■ BIOMECHANICAL TOXICITY OF ULTRAVIOLET RADIATION

Ultraviolet radiation (UV) from the sun is necessary for the synthesis of vitamin D in the skin, but it is also toxic, causingerythema (redness), photoaging, and cancer. Skin cancer is the most common form of cancer, causing 1.5 million disability-adjusted life-years and 60,000 premature deaths per year around the world. The mechanisms by which UV causes DNA damagehave been extensively studied, but we know little about its effects on the protective, barrier function of the skin. Now, Biniek et al.[(2012) Proc. Natl. Acad. Sci. U.S.A., 109, 17111] address this question.Forming the surface of the epidermis, the stratum corneum (SC) comprises multiple layers of dead cells that contain networks

of keratin fibers. The cells are held together by intercellular adhesive structures, the corneodesmosomes, and lipids. Biniek et al.isolated SC from the skin of human cadaver donors and exposed it to different doses of UV. Microtension testing revealed thatUV exposure had little effect on stiffness but caused reductions in fracture stress and strain. Bulge testing indicated a decrease inbiaxial stiffness and peak stress as a result of UV treatment. Biniek et al. went on to show that UV exposure lowers the energyrequired to delaminate the layers of the SC and that this effect became more pronounced with increasing depth. Attenuated totalreflectance Fourier transform infrared spectroscopy indicated that UV treatment resulted in reduced or modified lipid content,with increased lipid fluidity at the SC surface and decreased fluidity in deeper layers. UV exposure also increased hydration ofthe SC.The retention of stiffness by UV-treated skin suggests that keratin was not significantly affected. However, the loss of fracture

strength and strain combined with the decrease in delamination energy all suggest damage to the corneodesmosomes. Thesechanges are also consistent with alterations in lipid content and composition. The decrease in bulge stiffness can be explained bythe changes in lipid composition and the increase in hydration.Biniek et al. developed a mathematical model to relate the effects of UV on the biomechanical properties of the skin. The

results show that UV increases the mechanical driving force for cracking while decreasing the skin’s natural ability to resistcracking. They suggest that these are important toxic effects of UV that are not currently considered in the development andevaluation of sunscreens. Carol A. Rouzer

■ DISCRIMINATING ARSENATE FROM PHOSPHATE

Reprinted by permission from Macmillan Publishers Ltd from Elias et al. (2012) Nature, 491, 134.Copyright 2012.

Phosphate plays multiple critical roles in the biochemistry oflife, including energy metabolism, cell signaling, and nucleicacid structure. Thus, the fact that arsenate is almost structurallyidentical to phosphate in terms of pKa, oxygen atom charge,and thermochemical radius (only 4% larger) poses a dilemma.Failure of some enzymes of energy metabolism to discriminatebetween the two anions can lead to substitution of arsenate forphosphate and an uncoupling of oxidation from energy storage,a key mechanism of arsenic toxicity. Clearly, a cell’s ability to ex-clude arsenate is important, leading Elias et al. [(2012) Nature,491, 134] to explore the mechanism of phosphate/arsenate dis-crimination by bacterial periplasmic phosphate binding proteins(PBPs).Under phosphate-limited conditions, PBPs bind the anion in

the periplasmic space and deliver it to an ATP-dependent trans-porter for uptake into the cell. Elias et al. cloned, expressed,and purified one PBP each from E. coli, P. f luorescens, andK. variicola, and two PBPs from Halomonas sp. GFAJ-1. Com-petitive binding studies revealed 500- to 850-fold greaterbinding affinity for phosphate over arsenate in the case of 4

of the proteins. PBP-2 from Halomonas sp. GFAJ-1 exhibited a4500-fold discrimination. PBP-2 is highly expressed underconditions of low phosphate and high arsenate to which thisbacterium is well adapted.To understand the structural basis of the anion discrimina-

tion, Elias et al. obtained a high quality sub-Ångstrom crys-tal structure of the PBP from P. f luorescens complexed toarsenate. Comparison to comparable structures of the protein−phosphate complex revealed that the two anions are bound inexactly the same mode. Both are held in place by 12 H-bonds, 9of which are ion−dipole interactions. The difference in As−Oversus P−O bond lengths has very little effect on the bonddistances or angles of any of these interactions with the exceptionof one very short distance H-bond between O2 of the anion andthe carboxylate of Asp62. In the case of phosphate, this specialinteraction, classified as a heteromolecular negative-charge-assisted H-bond ((−)CAHB), exhibits nearly canonical bondlengths and angles. In contrast, for arsenate, the geometry issuboptimal. Also notable are very tight van der Waalsinteractions between each anion and Ala7 and Leu9 of theprotein. The steric clash with these residues resulting fromarsenate’s larger size is channeled to the CAHB.Mutation of Asp62, Ala7, or Leu9 resulted in a loss of

discrimination between arsenate and phosphate while havinglittle effect on phosphate affinity. The results suggest thatbinding and discrimination have distinct structural determi-nants. The basis for the extremely high discrimination ofHalomonas sp. GFAJ-1 PBP-2 will be the interesting focus offuture work. Carol A. Rouzer

Published: December 17, 2012

Reprinted by permission from Macmillan Publishers Ltd fromElias et al. (2012) Nature, 491, 134. Copyright 2012.

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■ NANOTOXICITY ACROSS THE GENOME

Reprinted from Reyes et al. (2012) ACS Nano, published online Oct 6, DOI: 10.1021/nn302815w.Copyright 2012 American Chemical Society.

