Spotlight pubs.acs.org/acschemicalbiology
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MEMBRANE PROTEIN OLIGOMERS STABILIZED BY INTERFACIAL LIPIDS
GUT MICROBIOTA-DERIVED METABOLITES INHIBIT HOST PROTEASES
Reprinted by permission from Macmillan Publishers Ltd.: Nature Robinson et al., 541, 421. Copyright 2017.
Proteins embedded in the cell membrane have diverse and important functions that make membrane proteins popular targets for drug designers and structural biologists alike, the latter group rising to the challenge of establishing experimental conditions in which the native conformation of a membrane protein is preserved. This difficulty is further compounded by the fact that many membrane proteins form oligomers, which in turn can be affected by molecules that bind to and stabilize protein−protein interfacesa topic recently addressed in a breakthrough report by a research team led by Carol V. Robinson at the University of Oxford (Nature 2017, 541, 421). The team ranked the oligomeric stability of 125 structurally characterized α-helical oligomeric transmembrane proteins based on their buried interfacial surface area and number of salt-bridge interactions. Initial mass spectrometry studies indicated that proteins with high predicted oligomeric stability were devoid of additional small molecules, while protein complexes with low predicted oligomeric stability appeared to contain noncovalently bound lipids. To simultaneously identify these lipids while preserving the oligomeric state of the low-stability protein complexes, Robinson and co-workers developed a tandem mass spectrometry (MS) technique in which protein−lipid complexes are first isolated from the detergent micelles in which they are suspended by applying high energy at the source of sample injection; the liberated complexes are then separated from extraneous molecules by a quadrupole and finally are dissociated in a collision cell so that the constituent proteins and lips can be identified by the MS detector. Having determined the identity and stoichiometry of lipids bound to several transmembrane protein dimers and confirming their MS results with biochemical assays, the research team proposed that interfacial lipid binding stabilizes weak oligomers, a possible mechanism for controlling transmembrane protein oligomerization, and subsequently function, in cells.
Reprinted from Cell, 168, Chun-Jun Guo et al. Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases, pp 517−526.e18. Copyright 2017, with permission from Elsevier.
The mammalian gut microbiome, comprised of all microorganisms that reside in the intestinal tract, is known to influence host digestion and is linked to immune system function and cardiovascular and intestinal tract health. Researchers racing to elucidate the molecular mechanisms behind these effects are aided by the availability of the National Institutes of Health Human Microbiome Project (NIH HMP) database, which provides gastrointestinal microbial metagenomesthe combined genetic sequences of all microorganisms present in a samplederived from stool samples taken from hundreds of healthy volunteers. A research team headed by Michael A. Fischbach of the University of California, San Francisco observed that over 90% of the HMP stool samples contained gene clusters from a family of nonribosomal peptide synthases (NRPS) that appeared to almost exclusively reside in the gut microbiomes as opposed to other sites of host colonization. To determine the products of these synthases, which were confirmed to be actively transcribed in the gut but were mostly derived from bacteria that are difficult to manipulate in the lab, the research team expressed the synthases in E. coli and B. subtilis hosts instead. The NPRS clusters appeared to be producing cyclic amides known as pyrazinones, all of which could be derived from the cyclization and subsequent oxidation of dipeptide aldehyde
Heidi A. Dahlmann
Published: February 17, 2017 © 2017 American Chemical Society
313
DOI: 10.1021/acschembio.7b00095 ACS Chem. Biol. 2017, 12, 313−315
ACS Chemical Biology
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or pharmaceutical inhibition of ERK proteins in mouse models confirmed that the ERK pathway forms a critical link between wakefulness-induced neuronal gene expression and sleep duration and quality.
precursors initially released from the NPRS. Reasoning that in the anaerobic condition of the gut the irreversible oxidation of cyclized aldehydes would not occur, the research team hypothesized that the linear dipeptide aldehydes would be the predominant bioactive metabolites produced by the NPRSespecially because peptide aldehydes are well-known to be potent protease inhibitors. Indeed, four of the NPRSderived dipeptide aldehydes were found to inhibit cathepsin, a mammalian protease involved in antigen processing and presentation. These recently reported results (Cell 2017, 168, 517−526.e18) prompted the research team to speculate that the dipeptide aldehydes may have important immunomodulatory effects.
Heidi A. Dahlmann
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SENSING XENOESTROGENS
Heidi A. Dahlmann
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Reprinted from Furst et al., ACS Cent. Sci., DOI: 10.1021/ acscentsci.6b00322. Copyright 2017 American Chemical Society.
