Substitution Of Phenylalanine Research Articles

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non-receptor-type tyrosine kinase c-Abl. c-Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N- and C-terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C-terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c-Abl, enhanced the activation of estrogen signaling. In contrast, the c-Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild-type. These results suggest that c-Abl-mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453-1461, 2017. © 2016 Wiley Periodicals, Inc.

Molecular recognition at cholinergic synapses: acetylcholine versus choline.

Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H-bonds position ACh and choline differently in the aromatic cage to generate the different affinities. Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.

Sensitivity of Fusarium oxysporum f. sp. niveum to Prothioconazole and Thiophanate-Methyl and Gene Mutation Conferring Resistance to Thiophanate-Methyl

Fusarium wilt, incited by the fungus Fusarium oxysporum f. sp. niveum, is a soilborne disease that affects watermelon production worldwide. Approaches for effective management of Fusarium wilt in watermelon are limited. Studies conducted in recent years indicated that prothioconazole and thiophanate-methyl reduced the disease significantly under field conditions. However, effects of the fungicides on different life stages of F. oxysporum f. sp. niveum and potential existence of fungicide resistance in F. oxysporum f. sp. niveum populations are unknown. In the present study, effects of prothioconazole and thiophanate-methyl on mycelium growth and spore germination of F. oxysporum f. sp. niveum isolates collected in watermelon fields in Georgia were determined. In vitro mycelium growth studies indicated that all 100 isolates evaluated were sensitive to prothioconazole; the effective concentration that suppressed mycelium growth by 50% ranged from 0.75 to 5.69 μg/ml (averaged 1.62 μg/ml). In contrast, 33 and 4% of the isolates were resistant to thiophanate-methyl at 10 and 100 μg/ml, respectively. Microconidial germination assays showed that 36 and 64% of the isolates tested were sensitive or intermediately sensitive to prothioconazole at 100 μg/ml but the fungicide did not inhibit spore germination at 10 μg/ml. Sequencing a portion of the β-tubulin gene of eight isolates resistant or sensitive to thiophanate-methyl indicated that fungicide resistance was associated with a point mutation at nucleotide position 200, resulting in a substitution of phenylalanine by tyrosine. This is the first report of isolates of F. oxysporum resistant to thiophanate-methyl. Results of the research suggest that prothioconazole may be a viable option for management of Fusarium wilt of watermelon whereas thiophanate-methyl should be used judiciously due to the existence of isolates resistant to the fungicide.

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster.

The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo-, bromo-, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 μM (Kd ) and their efficiency of dehalogenation ranges from 2.4 - 9 x 103 M-1 s-1 (kcat /Km ). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta-genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis.

Characterization of additional vitamin D binding protein variants

The gene (GC) for the vitamin D binding protein (DBP) shows significant genetic variation. Two missense variants, p.D432E and p.T436K, are common polymorphisms and both may influence vitamin D metabolism. However, less common variants, identified biochemically, have been reported previously. This study aimed to identify the underlying mutations by molecular screening and to characterize the mutant proteins by mass spectrometry. Denaturing high performance liquid chromatography (DHPLC) was used for screening genetic variants in GC exons and exon/intron boundaries of genomic DNA samples. Sanger sequencing identified the specific mutations. An immuno-capture coupled mass spectrometry method was used to characterize protein variants in serum samples. Initial molecular screening identified 10 samples (out of 761) containing an alanine deletion at codon 246 in exon 7 (p.A246del, c.737_739delCTG), and 1 sample (out of 97) containing a cysteine to phenylalanine substitution at codon 311 in exon 8 (p.C311F, c.932G>T). The mutant allele proteins and posttranslational modified products were distinguishable from the wild-type proteins by mass spectrum profiling. Loss of a disulfide bond due to loss of cysteine-311 was accompanied by the appearance of a novel mixed disulfide species, consistent with S-cysteinylation of the remaining unpaired cysteine-299 in the mutant protein. We confirm earlier biochemical studies indicating that there are additional deleterious GC mutations, some of which may be low-frequency variants. The major findings of this study indicate that additional mutant proteins are secreted and can be identified in the circulation. By combining molecular screening and mass spectrometric methods, mutant DBP species can be identified and characterized.

