Membrane
proteins are present in a wide array of cellular processes
from primary and secondary metabolite synthesis to electron transport
and single carbon metabolism. A key barrier to applying membrane proteins
industrially is their difficult functional production. Beyond expression,
folding, and membrane insertion, membrane protein activity is influenced
by the physicochemical properties of the associated membrane, making
it difficult to achieve optimal membrane protein performance outside
the endogenous host. In this review, we highlight recent work on production
of membrane proteins in membrane augmented cell-free systems (CFSs)
and applications thereof. CFSs lack membranes and can thus be augmented
with user-specified, tunable, mimetic membranes to generate customized
environments for production of functional membrane proteins of interest.
Membrane augmented CFSs would enable the synthesis of more complex
plant secondary metabolites, the growth and division of synthetic
cells for drug delivery and cell therapeutic applications, as well
as enable green energy applications including methane capture and
artificial photosynthesis.
The
serotonin receptor 4b (5-HTR4b) is expressed throughout
the gastrointestinal tract, and its agonists are used in the treatment
of irritable bowel syndrome with constipation (IBS-C). Today, there
are no rapid assays for the identification of 5-HTR4b agonists.
Here, we developed a luciferase-based 5-HTR4b assay capable
of assessing one compound per second with a 38-fold dynamic range
and nM limit of detection for serotonin. We used the assay to screen
more than 1000 natural products and anti-infection agents and identified
five new 5-HTR4b ligands: hordenine, halofuginone, proflavine,
ethacridine, and revaprazan. We demonstrate that hordenine (antibiofilm),
halofuginone (antiparasitic), and revaprazan (gastric acid reducer)
activate 5-HTR4b in human colon epithelial cells, leading
to increased cell motility or wound healing. The 5-HTR4b assay can be used to screen larger pharmaceutical libraries to identify
novel treatments for IBS-C. This work shows that antimicrobials interact
not only with the gut microbiota, but also with the human host.
Olfactory
receptors are ectopically expressed (exORs) in more than
16 different tissues. Studying the role of exORs is hindered by the
lack of known ligands that activate these receptors. Of particular
interest are exORs in the colon, the section of the gastrointestinal
tract with the greatest diversity of microbiota where ORs may be participating
in host–microbiome communication. Here, we leverage a G-protein-coupled
receptor (GPCR)-based yeast sensor strain to generate sensors for
seven ORs highly expressed in the colon. We screen the seven colon
ORs against 57 chemicals likely to bind ORs in olfactory tissue. We
successfully deorphanize two colon exORs for the first time, OR2T4
and OR10S1, and find alternative ligands for OR2A7. The same OR deorphanization
workflow can be applied to the deorphanization of other ORs and GPCRs
in general. Identification of ligands for OR2T4, OR10S1, and OR2A7
will enable the study of these ORs in the colon. Additionally, the
colon OR-based sensors will enable the elucidation of endogenous colon
metabolites that activate these receptors. Finally, deorphanization
of OR2T4 and OR10S1 supports studies of the neuroscience of olfaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.