Engineered zinc nanomaterials are among the most widely usednanoparticles, with current applications in cosmetics, paints,plastics, sunscreens, and tires. These are also among the mosttoxic nanoparticles, with adverse effects observed in a broadrange of species. Current methods of testing fail to provide acomprehensive assessment of all possible toxic mechanisms.Some studies indicate a major role for Zn2+ ion resulting fromnanoparticle dissolution, while others suggest particle-specificmechanisms. To address these questions, Reyes et al. [(2012)ACS Nano, published online Oct. 6, DOI: 10.1021/nn302815w]employed a genome-wide high-throughput approach to evaluatethe toxicity of zerovalent zinc and zinc oxide nanoparticles(nZn and nZnO, respectively) and ZnCl2 as a source of Zn2+

ions.Reyes et al. used an E. coli library comprising 4000 clones,

each representing the knockout of a unique single gene in thebacterial genome. They exposed all clones to each material atthe concentration that caused 50% inhibition of growth ofwild-type bacteria (IC50) and monitored growth at four timepoints representing lag phase, exponential, and stationarygrowth. The results identified 173 clones that exhibitedincreased sensitivity to at least one of the materials at aminimum of one time point. These were subjected to twofurther testing events to elucidate the IC50 of all threematerials at two separate time points.Of the 173 clones, 112, 77, and 81 were sensitive to ZnCl2,

nZn, and nZnO, respectively, with 70, 17, and 19 respectiveclones being unique for each of the materials and 24 clonessensitive to all three. Cluster analysis indicated distinct groupsof clones that were sensitive at early (106 clones) versus late(91 clones) time points (24 shared clones) and distinguishedbetween groups of high (26 clones), intermediate (94 clones),and mild (53 clones) sensitivity. In general, IC50 values of thesensitive clones increased over time, though this trend was notfound in the high sensitivity clones, which tended to be late orshared time point responders.Functional annotation clustering of the sensitive clones

revealed 8 clusters in 5 general biological categories. Theseincluded metal homeostasis (transport and binding), centralmetabolism (monosaccharide synthesis), regulation and signal-ing (transcription and two component signaling), generaltransport, and membrane-related (lipopolysaccharide synthesisand membrane structure) categories. Clones bearing mutationsin Zn metabolism exhibited the lowest IC50’s at all time points.In general, persistent toxicity was associated with mutations in

membrane-related genes, while most other pathways led toeffects at early time points. The results revealed uniquemechanisms or patterns of mechanisms for each material thatwere only detected at one time point, confirming the value ofthis genome-wide, comprehensive approach for the assessmentof nanoparticle toxicity. Carol A. Rouzer

■ ROLE FOR TRNA MODIFICATION AFTER DNADAMAGE

In Saccharomyces cerevisiae, the enzyme tRNA methyltransferase9 (Trm9) catalyzes the methylation of uridines in the wobbleposition of tRNA codons to facilitate the synthesis of5-methoxycarbonylmethyluridine (mcm5U). The substratetRNAs code for Arg, Lys, Glu, or Gln. The presence ofmcm5U enhances binding to codons ending in A for Arg andGlu residues, and speeds translation. Cells deficient in Trm9(trm9Δ) are more sensitive to killing by ionizing radiation,hydroxyurea, and alkylating agents than wild-type (WT) cells,suggesting a role for Trm9-mediated translation regulation inthe DNA damage response. One possible mechanism by whichthis may occur is through regulation of translation of the Rnr1and/or Rnr3 subunits of the ribonucleotide reductase (RNR)complex, a key enzyme in the biosynthesis of dNTPs. RNRactivity markedly increases during DNA synthesis and afterDNA damage in order to provide adequate dNTPs for repair.Prior studies show decreased levels of Rnr1 and Rnr3 in trm9Δcells, which cannot be explained by reduced transcription oftheir respective genes. This led Patil et al. [(2012) Cell Cycle,11, 3656] to investigate the role of Trm9 in RNR regulation ingreater detail.When cells were synchronized in various stages of the cell

cycle, trm9Δ cells exhibited lower levels of Rnr1 protein thanWT cells. Analysis by chromatography-coupled MS revealeddifferences in levels of specific tRNA modifications in S-phasesynchronized versus asynchronous cells, including an ∼2-foldincrease in the level of mcm5U in S-phase. The resultssuggested a role for Trm9 in Rnr1 translation.After treatment with the DNA alkylating agent methyl

methanesulfonate (MMS), trm9Δ cells exhibited a delayedprogression to S-phase relative to WT cells. This delay wasalleviated by re-expressing TRM9, overexpressing RNR1, ordeleting the gene for the RNR inhibitor Sml1 in trm9Δ cells. Aluciferase reporter construct designed to measure translation oftranscripts containing a 12 base pair sequence of mcm5U-dependent codons demonstrated decreased translation of thesecodons in trm9Δ cells as compared to WT cells. This couldexplain low levels of Rnr1 in trm9Δ cells since the transcript ofRNR1 shows biased use of the mcm5U-dependent codons AGAfor Arg and GAA for Glu. To test this hypothesis, Patil et al.replaced the RNR1 in WT and trm9Δ cells with opt-RNR1, agene using all high-usage codons optimized for rapid, mcm5U-independent translation. Expression of opt-RNR1 resulted inhigher Rnr1 protein levels and a more rapid transition toS-phase following MMS treatment in both WT and trm9Δ cells.These results indicate that Rnr1 protein levels are rate-limitingfor the S-phase transition following DNA damage and providefurther support for the hypothesis that selective translation of RNR1transcripts is regulated by Trm9-dependent tRNA modificationsduring the DNA damage response. Carol A. Rouzer

Reprinted from Reyes et al. (2012) ACS Nano, published onlineOct 6, DOI: 10.1021/nn302815w. Copyright 2012 AmericanChemical Society.

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