Rapid Formation of Peptide/Lipid Coaggregates by the Amyloidogenic Seminal Peptide PAP248-286.

Protein/lipid coassembly is an understudied phenomenon that is important to the function of antimicrobial peptides as well as the pathological effects of amyloid. Here, we study the coassembly process of PAP248-286, a seminal peptide that displays both amyloid-forming and antimicrobial activity. PAP248-286 is a peptide fragment of prostatic acid phosphatase and has been reported to form amyloid fibrils, known as semen-derived enhancer of viral infection (SEVI), that enhance the viral infectivity of human immunodeficiency virus. We find that in addition to forming amyloid, PAP248-286 much more readily assembles with lipid vesicles into peptide/lipid coaggregates that resemble amyloid fibrils in some important ways but are a distinct species. The formation of these PAP248-286/lipid coaggregates, which we term "messicles," is controlled by the peptide:lipid (P:L) ratio and by the lipid composition. The optimal P:L ratio is around 1:10, and at least 70% anionic lipid is required for coaggregate formation. Once formed, messicles are not disrupted by subsequent changes in P:L ratio. We propose that messicles form through a polyvalent assembly mechanism, in which a critical surface density of PAP248-286 on liposomes enables peptide-mediated particle bridging into larger species. Even at ∼50-fold lower PAP248-286 concentrations, messicles form at least 10-fold faster than amyloid fibrils. It is therefore possible that some or all of the biological activities assigned to SEVI, the amyloid form of PAP248-286, could instead be attributed to a PAP248-286/lipid coaggregate. More broadly speaking, this work could provide a potential framework for the discovery and characterization of nonamyloid peptide/lipid coaggregates by other amyloid-forming proteins and antimicrobial peptides.

A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers.

Prior studies in both budding yeast (Saccharomyces cerevisiae) and in human cells have established that septin protomers assemble into linear hetero-octameric rods with 2-fold rotational symmetry. In mitotically growing yeast cells, five septin subunits are expressed (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) and assemble into two types of rods that differ only in their terminal subunit: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. EM analysis has shown that, under low salt conditions, the Cdc11-capped rods polymerize end to end to form long paired filaments, whereas Shs1-capped rods form arcs, spirals, and rings. To develop a facile method to study septin polymerization in vitro, we exploited our previous work in which we generated septin complexes in which all endogenous cysteine (Cys) residues were eliminated by site-directed mutagenesis, except an introduced E294C mutation in Cdc11 in these experiments. Mixing samples of a preparation of such single-Cys containing Cdc11-capped rods that have been separately derivatized with organic dyes that serve as donor and acceptor, respectively, for FRET provided a spectroscopic method to monitor filament assembly mediated by Cdc11-Cdc11 interaction and to measure its affinity under specified conditions. Modifications of this same FRET scheme also allow us to assess whether Shs1-capped rods are capable of end to end association either with themselves or with Cdc11-capped rods. This FRET approach also was used to follow the binding to septin filaments of a septin-interacting protein, the type II myosin-binding protein Bni5.