Immunomodulatory and Anti-Inflammatory Activities of Chicken Cathelicidin-2 Derived Peptides.

Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that full-length CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives.

Functional Modification of Bacterial Voltage-Gated Sodium Channels by Batrachotoxin

Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.

Clonal Evolution and Lack of BCR-ABL1 Mutations in Pediatric Ph+ ALL Patients Resistant/Refractory to Imatinib Treatment

Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL) is characterized by the translocation t(9;22), resulting in the BCR-ABL1 fusion gene. It occurs in 2-3% of pediatric ALL and is associated with poor prognosis. Despite the introduction of tyrosine kinase inhibitors (TKIs), such as Imatinib, resulting in a significant increase in the cure rate, a consistent number of patients show resistance to treatment and subsequently relapse. In an adult cohort, more than 70% of relapses during TKI treatment involve selection of tyrosine kinase domain (TKD) mutations.In order to better understand the mechanism of Imatinib resistance in pediatric Ph+ ALL, we selected as our cohort a subgroup of patients treated with Imatinib on top of chemotherapy, according to the EsPhALL clinical trial and enrolled in Italy from January 2004 to December 2014 by the AIEOP study group. Patients (n=10) either didn't achieve remission or experienced early relapse by the end of 2014. Patients' samples have been screened for mutations in the ABL1 tyrosine kinase domain (TKD). Nine out of ten BM samples collected at relapse showed wild type TKD. Accordingly, no subsequent relapses showed the appearance of an ABL1 mutated clone. Only one patient carried the tyrosine 243 to phenylalanine substitution (Y253F) at the first and second relapses, but not at diagnosis, demonstrating the expansion of a resistant minor clone at diagnosis, following TKI treatment.In addition, we monitored the BCR-ABL1 transcript level by RQ-PCR at diagnosis, relapse and multiple follow up points. In nine out of ten patients the expression of BCR-ABL1 was highly positive at the time of the relapse, and in five out of six cases the increase of the transcript level was predictive of a subsequent clinical relapse.Moreover, we investigated the cooperative copy number alterations by MLPA analysis in 6 matched diagnosis and relapse(s) samples. MLPA data showed a heterogeneous scenario of clonal evolution. In three patients, the relapse clone shared only a minor genetic identity with the predominant clone at diagnosis and wasn't the product of its direct evolution. In one case, the clone at relapse was identical to that at diagnosis, while in two cases the predominant clone at relapse was the product of the direct clonal evolution of the main clone at diagnosis. In 4 cases, the dynamics of clonal evolution were confirmed by the analyses of the IG/TCR rearrangements.In conclusion, our data depict a very different scenario of resistance in childhood Ph+ ALL compared to the adult cohort. Indeed, Imatinib resistance doesn't seem to be dependent on ABL1 mutations in pediatric Ph+ ALL. Moreover, the MLPA results contribute to the description of clonal heterogeneity in Ph+ ALL, and may partially explain the different response to treatment. DisclosuresNo relevant conflicts of interest to declare.

Modal affinities of endplate acetylcholine receptors caused by loop C mutations.

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes"). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5-β5' linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

Abstract PR06: Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice.

