Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

Abstract

The occurrence of highly conserved amyloid-forming sequences in Candida albicansProtein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4,11,17,38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1,33,39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five-to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of ␤-aggregates, according to the protein state prediction program TANGO (13,27,31). Such ␤-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel ␤-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have ␤-aggregate potentials greater than 20%, an insoluble ␤-aggregate state is likely to form. These ␤-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13,27,31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have ␤-aggregation potentials of Ͼ90% (27). An oligopeptide with this sequence, as well as 412-and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the ␤-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9,21,41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300-to

Force-induced formation and propagation of adhesion nanodomains in living fungal cells

“…Similar data were obtained using different tips and cells (10 tips, 6 cells). Als proteins expressed on S. cerevisiae exhibit the same activities as they do in C. albicans (13,14). 1 μm) recorded with an anti-V5 tip on a given target area of the native cells, i.e., cells that were never subjected to force (map 1; recorded on the dashed squares in A).…”

“…Als5p and other yeast adhesion proteins have conserved amyloid-forming sequences, and self-propagating amyloid formation is important for formation of robust cellular aggregates (13,14,16). The V326N single-site mutation in the amyloid-forming region of Als5p abrogates amyloid formation (13).…”

“…The results indicate that the adhesion function of Als5p is coupled to its local assembly within adhesion nanodomains, for which we propose the term "nanoadhesomes." Comparative genomics shows similar protein design in other fungal adhesins (14), suggesting that clustering of cell adhesion proteins in response to mechanical stimuli may be a general mechanism for activating cell adhesion in eukaryotes.…”

“…Als proteins are covalently attached to the cell wall and consist of an N-terminal Ig-like region, which initiates cell adhesion, followed by a threonine-rich region with an amyloid-forming sequence (T), a tandem repeat (TR) region that participates in cellcell aggregation, and a stalk region projecting the molecule away from the cell surface (Fig. 1A) (13)(14)(15). Previous studies in C. albicans and in a Saccharomyces cerevisiae surface display model showed that Als5p-mediated adhesion involves a rapid cellto-ligand adhesion step, followed by slower cell-to-cell aggregation mediated by the T and TR regions (13,15).…”

A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

“…MUC1 expression is subject to extensive regulatory control; the PKA, Kss1p MAPK, and Snf1p pathways described above all regulate MUC1 transcription, and epigenetic silencing through the histone deacetylase Hda1p further controls MUC1 expression (34,42,43). Muc1p contributes to cell-substrate adhesion in vivo, exhibits homotypic adhesion in vitro, and is the only flocculin family member expressed in filamentous strains of S. cerevisiae (37,44); however, additional cell wall proteins contribute to cell adhesion (45) and are expected to play essential roles during the pseudohyphal growth response. In addition, membrane transporters such as Mep2p and the amino acid transporter homolog Ssy1p regulate filamentous growth (33,46).…”