2 research outputs found
A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering
The rapid development of new small
molecule drugs, nanomaterials,
and genetic tools to modulate cellular function through cell surface
manipulation has revolutionized the diagnosis, study, and treatment
of disorders in human health. Since the cell membrane is a selective
gateway barrier that serves as the first line of defense/offense and
communication to its environment, new approaches that molecularly
engineer or tailor cell membrane surfaces would allow for a new era
in therapeutic design, therapeutic delivery, complex coculture tissue
construction, and <i>in situ</i> imaging probe tracking
technologies. In order to develop the next generation of multimodal
therapies, cell behavior studies, and biotechnologies that focus on
cell membrane biology, new tools that intersect the fields of chemistry,
biology, and engineering are required. Herein, we develop a liposome
fusion and delivery strategy to present a novel dual receptor and
reporter system at cell surfaces without the use of molecular biology
or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal
functional groups that can conjugate a range of ligands while simultaneously
reporting the conjugation through the emission of fluorescence. We
demonstrate this dual receptor and reporter system by conjugating
and tracking various cell surface ligands for temporal control of
cell fluorescent signaling, cell–cell interaction, and tissue
assembly construction
Host–Pathogen Interaction Profiling Using Self-Assembling Human Protein Arrays
Host–pathogen protein interactions
are fundamental to every
microbial infection, yet their identification has remained challenging
due to the lack of simple detection tools that avoid abundance biases
while providing an open format for experimental modifications. Here,
we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo
ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA)
with 10 000 unique human proteins. We identified known targets
of these L. pneumophila proteins and
potentially novel interaction candidates. In addition, we applied
our Click chemistry-based NAPPA platform to identify the substrates
for SidM, an effector with an adenylyl transferase domain that catalyzes
AMPylation (adenylylation), the covalent addition of adenosine monophosphate
(AMP). We confirmed a subset of the novel SidM and LidA targets in
independent in vitro pull-down and in vivo cell-based assays, and
provided further insight into how these effectors may discriminate
between different host Rab GTPases. Our method circumvents the purification
of thousands of human and pathogen proteins, and does not require
antibodies against or prelabeling of query proteins. This system is
amenable to high-throughput analysis of effectors from a wide variety
of human pathogens that may bind to and/or post-translationally modify
targets within the human proteome