Amino Acid Sequence Motifs Research Articles

Identification of common and cell type specific LXXLL motif EcR cofactors using a bioinformatics refined candidate RNAi screen in Drosophila melanogastercell lines

BackgroundDuring Drosophila development, titers of the steroid ecdysone trigger and maintain temporal and tissue specific biological transitions. Decades of evidence reveal that the ecdysone response is both unique to specific tissues and distinct among developmental timepoints. To achieve this diversity in response, the several isoforms of the Ecdysone Receptor, which transduce the hormone signal to the genome level, are believed to interact with tissue specific cofactors. To date, little is known about the identity of these cofactor interactions; therefore, we conducted a bioinformatics informed, RNAi luciferase reporter screen against a subset of putative candidate cofactors identified through an in silico proteome screen. Candidates were chosen based on criteria obtained from bioinformatic consensus of known nuclear receptor cofactors and homologs, including amino acid sequence motif content and context.ResultsThe bioinformatics pre-screen of the Drosophila melanogaster proteome was successful in identifying an enriched putative candidate gene cohort. Over 80% of the genes tested yielded a positive hit in our reporter screen. We have identified both cell type specific and common cofactors which appear to be necessary for proper ecdysone induced gene regulation. We have determined that certain cofactors act as co-repressors to reduce target gene expression, while others act as co-activators to increase target gene expression. Interestingly, we find that a few of the cofactors shared among cell types have a reversible roles to function as co-repressors in certain cell types while in other cell types they serve as co-activators. Lastly, these proteins are highly conserved, with higher order organism homologs also harboring the LXXLL steroid receptor interaction domains, suggesting a highly conserved mode of steroid cell target specificity.ConclusionsIn conclusion, we submit these cofactors as novel components of the ecdysone signaling pathway in order to further elucidate the dynamics of steroid specificity.

Complete genome sequence of the first endornavirus from the ascocarp of the ectomycorrhizal fungus Tuber aestivum Vittad.

The genus Endornavirus was recently approved by the International Committee on Taxonomy of Viruses (ICTV) [1, 10] to be separated from the family Partitiviridae, and endornaviruses have only been described in plants and fungi [4, 15]. They share common properties such as symptom-free infection of their host, occurrence in lowcopy number in host cells, lack of true virions [7], and an efficient vertical transmission in their hosts [16]. Endornaviruses have unique plasmid-like properties that differ markedly from those of other conventionally encapsidated viruses [5, 12, 16]. The genomes of endornaviruses consist of linear double-stranded RNA (dsRNA) with a characteristic, single open reading frame (ORF) of up to 18 kb in length, often preceded by a site-specific nick at the 5’ end [5, 7, 8, 12, 18]. The predicted protein from this ORF harbours in its N-terminal part characteristic amino acid (aa) sequence motifs for viral RNA helicases (VHel) or viral methyltransferases (VMet), while aa sequence motifs for UDP-glycosyltransferases (UGT) and RNA-dependent RNA polymerases (RdRp) are located at close proximity to the 30 end [7]. The remaining polyprotein is unrelated to any known functional proteins in the database. Currently, the INSDC databases contain complete sequences of seven endornaviruses. Three are plant-associated viruses infecting cultivated and wild rice (Oryza sativa endornavirus [OsEV] and Oryza rufipogon endornavirus [OrEV]) [5, 11], respectively), as well as broad bean (Vicia faba endornavirus [VFV]) [13]. In addition, four fungal isolates are known. One isolate each was reported from the mycelia of the violet root rot fungus (Helicobasidium mompa endornavirus 1-670 [HmEV1-670]) [12] and from an informally designated plant-pathogenic oomycete Phytophtora spp. (Phytophtora endornavirus 1 [PEV1]) [6], while two isolates originate from the pathogenic ascomycete Gremmeniella abietina (Gremmeniella abietina type B RNA virus) [15]. Here, we report the complete nucleotide sequence of an isolate of a putatively novel species in the genus Endornavirus from an ectomycorrhizal fungus, isolated from the hypogeous ascomata of a black truffle (Tuber aestivum Vittad.). Accordingly, we propose the name Tuber aestivum endornavirus (TaEV). The TaEV genome structure is highly similar to that of other Ascomycetes-infecting endornaviruses, such as Gremmeniella abietina type B RNA virus [15], containing characteristic motifs for the DEAD-like helicase (DEXDc), VMet and RdRp.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Turning the RING Domain Protein MdmX into an Active Ubiquitin-Protein Ligase*

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.