SLEEP DURATION REGULATED BY ERK SIGNALING PATHWAY
Many pathways in the human body are finely controlled by low concentrations of hormones produced by the endocrine glands. Processes from basal metabolism to fertility are tuned by hormone levels, so disorders that alter the complex endocrine system can have profound consequences. Complicating the situation further, endocrine disrupting compounds (EDCs) found in the environment can produce off-target effects which potentially impact human health and reproduction. One example of an EDC is bisphenol A (BPA), commonly used in the production of epoxy resins and polycarbonate plastics over the past half century. BPA exhibits estrogen-like properties, and though it is far less potent than the natural hormone, growing concerns about its long-term effects have inspired ban legislation and coaxed some plastic manufacturers to remove it from food and drink packaging. Today, the identities and effects of many EDCs are not fully tractable, so there is a growing need for assays that can both hunt for EDCs and quantify their activity on natural receptors of interests. In a recent study, Furst et al. (ACS Cent. Sci., DOI: 10.1021/ acscentsci.6b00322) built a disposable chip capable of detecting soluble EDCs that bind to the human estrogen receptor (ERα). Specificity was mediated by an ERα-specific monobody, a synthetic fibronectin domain-based protein previously selected for affinity to the ligand-bound form of ERα. When a ligand or EDC-bound receptor is present, it binds the monobody immobilized to the gold electrode surface, and electrochemical detection is achieved through impedance spectroscopy. Since the receptor is not enough to produce a significant signal, the researchers used freeze-dried E. coli expressing the estrogen receptor on the bacterial surface to dramatically increase the mass and in turn the impedance change. The results showed that with a sample volume as low as 10 μL, the chip could detect natural estrogen down to the femtomole level and detect numerous ERα-binding EDCs either diluted into a simple buffer or in the relevant and far more complex solution, infant formula.
From Mikhail et al., Sci. Signal, 2017, 10, eaai9219. Reprinted with permission from AAAS.
Life forms spanning the scale of complexity from tiny nematodes to mammals all share a very important behavior: we all sleep. Broadly defined as a reversible period of immobility and reduced ability to respond to external stimuli, sleep is also recognized to be homeostatically regulated. To illustrate, anyone who has experienced a long period of activity- or stressinduced wakefulness looks forward to being compensated with an extended, deeper period of sleep. The biological effects of sleep deprivation go beyond the symptoms of physical and psychological burnout. At the molecular level, extended wakefulness is associated with upregulation of certain genes including Arc, Bdnf, Egr, and Homer1a, which are involved in synaptic activity (i.e., neurotransmission) and neuronal plasticity (the ability of the brain to remodel itself in response to external stimuli, a crucial process in learning and forming memories). Other genes such as Cirbp and Dbp, which are involved in cell stress responses and circadian rhythm regulation, respectively, are notably downregulated during extended wakefulness. Considering the distribution of sleep behavior across the animal kingdom as well as the defined pattern of gene regulation associated with sleep deprivation, a research team led by Mehdi Tafti at the University of Lausanne sought to determine if a single, conserved signaling pathway was playing a role in sleep homeostasis (Sci. Signal. 2017, 10, eaai9219). Using a previously validated cell culture model that mimicked the synchronous firing of cortical neurons in nonrapid eye movement sleep, they determined that the ERK pathway was the only major signaling pathway that could be induced to simultaneously upregulate Arc, Bdnf, and Homer1a and downregulate Dbp. Further experiments involving knockdown
Jason G. Underwood 314
DOI: 10.1021/acschembio.7b00095 ACS Chem. Biol. 2017, 12, 313−315
ACS Chemical Biology
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Spotlight
PROBING HYPOXIA WITH AZIDES
Reprinted with permission from O’Connor et al. ACS Cent Sci., 3, 20−30. Copyright 2017 American Chemical Society.
Azides have become a darling of chemical biology, both as a tool for facilitating selective reactions of biological probes and for detecting hydrogen sulfide. But now researchers have reported that these nitrogen moieties can also serve as a sensitive way to follow hypoxia (O’Connor, L. J., et al. ACS Cent Sci. 2017, 3, 20−30). Physiological data on drugs containing azides, such as the antiviral drug AZT, have suggested that oxygen could play a role in their metabolism. O’Connor et al. probed this question using CH-02, a biological probe containing an azide in conjugation with a system of π electrons. CH-02 is not fluorescent, but the corresponding amine CH-02F is highly fluorescent at 625 nm. The researchers started with a simple test in several cell types: at 21% oxygen, CH-02 was not reactive after 3 h, but under hypoxic conditions (
