■ READING RNAS IN THE CELL

From Lee, J. H., et al., Science, 2014, 343, 1360. Reprinted withpermission from AAAS.

In the past decade, high-throughput sequencing technologieshave inspired a wide range of novel techniques to interrogatethe genome and transcriptome. By altering the biochemicalsteps of sample preparation, sequencing data is now used todetermine the structure of genomes or RNAs, observe ribosomepositioning on the mRNA, or determine the methylation statusof the DNA. These and many other “seq” methods haveemerged during the recent technology boom, but they all sharethe common thread of observing sequences as they are copiedin the instrument. Now, a group has taken that observation backto the cell by performing fluorescent in situ RNA sequencing, orFISSEQ.Instead of the usual flavor of RNA-seq where cDNA is read

while immobilized on a solid support in the sequencer, Leeet al. (Science, 2014, 343, 1360−1363) generated the cDNAsegments inside of cells by cross-linking, circularization, androlling circle amplification. The resulting cells were subjected tothe same ligation-mediated sequencing chemistry used by theApplied Biosystems SOLiD sequencer. Instead of imaging onthe usual instrument, however, confocal fluorescence micro-scopy was used to see 27−30 base pair reads growing into smallfoci approximately 600 nm in diameter. Analysis of the dataconfirmed the known subcellular locations for both cytoplasmicand nuclear RNAs while profiling expression of thousands ofgenes. Using human primary fibroblasts, an impressive 43% of

the in situ reads mapped to mRNAs. As expected, many of thesewere highly abundant genes and were markers for fibroblasts.In this proof-of-principal study, the authors showed thattheir sequencing preparation method could work in tissuessuch as brain slices or mouse embryos. This opens the doorfor the FISSEQ technique to take on challenging problems incomplex organisms like cell-type specific transcription or RNAprocessing.

Jason G. Underwood,, Ph.D.

■ A POWERFUL AND SELECTIVE TYPE I TYROSINEKINASE INHIBITOR

Smith, C. C., et al., Proc. Natl. Acad. Sci. U.S.A. DOI: 10.1073/pnas.1320661111. Copyright 2014 National Academy ofSciences, U.S.A.

Targeted cancer therapies are critical tools for the treatment ofspecific cancers. Tyrosine kinase inhibitors (TKIs) make up animportant class of these treatments because they antagonizeenzymes that promote cellular growth. But developing thesedrugs is difficult, and TKIs typically show at least one of twocommon flaws: they may interact with enzymes other than theirintended targets and thereby cause toxicity, or cancer cells mayeasily acquire resistance to them. Now Smith et al. have shownthat a new TKI for acute myeloid leukemia (AML), crenolanib,is both selective for its target, FLT3, and is invulnerable to themost common mechanism of resistance (Proc. Natl. Acad. Sci.U.S.A., 2014, DOI: 10.1073/pnas.1320661111).Researchers have typically found that TKIs that bind to

inactive forms of their tyrosine kinase target (called “type II”inhibitors) are the most selective, and most of the mostsuccessful TKIs used in cancer therapy fall into this category,with imatinib representing the prototypic example. Althoughtype II inhibitors can have very selective binding profiles, cancer

Published: April 18, 2014

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cells can often develop resistance to these drugs over time byacquiring mutations that favor the active kinase confirmation.Therefore, small molecules that target the active conformationof the kinase (“Type I” inhibitors) yet retain a high degreeof selectivity are particularly appealing. Crenolanib is a type Iinhibitor, that binds to and inhibits the active conformationof select class III receptor tyrosine kinases of which FLT3,the most commonly mutated gene in AML, is a member. Smithand Lasater et al. investigated the specificity and activity ofcrenolanib toward FLT3.Initial studies demonstrated that crenolanib selectively

inhibited FLT3 over KIT, a closely structurally related classIII RTK. Given its specificity for FLT3 inhibition, crenolanibwas screened against a panel of FLT3 mutants known to conferresistance to other FLT3 TKIs and was found to maintainactivity against all of these mutants. Crenolanib’s lack ofvulnerability to known TKI resistant mutations prompted theauthors to search broadly for any possible drug-resistantmutations. Crenolanib showed remarkably limited vulnerabilityto mutations in FLT3 that could lead to resistance, therebywarranting its designation as a “pan-FLT3” inhibitor. Therefore,crenolanib may represent an important step forward forachieving deep and durable remissions in AML patients whohave activating FLT3 mutations.

Sarah A. Webb,, Ph.D.

■ PROBING PROTEIN FOLDING IN CELLS

Liu, Y., et al., Proc. Natl. Acad. Sci. U.S.A., 111, 4449−4454.Copyright 2014 National Academy of Sciences, U.S.A.