Chlamydia trachomatisSerovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

Lipoic acid is an essential protein bound cofactor that is vital for the functioning of several important enzymes involved in central metabolism. Genomes of all sequenced chlamydiae show the presence of two genes encoding lipoic acid ligases and one gene encoding a lipoate synthase. However, the roles of these proteins in lipoic acid utilization or biosynthesis have not yet been characterized. The two distinct lipoic acid ligases in Chlamydia trachomatis serovar L2, LplA1(Ct) and LplA2(Ct) (encoded by the open reading frames ctl0537 and ctl0761) display moderate identity with Escherichia coli LplA (30 and 27%, respectively) but possess amino acid sequence motifs that are well conserved among all lipoyl protein ligases. The putative lipoic acid synthase LipA(Ct), encoded by ctl0815, is ca. 43% identical to the E. coli LipA homolog. We demonstrate here the presence of lipoylated proteins in C. trachomatis serovar L2 and show that the lipoic acid ligase LplA1(Ct) is capable of utilizing exogenous lipoic acid for the lipoylation Therefore, host-derived lipoic acid may be important for intracellular growth and development. Based on genetic complementation in a surrogate host, our study also suggests that the C. trachomatis serovar L2 LipA homolog may not be functional in vivo.

Interaction prediction and classification of PDZ domains

BackgroundPDZ domain is a well-conserved, structural protein domain found in hundreds of signaling proteins that are otherwise unrelated. PDZ domains can bind to the C-terminal peptides of different proteins and act as glue, clustering different protein complexes together, targeting specific proteins and routing these proteins in signaling pathways. These domains are classified into classes I, II and III, depending on their binding partners and the nature of bonds formed. Binding specificities of PDZ domains are very crucial in order to understand the complexity of signaling pathways. It is still an open question how these domains recognize and bind their partners.ResultsThe focus of the current study is two folds: 1) predicting to which peptides a PDZ domain will bind and 2) classification of PDZ domains, as Class I, II or I-II, given the primary sequences of the PDZ domains. Trigram and bigram amino acid frequencies are used as features in machine learning methods. Using 85 PDZ domains and 181 peptides, our model reaches high prediction accuracy (91.4%) for binary interaction prediction which outperforms previously investigated similar methods. Also, we can predict classes of PDZ domains with an accuracy of 90.7%. We propose three critical amino acid sequence motifs that could have important roles on specificity pattern of PDZ domains.ConclusionsOur model on PDZ interaction dataset shows that our approach produces encouraging results. The method can be further used as a virtual screening technique to reduce the search space for putative candidate target proteins and drug-like molecules of PDZ domains.

Infectivity of Asia1 type foot-and-mouth disease virus was increased via an alternative RDD motif

The amino acid sequence motif Arg-Gly-Asp (RGD), located in the surface-exposed βG-βH loop of the 1D protein of different serotypes and subtypes of foot-and-mouth disease virus (FMDV), is highly conserved and participates in binding of FMDV to susceptible cells. Previous sequence analyses of the 1D-encoding region of a FMDV serotype Asia1 field isolates from China indicated the presence of an alternative RDD motif instead of the conserved βG-βH loop RGD. The role of the RGD and RDD sequences in virus infection was investigated by recovering viruses with the βG-βH loop RGD (named rRGD-Asia1) and a RDD tripeptide (named rRDD-Asia1), respectively, using a genome-length infectious chimeric cDNA clone. The results show that the infectivity of the rRDD-Asia1 virus for baby hamster kidney cells (BHK-21) and baby hamsters was higher as 10 times. These results also demonstrate that field isolates with RGD and an alternative RDD receptor recognition site have infectivity, and RDD motif can increase the infectivity of the viruses. The study lays a foundation for further study of the existence of other receptor molecules and alternative mechanisms for FMDV entry into cells.

The rheumatoid arthritis shared epitope is a potent helper T cell-polarizing ligand (98.8)

Abstract Recent evidence strongly implicates IL17-producing helper T (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) and other autoimmune conditions. Susceptibility to RA and disease severity have long been shown to associate with a HLA-DRB1-encoded 5 amino acid sequence motif, called the shared epitope (SE), however, the mechanistic basis of SE-RA association remains unknown. We have previously sown that the SE acts as an allele-specific ligand that triggers innate immune signaling in other cells. In order to gain better insights into the impact of the SE on immune regulation, we have studied its effect on helper T cell polarization in vitro. Here we show that when added to the CD8+ subset of dendritic cells (DCs), the SE ligand inhibits the enzymatic activity of indoleamine 2,3 dioxygenase (IDO), a key tolerogenic enzyme. When SE-activated DCs were co-cultured with naïve CD4+ T cells, they enhanced the differentiation to Th17 cells, and inhibited generation of Foxp3+ T regulatory cells (Tregs). The SE effect could be seen with peptidic SE ligands, but even more potently, when the SE was presented in its natural tri-dimensional conformation as part of HLA-DR tetrameric proteins. Our data demonstrate a previously unknown immune regulatory effect of the SE ligand that may provide important clues about its functional role in RA.

