiquitin-activating enzymes), E2s (ubiquitin-conjugation enzymes or UBCs), and E3s (ubiquitin-protein Ugases) (12,257) (see Fig. 10.4). 

FIGURE 4-11 Structure of hair, (a) Hair a-keratin is an elongated a helix with somewhat thicker elements near the amino and carboxyl termini. Pairs of these helices are interwound in a left-handed sense to form two-chain coiled coils. These then combine in higher-order structures called protofilaments and protofibrils. About four protofibrils—32 strands of a-keratin altogether—combine to form an intermediate filament. The individual two-chain coiled coils in the various substructures also appear to be interwound, but the handedness of the interwinding and other structural details are unknown, (b) A hair is an array of many a-keratin filaments, made up of the substructures shown in (a). 

The assembly of hair a-keratin from one a helix to a protofibril, to a microfibril, and finally, to a single hair. (Illustration copyright by Irving Geis. Reprinted by permission.)... 

---