Though strung together as a linear collection of amino acids,proteins must fold into intricate three-dimensional structures inorder to function properly. Through this process, various statesof properly folded, misfolded, and aggregated proteins cancoexist. It is relatively simple to distinguish aggregated, insolubleprotein from soluble protein; it is less straightforward to discernbetween soluble folded and soluble misfolded protein species incomplex cellular environments. To gain insight into this tangleddynamic, Liu et al. (Proc. Natl. Acad. Sci. U.S.A., 2014, 111,4449−4454) present the design, synthesis, and utility offluorescent small molecule probes that are highly selective fora folded and functional protein-of-interest over their soluble, butmisfolded, counterparts.The authors test their approach with two proteins, a de novo

designed retroaldolase and the thyroid hormone bindingprotein transthyretin. They use mutated, destabilized versionsof these proteins, because an increased proportion of the protein-of-interest is present in a soluble misfolded state−facilitating aninvestigation of the distribution of folded and soluble misfoldedproteins in cells. Using small molecule fluorescent folding probes,they were able to quantitate the fraction of folded and functionalprotein in cell lysates at a given point in time and discover thedependence of this fraction on the cellular folding environment.

Key to the success of their approach was the presence ofsufficient cellular holdase activity, created by ATP depletion ofthe lysed cell, which converts chaperones to holdases that bindavidly to protein folding intermediates. Cellular holdase activityprevents folding probe-associated folding equilibrium shifts.This study offers insight into the intricacies that govern cellularproteostasis and provides a blueprint for creating fluorogenicfolding probes, probes that can be used in a plate reader toquantify the folded fraction of a protein-of-interest to explorehow protein folding dynamics influence cellular function.

Eva J. Gordon,, Ph.D.

■ HARNESSING THE HEXOSAMINE PATHWAY

Reprinted from Cell, 156, Denzel, M. S., et al., HexosaminePathway Metabolites Enhance Protein Quality Control andProlong Life, 1167−1178. Copyright 2014, with permissionfrom Elsevier.

Aging, the extraordinarily complex but inevitable process thatso many seek to elude, devolves through an accumulateddecline in function at the cellular, organ, and whole organismlevel. Protein quality control systems, which regulate proteinsynthesis, folding, maturation, and removal in the cell, playa key role in longevity, and misregulation in these systems isassociated with a variety of age-related diseases. The nematodeCaenorhabditis elegans has been a pioneering model system tostudy the influence of protein quality control on longevity,as exemplified by numerous long-lived mutant strains that areable to sustain protein homeostasis to older ages. Exploringthis remarkable phenomenon further, Denzel et al. (Cell, 2014,156, 1167−1178) discover that certain mutations in an enzymecalled GFAT-1 lead to diminished susceptibility to agingdisorders and increased lifespan.The authors developed a screen to identify C. elegans mutants

resistant to treatment with tunicamycin, an inhibitor of thesynthesis of N-glycans, which are essential for the proper matura-tion and folding of many secreted proteins. Certain mutationsthat activate GFAT-1, a key enzyme in the hexosamine pathwayresponsible for synthesizing precursors for N-linked and O-linked

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glycans, conferred resistance to tunicamycin. Activation of thehexosamine pathway, as well as supplementation with theN-glycan precursor N-acetylglucsoamine, led to lifespan extensionand protection from certain age-related neurotoxicities. Thoughan association between protein quality control and longevityis well established, this study uncovers an unexpected linkbetween hexosamine metabolites and the protein quality controlsystem that was previously unappreciated. These findings suggestthat enhancing the capacity of the cell’s protein quality controlmechanisms through hexosamine pathway activation or supple-mentation with N-acetylglucosamine may be a therapeuticstrategy for various diseases related to aging or protein misfolding.

Eva J. Gordon,, Ph.D.

■ A PHOTO-OP FOR GPCRSApproximately one-quarter of marketed drugs target G-proteincoupled receptors (GPCRs), underscoring their value (boththerapeutic and financial) in medicine. New molecular tools forinvestigating GPCR biology will deepen our understanding ofhow these proteins function and could lead to the developmentof new and improved therapeutics for a variety of conditions.Opioid receptors, which control the perception of varioussensations including pain and euphoria, belong to Family A ofthe GPCRs. Also in this family are the opsin photoreceptors,such as rhodopsin, which control vision. Rhodopsin is activatedupon a light-induced cis−trans isomerization of a covalentlybound ligand, retinal. Schonberger et al. (Angew. Chem., Int. Ed.,2014, 53, 3264−3267) cleverly impart this light-sensitive propertyonto the u-opioid receptor (MOR) by designing and synthesizinga photochromic ligand derived from the MOR agonist fentanyl.The photochromic ligand, referred to as PF2, undergoes a

double-bond isomerization upon exposure to light. Specifically,in the dark or exposure to 420−480 nm light, the trans-configuration predominates, but irradiation with 360 nm lightinduces conversion to the cis-isomer. trans-PF2 is an effectiveopioid receptor agonist, but cis-PF2 is much less active. Thus,receptor activity can be manipulated by switching between 360and 480 nm light. Indeed, using electrophysiology to measurereceptor activity, the authors show that under 480 nm light,a potassium influx is triggered, but the influx can be stoppedby switching to 360 nm light. This photoactivation was stableand repeatable. This method for optically controlling MORactivity offers a new approach for exploring Family A GPCRs.Moreover, it provides a blueprint for pharmacological develop-ment of light-activated drugs.

Eva J. Gordon,, Ph.D.

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