Abstract Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3'-splice site recognition, in 11% of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased frequency of Annexin V+ staining (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency in bone marrow and spleen of mice. U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+ Lineage - (KL) cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for KLS (c-Kit+ Lineage- Sca-1+) cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as an increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice at 5 days is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to controls (p≤0.05, n=6-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice. We identified 402 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) (FDR <10%, >2 |log2| fold change). We also observed a preference of the mutant U2AF1(S34F) to skip exons (n=72) and alternative splice sites (n=45) with a T in the -3 position relative to the AG splice acceptor site. We intersected our junction results with RNA sequencing from AML patient samples with U2AF1 mutations and human CD34+ cells expressing U2AF1(S34F), and we identified homologous dysregulated junctions in all 3 datasets that are currently being tested for their functional significance. Together, these results suggest that mutant U2AF1 expression contributes to the altered pre-mRNA splicing and hematopoiesis observed in patients with U2AF1 mutations. Citation Format: Cara Lunn Shirai, James N. Ley, Brian White, Justin Tibbitts, Jin Shao, Matthew Ndonwi, Sanghyun Kim, Theresa Okeyo-Owuor, Timothy A. Graubert, Matthew J. Walter. Mutant U2AF1 alters hematopoiesis and pre-mRNA splicing in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR06.

Heterologous expression of the Monilinia fructicola CYP51 (MfCYP51) gene in Pichia pastoris confirms the mode of action of the novel fungicide, SYP-Z048.

The novel agricultural fungicide 3-[5-(4-chlorophenyl)-2,3-dimethyl-3-isoxazolidinyl] pyridine (SYP-Z048) developed by China Shenyang Research Institute of Chemical Industry has been confirmed to be an ergosterol biosynthesis inhibitor (EBI). Previous studies have shown that EBIs target the proteins from a range of genes, including CYP51, ERG2 and/or ERG24, and ERG27, which are involved in the ergosterol biosynthesis pathway. In the current study the ERG2, ERG24, and ERG27 genes were cloned from wild type and resistant mutants of Monilinia fructicola in an attempt to clarify the target site of SYP-Z048. Comparative analysis of the deduced aa sequence of these genes, as well as CYP51, revealed several point mutations that resulted in amino acid variation among the sensitive and resistant isolates. However, sensitivity assays indicated that only one, the substitution of phenylalanine (F) for the tyrosine (Y) at 136 in CYP51, was correlated with reduced sensitivity to SYP-Z048. Heterologous expression of MfCYP51-136Y (MfCYP136Y) and MfCYP51-136F (MfCYP136F) in Pichia pastoris revealed that MfCYP136F significantly reduced sensitivity to SYP-Z048, increasing the average EC50 of the transformants 11-fold relative to those carrying MfCYP136Y. However, neither the additional copy of MfCYP136Y nor multiple copies of MfCYP136F were found to reduce sensitivity relative to the empty vector control or single copy transformants, respectively. Molecular docking experiments using SYP-Z048 with HsCYP145Y and the mutated version HsCYP145F as substitutes for MfCYP136Y and MfCYP136F, respectively, indicated that the reduced affinity of HsCYP145F for SYP-Z048 resulted from the loss of a hydrogen bond between the fungicide and the active site. Taken together these results indicate that MfCYP51 is the major target site of SYP-Z048 in M. fructicola, which has important implications for the resistance management of this fungicide in the field.

Glia maturation factor-γ phosphorylation at Tyr-104 regulates actin dynamics and contraction in human airway smooth muscle.

Actin dynamics plays an essential role in regulating airway smooth muscle contraction. The mechanisms that regulate actin dynamics in smooth muscle are not completely understood. Glia maturation factor (GMF) is a protein that has been reported to inhibit actin nucleation and to induce actin network debranching in vitro. The role of GMF in human smooth muscle cells and tissues has not been investigated. In this study, knockdown of GMF-γ by RNA interference enhanced actin polymerization and contraction in human airway smooth muscle (HASM) cells and tissues without affecting myosin phosphorylation (another important biochemical change during contractile activation). Activation of HASM cells and tissues with acetylcholine induced dissociation of GMF-γ from Arp2 of the Arp2/3 complex. Acetylcholine stimulation also increased GMF-γ phosphorylation at Tyr-104. GMF-γ phosphorylation at this residue was mediated by c-Abl tyrosine kinase. The GMF-γ mutant Y104F (phenylalanine substitution at Tyr-104) had higher association with Arp2 in HASM cells upon contractile activation. Furthermore, expression of mutant Y104F GMF-γ attenuated actin polymerization and contraction in smooth muscle. Thus, we propose a novel mechanism for the regulation of actin dynamics and smooth muscle contraction. In unstimulated smooth muscle, GMF-γ binds to the Arp2/3 complex, which induces actin disassembly and retains lower levels of F-actin. Upon contractile stimulation, phosphorylation at Tyr-104 mediated by c-Abl tyrosine kinase leads to the dissociation of GMF-γ from Arp2/3, by which GMF-γ no longer induces actin disassembly. Reduced actin disassembly renders F-actin in higher level, which facilitates smooth muscle contraction.

