Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1−40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed

Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase enzyme.

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site

Mapping Roles of Active Site Residues in the Acceptor Site of the PA3944 Gcn5-Related N-Acetyltransferase Enzyme

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site

Understanding the Roles of Bicoid and Broad in Lysozyme Production

This poster is the presentation of the outline for an ongoing project which evaluates the genes Bicoid and Broad and their effects on the production of lysozymes as potential transcription factors. This research project involves the use of a temperature-sensitive GAL system which localizes the expression of double-stranded RNA to the gut. This method of RNA interference is used to knockdown Bicoid and Broad expression to determine their effects on lysozyme expression. Lysozyme expression is to be measured via RNA extraction and analysis. It is expected that knocking down Bicoid will lead to higher expression of lysozymes and knocking down Broad will lead to a lower expression of lysozymes.Biology and Biochemistry, Department ofHonors Colleg

The design, synthesis, and evaluation of compounds that bind to Alzheimer's-related and HIV-1-related amyloids

Amyloids--misfolded, aggregated peptides--have been implicated in over thirty human diseases. This thesis focused on the study of two different amyloids--A[beta](1- 42) and SEVI (semen-derived enhancer of virus infection)-- associated with two distinct conditions--Alzheimer's Disease (AD) and Acquired Immune Deficiency Syndrome (AIDS), respectively. A[beta] aggregates are a hallmark of AD and may play a central, causative role in the pathogenesis of this disease. A[beta]-amyloid-targeting small molecules have, therefore, attracted wide interest as potential agents for the treatment or diagnosis of AD. This thesis describes the development of a general method to evaluate small molecule-A[beta]-amyloid binding interactions via a modified quantitative ELISA protocol. The implementation of an in vitro model to evaluate the blood-brain barrier permeability of [beta]-amyloid- targeting compounds is also discussed in this thesis. The design and evaluation of a new class of fluorescent probes that bind to A[beta] aggregates is described in this thesis. The advantage of these compounds is that their spectroscopic properties can be altered and fine-tuned via simple synthetic methods. The second portion of this thesis discusses the study of small molecules that bind to SEVI, a naturally abundant amyloid found in semen. SEVI can potentially increase the infectivity of HIV-1 in cells by up to 400,000-fold. Although the mechanism of SEVI- mediated transmission of HIV-1 remains poorly understood, evidence suggests that SEVI binds to both HIV-1 virions and cell membranes, thereby facilitating viral infection. We hypothesized that BTA-EG₆, a derivative of the well- known amyloid-binding compound Thioflavin T, could coat SEVI fibrils, thereby inhibit HIV-1 interactions with SEVI fibrils, and thus, reduce SEVI-mediated enhancement of HIV -1 infectivity. The results of these investigations are presented in this thesis. The final project described in this thesis is the design, synthesis, and evaluation of multivalent analogs of BTA-EG6. The goals of this project were 2-fold : 1) Create compounds that bind with high affinity to both A[beta] fibrils and SEVI fibrils based on the multivalent design strategy and 2) evaluate whether oligomers of the BTA moiety exhibit improved ability over the BTA monomer to inhibit SEVI-mediated enhancement of HIV-1 infectivity. The results of this project are presented in this thesi

Acute Oral Toxicity of the Crude Aqueous Extract of the Whole Plant of Euphorbia hirta L. (Family Euphorbiaceae)

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