EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World

A strain of the whitefly Bemisia tabaci (Gennadius) possessing unusually high levels of resistance to a wide range of insecticides was discovered in 2004 in the course of routine resistance monitoring in Arizona. The multiply resistant insects, collected from poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch) plants purchased at a retail store in Tucson, were subjected to biotype analysis in three laboratories. Polyacrylamide gel electrophoresis of naphthyl esterases and sequencing of the mitochondrial cytochrome oxidase I gene (780 bp) confirmed the first detection of the Q biotype of B. tabaci in the New World. This U.S. Q biotype strain, referred to as Poinsettia'04, was highly resistant to two selective insect growth regulators, pyriproxyfen and buprofezin, and to mixtures of fenpropathrin and acephate. It was also unusually low in susceptibility to the neonicotinoid insecticides imidacloprid, acetamiprid, and thiamethoxam, relative to B biotype whiteflies. In 100 collections of whiteflies made in Arizona cotton (Gossypium spp.), vegetable, and melon (Cucumis melo L.) fields from 2001 to 2005, no Q biotypes were detected. Regions of the United States that were severely impacted by the introduction of the B biotype of B. tabaci in the 1980s would be well advised to promote measures that limit movement of the Q biotype from controlled environments into field systems and to formulate alternatives for managing this multiply-resistant biotype, in the event that it becomes more widely distributed.     

Protein Folding Requires Crowd Control in a Simulated Cell

Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have computationally studied the effect of crowding upon protein folding for 12 small domains in a simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics, designed to unify the often disjoint goals of protein folding simulation and structure prediction. The model can make predictions of native conformation with accuracy comparable with that of the best current template-free models. It is fast enough to enable a more extensive analysis of crowding than previously attempted, studying several proteins at many crowding levels and further random repetitions designed to more closely approximate the ensemble of conformations. We found that when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in extended conformations. These results suggest that the ability of small protein domains to fold without the help of chaperones may be an important factor in limiting the degree of macromolecular crowding in the cell. Here, we discuss the possible implications regarding the relationship between protein expression level, protein size, chaperone activity and aggregation.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Complete Genome Sequence of Staphylococcus aureus Strain JKD6159, a Unique Australian Clone of ST93-IV Community Methicillin-Resistant Staphylococcus aureus

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.Staphylococcus aureus is a major cause of both hospital- and community-acquired infections, with rapid emergence of antibiotic resistance, in particular methicillin resistance, adding complexity to the treatment of this organism (3). While previously a hospital problem, methicillin-resistant S. aureus (MRSA) is now being increasingly documented in healthy patients in the community, and these isolates are termed “community MRSA” (cMRSA). A number of cMRSA genomes have been sequenced; however, these are phylogenetically closely related to each other. In contrast, ST93-MRSA-IV, a unique Australian clone, is a singleton by multilocus sequence typing (MLST) eBURST analysis (4). It is now the dominant cMRSA clone in Australia and is associated with both skin infection and severe invasive infection, including necrotizing pneumonia, deep-seated abscesses, and septicemia (5, 10). JKD6159 is a representative ST93-MRSA-IV clinical isolate which caused septicemia and multifocal pulmonary and musculoskeletal abscesses in a previously well intravenous drug user and also resulted in a familial outbreak of skin infection.The genome sequence of S. aureus strain JKD6159 was determined by high-throughput whole-genome shotgun sequencing, using both Illumina GAII (Illumina, CA) and Roche GS FLX Titanium (Roche Diagnostics, Basel, Switzerland) sequencing technologies, producing approximately 164× and 32× coverage of the genome, respectively. The GS FLX Titanium reads were assembled using Newbler 2.0.01.12, resulting in 56 contigs totaling 2.8 Mbp (9). The paired GAII reads were aligned to the contigs using SHRiMP 1.3.2 to identify and correct 74 homopolymeric sequencing errors (11). Optical mapping was used to produce a high-resolution XbaI chromosome restriction map, and the contigs were ordered and oriented against this map using MapSolver 2.1.1 (Opgen). Gap closures were performed by PCR followed by Sanger sequencing of amplification products (3730S genetic analyzer sequencer; Applied Biosystems, CA). The finished sequence was validated by reference to the XbaI optical map, Roche GS FLX Titanium mate pair analysis, and Illumina paired-end-read analysis.Protein coding regions were predicted using GeneMarkS 4.6b, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 7, 8). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq Proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 6). These automated analyses were followed by manual curation, including comparison with other completed S. aureus genomes.The genome of S. aureus strain JKD6159 consists of a circular 2,811,435-bp chromosome with 33% G+C content—similar to those of other staphylococci—and one circular plasmid of 20,730 bp. A total of 2,605 coding regions, 57 tRNA genes, and 5 rRNA loci were detected. Over 67% of genes were assigned to specific Clusters of Orthologous Groups (COG) Database functional groups, and 40% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6159 confirms that ST93-MRSA-IV is distantly related to other previously sequenced S. aureus genomes. ST93-MRSA-IV has a distinct accessory genome. There were a number of regions of difference in JKD6159 that contain coding sequences (CDS) not present in any other published S. aureus genomes. Additionally, the ssl gene cluster in JKD6159 appears distinct from other sequenced S. aureus isolates. Comparison with other S. aureus genomes also shows that although JKD6159 carries lukSF-PV (the genes encoding Panton-Valentine leukocidin), there is a relative paucity of virulence factors such as tst-1, genes encoding staphylococcal enterotoxins A to U, and the ACME locus. Further analysis of the genome is now under way to identify factors that might explain the emergence of this MRSA strain in the community.     

Syntheses and characterization of the W(II) and W(III) N2 complexes, [WCl2(PMe3)3]2N2 and [WCl3(PMe3)2]2N2

Refluxing WCl₄(PMe₃)₃ under a nitrogen atmosphere in the presence of two equivalents of sodium amalgam leads to a reduction to the W(II) complex [cis,mer-WCl₂(PMe₃)₃]₂N₂ (1), which can be converted to [mer,trans-WCl₃(PMe₃)₂]₂N₂ (2) via appropriate oxidation/chlorination. Structural data have been obtained for both complexes, and demonstrate significantly increased steric crowding in 1 due to PMe₃/PMe₃ interactions. The N-N bond distances in the two compounds are similar, at 1.279(4) and 1.243(18) Å, respectively.     

Notes on <Emphasis Type="Italic">Swartzia</Emphasis> (Leguminosae) in Central America preliminary to the Flora Mesoamericana,with descriptions of two new species from Costa Rica

In preparation of a treatment of Swartzia (Leguminosae) for the Flora Mesoamericana, recent updates to the taxonomy of the genus in Central America are discussed, and two new species from the Pacific slope and lowlands of central and southern Costa Rica are described and illustrated. One of them, S. picramnioides, is a member of the section Possira and is closely related to S. standleyi of Guatemala and Belize and to the South American species S. myrtifolia. The other, S. zeledonensis, belongs to the Central American apetalous clade of section Terminales. It is probably most closely related to the Panamanian species, S. nuda. We conclude that Swartzia is represented in Mexico and Central America by 14 species and provide a key for their identification.     

Long range dynamic effects of point-mutations trap a response regulator in an active conformation

When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This ‘conformational trapping’ results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Structural bases of PAS domain-regulated kinase (PASK) activation in the absence of activation loop phosphorylation

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.