3 research outputs found
Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle
Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme–nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector
A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins
Using a chemical approach to crosslink functionally versatile
bioeffectors
(such as peptides) to native proteins of interest (POI) directly inside
a living cell is a useful toolbox for chemical biologists. However,
this goal has not been reached due to unsatisfactory chemoselectivity,
regioselectivity, and protein selectivity in protein labeling within
living cells. Herein, we report the proof of concept of a cytocompatible
and highly selective photolabeling strategy using a tryptophan-specific
Ru-TAP complex as a photocrosslinker. Aside from the high selectivity,
the photolabeling is blue light-driven by a photoinduced electron
transfer (PeT) and allows the bioeffector to bear an additional UV-responsive
unit. The two different photosensitivities are demonstrated by blue
light-photocrosslinking a UV-sensitive peptide to POI. Our visible
light photolabeling can generate photocaged proteins for subsequent
activity manipulation by UV light. Cytoskeletal dynamics regulation
is demonstrated in living cells via the unprecedented POI photomanipulation
and proves that our methodology opens a new avenue to endogenous protein
modification
Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking
The abnormal assembly
of misfolded proteins into neurotoxic aggregates
is the hallmark associated with neurodegenerative diseases. Herein,
we establish a photocontrollable platform to trigger amyloidogenesis
to recapitulate the pathogenesis of amyotrophic lateral sclerosis
(ALS) by applying a chemically engineered probe as a “switch”
in live cells. This probe is composed of an amyloidogenic peptide
from TDP-43, a photolabile linker, a polycationic sequence both to
mask amyloidogenicity and for cell penetration, and a fluorophore
for visualization. The photocontrollable probe can self-assemble into
a spherical vesicle but rapidly develops massive nanofibrils with
amyloid properties upon photoactivation. The photoinduced <i>in vitro</i> fibrillization process is characterized by biophysical
techniques. In cellular experiments, this cell-penetrable vesicle
was retained in the cytoplasm, seeded the mislocalized endogenous
TDP-43 into aggregates upon irradiation, and consequently initiated
apoptosis. In addition, this photocontrollable vesicle interfered
with nucleocytoplasmic protein transport and triggered cortical neuron
degeneration. Our developed strategy provides <i>in vitro</i> and <i>in vivo</i> spatiotemporal control of neurotoxic
fibrillar aggregate formation, which can be readily applied in the
studies of protein misfolding, aggregation-induced protein mislocalization,
and amyloid-induced pathogenesis in different diseases