8 research outputs found
Helical Conformation of the SEVI Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion
AbstractIn previous in vivo studies, amyloid fibers formed from a peptide ubiquitous in human seminal fluid (semen-derived enhancer of viral infection (SEVI)) were found to dramatically enhance the infectivity of the HIV virus (3–5 orders of magnitude by some measures). To complement those studies, we performed in vitro assays of PAP248-286, the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP248-286 promotes the interaction with lipid bilayers. At acidic (but not at neutral) pH, freshly dissolved PAP248-286 catalyzes the formation of large lipid flocculates in a variety of membrane compositions, which may be linked to the promotion of convective transport in the vaginal environment rather than transport by a random Brownian motion. Furthermore, PAP248-286 is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. An α-helical conformation of PAP248-286, lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests that nonspecific binding of monomeric or small oligomeric forms of SEVI in a helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus
The Sevi Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion
The Sevi Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion
Ligands for Glaucoma-Associated Myocilin Discovered by a Generic Binding Assay
Mutations in the olfactomedin domain of myocilin (myoc-OLF) are the strongest link to inherited primary open angle glaucoma. In this recently identified protein misfolding disorder, aggregation-prone disease variants of myocilin hasten glaucoma-associated elevation of intraocular pressure, leading to vision loss. Despite its well-documented pathogenic role, myocilin remains a domain of unknown structure or function. Here we report the first small-molecule ligands that bind to the native state of myoc-OLF. To discover these molecules, we designed a general label-free, mix-and-measure, high throughput chemical assay for restabilization (CARS), which is likely readily adaptable to discover ligands for other proteins. Of the 14 hit molecules identified from screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically Active Compounds using CARS, surface plasmon resonance binding studies reveal three are stoichiometric ligand scaffolds with low micromolar affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid formation in vitro. Structure–activity relationship-based soluble derivatives reduce aggregation in vitro as well as enhance secretion of full-length mutant myocilin in a cell culture model. Our compounds set the stage for a new chemical probe approach to clarify the biological function of wild-type myocilin and represent lead therapeutic compounds for diminishing intracellular sequestration of toxic mutant myocilin
Ligands for Glaucoma-Associated Myocilin Discovered by a Generic Binding Assay
Mutations in the olfactomedin domain
of myocilin (myoc-OLF) are
the strongest link to inherited primary open angle glaucoma. In this
recently identified protein misfolding disorder, aggregation-prone
disease variants of myocilin hasten glaucoma-associated elevation
of intraocular pressure, leading to vision loss. Despite its well-documented
pathogenic role, myocilin remains a domain of unknown structure or
function. Here we report the first small-molecule ligands that bind
to the native state of myoc-OLF. To discover these molecules, we designed
a general label-free, mix-and-measure, high throughput chemical assay
for restabilization (CARS), which is likely readily adaptable to discover
ligands for other proteins. Of the 14 hit molecules identified from
screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically
Active Compounds using CARS, surface plasmon resonance binding studies
reveal three are stoichiometric ligand scaffolds with low micromolar
affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid
formation <i>in vitro</i>. Structure–activity relationship-based
soluble derivatives reduce aggregation <i>in vitro</i> as
well as enhance secretion of full-length mutant myocilin in a cell
culture model. Our compounds set the stage for a new chemical probe
approach to clarify the biological function of wild-type myocilin
and represent lead therapeutic compounds for diminishing intracellular
sequestration of toxic mutant myocilin