Biology subjects

Peruzzi, J. A.

Publications and source records attributed to Peruzzi, J. A..

7 recordsLinked to original sources

bioRxiv2023

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

bioengineering↗

bioRxiv2023

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

synthetic biology↗

bioRxiv2022

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step towards this goal, we investigated how bacterial two-component systems can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the two-component system can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing. Significance StatementCells detect and respond to environmental and chemical information by using a combination of membrane proteins and genetic polymers. Recapitulation of this behavior in synthetic systems holds promise for engineering biosensors and therapeutics. Using the nitrate-sensing bacterial two-component system as a model, we demonstrate methods to reproduce and tune transmembrane signaling in synthetic lipid membranes, leading to the synthesis of genetically programmed proteins. Through this study, we gain insight into how membrane augmented cell-free systems can be used as a platform to characterize membrane-receptor interactions and engineer new biosensors.

synthetic biology↗

bioRxiv2022

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The extent to which membrane biophysical properties, such as hydrophobic thickness, can drive membrane protein organization remains unknown. Inspired by this question, we used de novo protein design, molecular dynamic simulations, and cell-free systems to elucidate how membrane-protein hydrophobic mismatch affects protein integration and organization in synthetic lipid membranes. We found that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We used this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to self-organizing membrane-based materials with new functions.

synthetic biology↗

bioRxiv2021

Lipid phase separation in vesicles enhances TRAIL-mediated cytotoxicity

Ligand spatial presentation and density play important roles in many signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Currently, Janus particles are most often used to spatially control ligand conjugation, but the technological challenge of manufacturing Janus particles limits adoption for translational applications. Here, we demonstrate that lipid phase separation can be used to spatially control protein presentation onto lipid vesicles. We used this system to study the density dependence of TNF-related apoptosis inducing ligand (TRAIL), a model therapeutic protein that exhibits greater cytotoxicity to cancer cells when conjugated onto a vesicle surface than when administered as a soluble protein. Using assays for apoptosis and caspase activity, we show that phase separated TRAIL vesicles induced higher cytotoxicity to Jurkat cancer cells than uniformly-conjugated TRAIL vesicles, and enhanced cytotoxicity was dependent on the TRAIL domain density. We then assessed this relationship in other cancer cell lines and demonstrated that phase separated TRAIL vesicles only enhanced cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, was unaffected by the TRAIL density. These results indicate unique signaling requirements for each TRAIL receptor and how TRAIL therapy could be tailored depending on the relative levels of expression for cancer receptors of interest. Overall, this work demonstrates a readily adoptable method to control protein conjugation and density on bilayer vesicles that can be easily adopted to other therapeutic nanoparticle systems to improve receptor signaling of nanoparticles targeted to cancer and diseased cells.

bioengineering↗

bioRxiv2020

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

synthetic biology↗

bioRxiv2019

Barcoding biological reactions with DNA-functionalized vesicles

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

synthetic biology↗