Geographical spread of highly pathogenic avian influenza virus H5N1 during the 2006 outbreak in Austria.

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Austria in 119 dead wild birds. The hemagglutinin cleavage site showed that the amino acid sequence motif was identical to that of the Qinghai lineage. For detailed analysis, the hemagglutinin (HA) and neuraminidase (NA) genes of 27 selected Austrian H5N1 viruses originating from different regions and wild bird species were analyzed phylogenetically, which revealed two clearly separated Austrian subclusters, both belonging to European cluster EMA-1. Subcluster South (SCS) contains virus isolates from the south of Austria as well as from Slovenia, Turkey, Egypt, and Nigeria. The second subcluster, Northwest (SCN), covered a larger group of viruses originating from different locations and wild bird species in the northern and very western parts of Austria, as well as from Bavaria and Switzerland. Surprisingly, virus isolates originating from two mute swans and one wild duck found on the north side of the Alps did not cluster with SCN but with SCS. Together with isolates from Bavarian, the Czech Republic, Italy, and Slovakia, they form a genuine subgroup, named subgroup Bavaria (SGB). This subgroup forms a link to SCN, indicating a spread of the virus from south to north. There has been a general assumption that the generic HPAI introduction route into Europe was from Russia to north Germany, introducing cluster EMA-2 into Europe. Interestingly, our findings support the assumption of an alternative introduction of the HPAI H5N1 virus from Turkey to central Europe, where it spread as cluster EMA-1 during the outbreak of 2006.

Identification of the Amino Acid Sequence Motif for Conventional PKC-Mediated Regulation of NKp46 Surface Expression

A number of surface receptors expressed on NK cells are related to the regulation of NK cell activity and characterized by either inhibitory or activating properties. Natural cytotoxicity receptors (NCR) are one major family of activating receptors involved in NK cytotoxicity. The three family members of NCR are NKp46, NKp44, and NKp30. The surface density of these receptors might vary with the activation state of NK cells, and their density may directly correlate with their natural cytotoxicity. In this study, we investigated the regulation of NKp46 expression and determined the amino acid sequence motif for protein kinase C (PKC)-mediated regulation of its surface expression. We produced stable cell lines expressing full-length NKp46 and investigated the change in expression after PMA or IL-2 treatment using flow cytometry, RT-PCR, and immunoblotting methods. Expression of NKp46 on Jurkat T-cell transfectants appeared to be induced by PMA treatment until 8-h post-treatment and then gradually decreased afterwards to levels that were less than those measured at pretreatment. Parallel to surface expression of NKp46, total NKp46 protein expression also appeared to fluctuate after PMA treatment, but the expression of mRNA transcripts was not significantly affected. Experiments with mutant NKp46-expressing stable cell lines demonstrated that Ser288 might be critical for the surface expression of NKp46 and the PKC-mediated regulation of NKp46 expression. However, NKp46 surface expression was not influenced by IL-2 in stable cell lines expressing wild-type and mutant NKp46.

Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways?

Intracellular malaria parasites export numerous proteins into their host cell, a process essential for parasite survival and virulence. Many of these proteins are defined by a short amino acid sequence motif termed PEXEL or VTS that mediates their export, suggesting a collective trafficking route. The existence of several PEXEL-negative exported proteins (PNEPs) indicates that alternative export pathways might also exist. We review recent data on the sequences mediating export of PNEPs and compare this process to PEXEL export taking into account novel findings on the function of this motif. Based on this we propose that, despite the lack of a PEXEL in PNEPs, both groups of proteins might converge in a single export pathway on their way into the host cell.

Computational methods to identify novel methyltransferases

Background 1.2% of the yeast genes are estimated to encode enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to protein, nucleic acid, lipid, and small molecule substrates [1]. These enzymes function in biosynthesis, regulating metabolic pathways, and controlling gene expression, including writing the histone code. BLAST and MEME/MAST analysis using the amino acid sequence of motifs have previously generated a list of putative Class I methyltransferases [2]. Recently we have used a combination of a new search algorithm and structural information to refine this analysis [3]. This study utilizes these updated methods of identifying motifs and scanning the proteome to predict new members of the different families of methyltransferases in different organisms. These new members may function in novel pathways or new modes of regulation.