RB family tumor suppressor activity may not relate to active silencing of E2F target genes.

The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1-S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRB(N750F) activity in the presence and absence of p130. The pRB-LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RAS(V12)- and radiation-induced cell-cycle arrest. Remarkably, the pRB-LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein-perturbed cells to mitogen deprivation and cell-cell contact seems a better predictor of tumor development than the response to ectopic RAS(V12) expression. Cancer Res; 74(18); 5266-76. ©2014 AACR.

A Compare-and-Contrast NMR Dynamics Study of Two Related RRMs: U1A and SNF

The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/β sandwich topology, and these RRMs use their β-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the β-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 β-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.

The ShcA adaptor activates AKT signaling to potentiate breast tumor angiogenesis by stimulating VEGF mRNA translation in a 4E-BP-dependent manner.

The ShcA adaptor protein is engaged by numerous receptor tyrosine kinases (RTKs) in breast cancer cells. Once activated, RTKs phosphorylate three key tyrosine phosphorylation sites (Y239, Y240 and Y317) within ShcA that creates a docking site for Grb2/SOS and Grb2/Gab-containing complexes to activate the MAPK and AKT signaling pathways, respectively. We previously demonstrated that a tyrosine to phenylalanine substitution of the ShcA tyrosine phosphorylation sites (Shc3F-Y239/240/313F) significantly impairs breast tumor growth and angiogenesis in transgenic mouse models, in part, through the regulation of vascular endothelial growth factor (VEGF) production. Despite this fact, the underlying molecular mechanisms by which ShcA transduces pro-tumorigenic signals in breast cancer cells remain poorly defined. In this study, we demonstrate that ShcA-dependent activation of AKT, but not the RAS/MAPK pathway, induces VEGF production by bolstering VEGF mRNA translation. Accordingly, ShcA drives breast tumor growth and angiogenesis in vivo in a 4E-BP-dependent manner. These findings establish ShcA as a biological bridge that links AKT activation downstream of RTKs to cap-dependent VEGF mRNA translation in order to promote mammary tumorigenesis.

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.

Involvement of Ectodomain Leu 214 in ATP Binding and Channel Desensitization of the P2X4 Receptor

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.

Genetic variation in coding region of Aurora kinase A gene leads to cancer susceptibility

Aurora kinase A (AURKA) gene, located on the frequently amplified chromosome 20q13 locus in human cancers, belongs to an evolutionarily conserved gene family and encodes a critical mitosis regulatory oncoprotein that is over expressed in a wide variety of human tumors [1]. In this issue of Cancer Epidemiology, Xu et al. performed a systematic and comprehensive metaanalysis to probe and unravel the true effect of a putatively functional AURKA rs2273535 SNP in cancer etiology using data from fifty-two published case–control studies with a total number of 74,896 subjects in the last decade [2]. The functional single nucleotide polymorphism (SNP), rs2273535 (T91A) leads to substitution of the amino acid phenylalanine with isoleucine at codon 31; and this allele ‘‘A’’ variant of AURKA was earlier identified as a low penetrance cancer susceptibility allele for skin tumor in mice and for multiple cancer types in humans associated with increased aneuploidy in colon tumors and cell transformation in vitro [3]. At the cellular level, AURKA amplification leads to deregulation of mitosis including defects in centrosome maturation and spindle assembly and correlates with activation of oncogenic as well as inactivation of multiple tumor suppressor pathways involving c-Myc, TP53, TP73 and BRCA1 [4]. Although several association analyses have reported correlation of allele ‘‘A’’ with different types of malignancies; a number of studies also failed to detect association of rs2273535 with cancers [2]. Epidemiological studies addressing the predisposing candidate genetic variations for complex phenotypes such as cancers may have limitations of power reducing the likelihood of detecting a significant association under certain parameters, and explanatory capability due to the variance of phenotype [5]. Approaches such as meta-analyses are useful for addressing these issues as these analyses increase the power by pooling the data from small scale studies and help identify the source(s) of heterogeneity as well as potential biases. Despite the conflicting results of individual association studies, this meta-analysis by Xu and colleagues [2]