Characteristics and function ofAlcaligenessp. NBRC 14130 esterase catalysing the stereo-selective hydrolysis of ethyl chrysanthemate

Alcaligenes sp. NBRC 14130 was found as a strain hydrolysing a mixture of (+/-)-trans- and (+/-)-cis ethyl chrysanthemates to (1R,3R)-(+)-trans-chrysanthemic acid. The Alcaligenes cells also have hydrolytic activity for 6-aminohexanoate-cyclic dimer (6-AHCD, 1,8-diazacyclotetradecane-2,9-dione). The correlation of function on the enzyme from the Alcaligenes strain with hydrolysis activities for both ethyl chrysanthemate and 6-AHCD was demonstrated. The esterase was purified to homogeneity. The purified esterase hydrolysed 20 mmol l(-1) ester including the four stereoisomers to the corresponding (+)-trans acid with a 37% molar conversion of ethyl (+)-trans chrysanthemate. The esterase showed high hydrolytic activity for various short-chain fatty acid esters, n-hexane amide and 6-AHCD. The amino acid sequence of the Alcaligenes esterase was identical to that of Arthrobacter 6-AHCD hydrolase (EC 3.5.2.12) and similar to that of fatty acid amide hydrolase (EC 3.5.1.4) from Rattus norvegicus, having both serine and lysine residues of the catalytic site and the consensus motif Gly-X-Ser-X-Gly. The stereo-selective hydrolytic activity was found in Alcaligenes sp. NBRC14130 by screening of ethyl chrysanthemate-hydrolysing activity in micro-organisms, and the purified esterase also acted on fatty acid esters and amides. This study has demonstrated that there are great differences in the enzymatic properties, amino acid sequence and catalytic motif of esterases in both Alcaligenes and Arthrobacter globiformis with excellent stereo-selectivity for (+)-trans-ethyl chrysanthemate, but the amino acid sequence of Alcaligenes esterase is identical to that of Arthrobacter 6-AHCD hydrolase.

Comparison of Embiopteran Silks Reveals Tensile and Structural Similarities across Taxa

Embioptera is a little studied order of widely distributed, but rarely seen, insects. Members of this group, also called embiids or webspinners, all heavily rely on silken tunnels in which they live and reproduce. However, embiids vary in their substrate preferences and these differences may result in divergent silk mechanical properties. Here, we present diameter measurements, tensile tests, and protein secondary structural analyses of silks spun by several embiid species. Despite their diverse habitats and phylogenetic relationships, these species have remarkably similar silk diameters and ultimate stress values. Yet, ultimate strain, Young's modulus, and toughness vary considerably. To better understand these tensile properties, Fourier transformed infrared spectroscopy was used to quantify secondary structural components. Compared to other arthropod silks, embiid silks are shown to have consistent secondary structures, suggesting that commonality of amino acid sequence motifs and small differences in structural composition can lead to significant changes in tensile properties.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Analysis of Foot-and-Mouth Disease Virus Integrin Receptor Expression in Tissues from Naïve and Infected Cattle

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals principally affecting cattle, pigs and sheep. FMD virus (FMDV) uses the alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(6), and alpha(V)beta(8) integrins as receptors in vitro via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. Immunofluorescence and confocal microscopy were used to study the expression of two major FMDV receptors, alpha(V)beta(3) and alpha(V)beta(6), within epithelial tissues from FMDV-infected and uninfected cattle in order to understand the role of these receptors in tissue tropism. Integrin alpha(V)beta(6) was expressed by epithelial cells in tissues that are important sites for FMDV replication (i.e. tongue and coronary band). Integrin alpha(V)beta(3) was detected in epithelium of all tissues examined except tongue. In addition, alpha(V)beta(3) expression was associated with blood vessels in all tissues examined. In infected tissues, alpha(V)beta(6) integrin was distributed on the surface of those epithelial cells also expressing FMDV antigen. Although integrin alpha(V)beta(3) has been shown to be a receptor for FMDV, no expression of alpha(V)beta(3) was associated with FMDV-positive keratinocytes in the tongue. In contrast, podal epithelial cells containing FMDV antigen also expressed alpha(V)beta(3) integrin. Thus, at the cellular level the expression of these two integrins correlates with susceptibility to infection and may contribute substantially to viral tropism in FMD pathogenesis.