Amyloid β-Protein: The Influence of Intrinsic and Extrinsic Factors on Fibril Formation

Aggregation of the amyloid β-protein (Aβ) is believed to be involved in Alzheimer's disease pathogenesis. The central hydrophobic region (CHR) and the Aβ42/Aβ40 ratio play key roles in Aβ aggregation. Studying intrinsic (amino acid substitutions) and extrinsic (temperature, other molecules) factors contributes to understanding the mechanisms that cause Aβ monomers to aggregate and form oligomers and fibrils. This could facilitate the development of agents that therapeutically target toxic assemblies or prevent their formation.In our studies we mainly used a highly reproducible thioflavin T assay to probe the aggregation kinetics. Substitution of phenylalanine with leucine at position 19 in the CHR significantly affected the aggregation rate. This is seen as an extended half-time and steeper concentration dependence suggesting a change in aggregation mechanism relative to the wild type protein. We also studied co-aggregation and cross-seeding between the kinetically faster Aβ42 and the slower Aβ40. The aggregation process starting from mixed monomers displays two transitions and our data imply that there is cross-reactivity between Aβ40 and Aβ42 at the level of primary nucleation only, while fibril elongation and surface-catalysed secondary nucleation are highly specific events. In contrast, co-aggregation of Aβ42 with the slower mutant F19L only displays a single sigmoidal transition and the cross-seeding is as efficient as the self-seeding. The main reason for the discrimination in the different pathways is the length at the C-terminus rather than the difference in intrinsic aggregation rates.To further investigate the relative role of intermolecular interactions we changed the temperature to provide information on energy barriers and their enthalpic and entropic components. With an analytically solved model we could do a global fitting to estimate the activation energy for Aβ42 for the primary nucleation, secondary nucleation and elongation.

Distinct Orai-Coupling Domains in Stim1 and Stim2 Define the Orai-Activating Site

The ER membrane-spanning STIM1 protein is a finely-tuned sensor of ER luminal Ca2+. Small changes in ER Ca2+ induce STIM1 to undergo an intricate self-triggering process, causing it to translocate into ER-PM junctions where it couples with and activates the highly Ca2+-selective family of Orai channels in the PM. The entering Ca2+ sustains Ca2+ oscillations, maintains Ca2+ homeostasis, and provides crucial long-term Ca2+ signals in many cell types which control gene expression and cellular growth. Similar in structure and also widely expressed among cells, the little-studied STIM2 protein is reported to differ subtly from STIM1 in its N-terminal domain, affecting luminal Ca2+-sensitivity and the rate of unfolding and self-activation. The STIM1 cytoplasmic C-terminus contains the STIM-Orai activating region (SOAR) which has been structurally resolved. While the corresponding SOAR sequence in STIM2 is highly conserved, we reveal it has a profoundly diminished interaction with and ability to gate Orai1 channels. We narrowed this distinction in Orai1 activation to a small sequence in SOAR, within which substitution of a single phenylalanine in STIM1 with leucine in STIM2 confers a severe decrease in Orai1 channel-gating efficacy. This residue is strategically positioned at the structural apex of the SOAR domain. Modification of this single residue within the intact STIM1 protein reveals its pivotal role in both interaction with and gating of the Orai1 channel. The results not only pinpoint a crucial locus of STIM-Orai coupling but also reveal a physiologically profound distinction between STIM1 and STIM2.