Is Runx a linchpin for developmental signaling in metazoans?

The Runt domain (Runx) is a 128 amino acid sequence motif that defines a metazoan family of sequence-specific DNA binding proteins, which appears to have originated in concert with the intercellular signaling systems that coordinate multicellular development in animals. In the model organisms where they have been studied (fruit fly, mouse, sea urchin, and nematode) Runx genes are essential for normal development, and in humans they are causally associated with a variety of cancers, manifesting both oncogenic and tumor suppressive attributes. During development Runx proteins support both cell proliferation and differentiation, and function in both transcriptional activation and repression. Runx function is thus context-dependent, with the context provided genetically by cis-regulatory sequence architecture and epigenetically by development. This context dependency makes it difficult to formulate reductionistic generalizations concerning Runx function in normal and carcinogenic development. However, a growing body of literature links Runx function to each of the major intercellular signaling systems in animals, suggesting that the general function of Runx transcription factors may be to potentiate and govern genomic responsiveness to developmental signaling.

Peptide Models of Membrane Protein Folding

Given the central roles of membrane proteins in cellular processes ranging from nutrient uptake to cell-cell communication, as well as the importance of these proteins as drug targets, efforts to understand and control their structures are vital in human health and disease. The rational design of membrane proteins with modified properties is thus a highly desirable goal in molecular medicine and biotechnology. However, experimental data showing how individual transmembrane (TM) residues and/or segments direct the packing and folding of membrane proteins into biologically functional entities remain sparse. To address these questions in a systematic manner, helix-helix interactions between two (or more) TM segments must be identified and analyzed. Here we present an overview of the utilization of peptides as models of the TM segments of alpha-helical membrane proteins in uncovering the amino acid sequence motifs and interactions that build these molecules. TM peptide design and production strategies are discussed, and specific examples of the application of TM peptides to the study of membrane proteins are presented. We demonstrate that TM peptides can be routinely produced in sufficient quantities for biophysical analysis, are amenable to a variety of experimental techniques, and can effectively replicate the native helix-helix contacts and key aspects of the natural biological structures of membrane proteins.

Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti

At the end of each developmental stage, the yellow fever mosquito Aedes aegypti performs the ecdysis behavioral sequence, a precisely timed series of behaviors that culminates in shedding of the old exoskeleton. Here we describe ecdysis triggering hormone-immunoreactive Inka cells located at branch points of major tracheal trunks and loss of staining coincident with ecdysis. Peptides (AeaETH1, AeaETH2) purified from extracts of pharate 4th instar larvae have—PRXamide C-terminal amino acid sequence motifs similar to ETHs previously identified in moths and flies. Injection of synthetic AeaETHs induced premature ecdysis behavior in pharate larvae, pupae and adults. Two functionally distinct subtypes of ETH receptors (AeaETHR-A, AeaETHR-B) of A. aegypti are identified and show high sensitivity and selectivity to ETHs. Increased ETHR transcript levels and behavioral sensitivity to AeaETHs arising in the hours preceding the 4th instar larva-to-pupa ecdysis are correlated with rising ecdysteroid levels, suggesting steroid regulation of receptor gene expression. Our description of natural and ETH-induced ecdysis in A. aegypti should facilitate future approaches directed toward hormone-based interference strategies for control of mosquitoes as human disease vectors.

Identification of the amino acid sequence motif of α-synuclein responsible for macrophage activation

α-Synuclein (Syn) is implicated in the pathogenesis of PD and related neurodegenerative disorders. Recent studies have also shown that α-synuclein can activate microglia and enhance dopaminergic neurodegeneration. The mechanisms of microglia activation by α-synuclein, however, are not well understood. In this study, we found that not only α-synuclein but also β- and γ-synucleins activated macrophages (RAW 264.7) in vitro. Macrophages treated with synuclein proteins secreted TNF-α in a dose-dependent manner. Synuclein family proteins also increased mRNA transcription of COX-2 and iNOS. Two α-synuclein deletion mutants, SynΔNAC and Syn61–140, activated macrophages, while deletion mutants Syn1–60 and Syn96–140 did not significantly activate them. Finally, we demonstrated that macrophage activation by α-synuclein was accompanied by phosphorylation of ERK. These results suggest that synuclein family proteins can activate macrophages, and that macrophage activation needs both the N-terminal and C-terminal domains of α-synuclein, but not the central NAC region.