Conserved Consensus Motif Research Articles

Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells.

Phosphatase and tensin homolog (PTEN) mutation is common in prostate cancer during progression to metastatic and castration resistant forms. We previously reported that loss of PTEN function in prostate cancer leads to increased expression and secretion of the Prorenin Receptor (PRR) and its soluble processed form, the soluble Prorenin Receptor (sPRR). PRR is an essential factor required for proper assembly and activity of the vacuolar-ATPase (V-ATPase). The V-ATPase is a rotary proton pump required for the acidification of intracellular vesicles including endosomes and lysosomes. Acidic vesicles are involved in a wide range of cancer related pathways such as receptor mediated endocytosis, autophagy, and cell signalling. Full-length PRR is cleaved at a conserved consensus motif (R-X-X-R↓) by a member of the proprotein convertase family to generate sPRR, and a smaller C-terminal fragment, designated M8.9. It is unclear which convertase processes PRR in prostate cancer cells and how processing affects V-ATPase activity. In the current study we show that PRR is predominantly cleaved by PACE4, a proprotein convertase that has been previously implicated in prostate cancer. We further demonstrate that PTEN controls PRR processing in mouse tissue and controls PACE4 expression in prostate cancer cells. Furthermore, we demonstrate that PACE4 cleavage of PRR is needed for efficient V-ATPase activity and prostate cancer cell growth. Overall, our data highlight the importance of PACE4-mediated PRR processing in normal physiology and prostate cancer tumorigenesis.

Isolation of a Peptide That Binds to Pseudomonas aeruginosa Lytic Bacteriophage.

Antimicrobial resistance is a global health threat that is exacerbated by the overuse and misuse of antibiotics in medicine and agriculture. As an alternative to conventional antimicrobial drugs, phage therapy involves the treatment of infected patients with a bacteriophage that naturally destroys bacterial pathogens. With the re-emergence of phage therapy, novel tools are needed to study phages. In this work we set out to screen and isolate peptide candidates that bind to phages and act as affinity tags. Such peptides functionalized with an imaging agent could serves as versatile tools for tracking and imaging of phages. Specifically, we screened a phage display library for peptides that bind to the Good Vibes phage (GV), which lyses the bacterial pathogen Pseudomonas aeruginosa. Isolated monoclonal library phages featured a highly conserved consensus motif, LPPIXRX. The corresponding peptide WDLPPIGRLSGN was synthesized with a GGGSK linker and conjugated to cyanine 5 or biotin. The specific binding of the LPPIXRX motif to GV in vitro was confirmed using an enzyme-linked immunosorbent assay. We demonstrated imaging and tracking of GV in bacterial populations using the fluorescent targeting peptide and flow cytometry. In conclusion, we developed fluorescent labeled peptides that can bind to bacteriophage GV specifically, which may enable real-time analysis of phage in vivo and monitor the efficacy of phage therapy.

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA.

A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-β-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52°C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

Identification and functional annotation of m6A methylation modification in granulosa cells during antral follicle development in pigs

The N6-methyladenosine (m6A) derivative has the capacity for ubiquitous epigenetic modification of messenger RNA (mRNA) that regulates gene expression through post-transcriptional mRNA modifications. Findings with mapping of m6A methylomes have indicated there are potential functions of this derivative in different cell types of several species. A profile of m6A methylomes and potential functions in granulosa cells of pigs during antral follicle development, however, has not yet occurred. In the present study, there was profiling of an epitranscriptome-wide map of m6A methylation in granulosa cells of pigs derived from small and large follicles using methylated RNA immunoprecipitation techniques, next-generation sequencing and further annotation of the potential functions of m6A utilizing bioinformatic analyses procedures. The m6A modification is abundant in granulosa cells of pigs, and there are dynamic changes in m6A methylomes during the developmental transition from small (< 3 mm) to large (> 5 mm) sized follicles. In particular, there was a prevalence of 7289 and 6882 m6A in granulosa cells from follicles of two different sizes. There was an increased prevalence of m6A in close proximity to the 5′ or 3′-untranslated coding regions and a shared conserved consensus motif. Results from further analysis indicated there was significant enrichment of differentially expressed m6A methylated genes in several signaling pathways associated with steroidogenesis, granulosa cell proliferation and follicular development. When considered as a whole, these results indicate there are differential m6A modifications in granulosa cells of pigs during follicle development that are potentially associated with steroidogenesis and folliculogenesis.

Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity

Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/Mn2+ transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn2+ sensitivity.Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn2+ sensitivity.

Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport.

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.

Characterization of Four Novel Caspases from Litopenaeus vannamei (Lvcaspase2-5) and Their Role in WSSV Infection through dsRNA-Mediated Gene Silencing

Apoptosis plays an important role in white spot syndrome virus (WSSV) pathogenesis, and caspases are central players in apoptosis. Here, we cloned four novel caspases (Lvcaspase2-5) from the Pacific white shrimp Litopenaeus vannamei, and investigated their potential roles in WSSV replication using dsRNA-mediated gene silencing. Lvcaspase2-5 have the typical domain structure of caspase family proteins, with the conserved consensus motifs p20 and p10. Lvcaspase2 and Lvcaspase5 were highly expressed in muscle, while Lvcaspase3 was highly expressed in hemocytes and Lvcaspase4 was mainly expressed in intestine. Lvcaspase2-5 could also be upregulated by WSSV infection, and they showed different patterns in various tissues. When overexpressed in Drosophila S2 cells, Lvcaspase2-5 showed different cellular localizations. Using dsRNA-medicated gene silencing, the expression of Lvcaspase2, Lvcaspase3, and Lvcaspase5 were effectively knocked down. In Lvcaspase2-, Lvcaspase3- or Lvcaspase5-silenced L. vannamei, expression of WSSV VP28 gene was significantly enhanced, suggesting protective roles for Lvcaspase2, Lvcaspase3 and Lvcaspase5 in the host defense against WSSV infection.

Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

BackgroundDNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding.ResultsUsing genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation.ConclusionsOur analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

Induction of Apoptosis in CLL by Peptides Binding the B-Cell Antigen Receptor in Vitro

IGHV mutated (M-CLL) and unmutated (U-CLL) CLL arise from clonal expansions of antigen-experienced B lymphocytes. As the specific IGHV genes coding the clone's B-cell antigen receptor (BCR) and the selection for particular genetic and amino acid structures shared by different patients are not random, a role for antigenic selection in biasing BCR structure is likely. However, the binding specificities of the BCR and the nature of the selecting antigens are yet to be identified. In order to characterize actual or mimetic ligands to BCRs in CLL patients, peptide phage display technologies were used. BCRs from 3 M-CLL and 2 U-CLL patients were expressed as IgG1 mAbs in human embryonic fibroblasts and used to probe a 12-mer peptide phage display library. For all the tested mAbs, ligands were isolated after 3 rounds of selection. M-CLL mAbs bound a large number of related, non-redundant phages carrying peptide inserts with conserved consensus motifs, whereas U-CLL mAbs reacted with multiple phages expressing identical 12-mer sequences for which no consensus or a consensus of shorter length and less definition than with M-CLL mAbs was found. Repeat probings with some of the same M-CLL or U-CLL mAb yielded identical and/or similar peptide-expressing phages. Ligands binding on the phage level were synthesized with a biotin-tag at the C-terminus and tested by direct ELISA for reactivity with the selecting mAbs as well as mAbs from different IGHV subgroups. In some instances, when synthesized as peptides, binding to the mAbs changed. For example, U-CLL peptides exhibited “polyreactitvity”, reacting with other U-CLL and M-CLL mAbs, although they still bound better to their selecting mAbs. For M-CLL, one “borderline” mutated mAb (2% difference from germline) had a peptide which bound other M-CLL and U-CLL mAbs. Two peptides derived from phages specific for another M-CLL mAb didn't react with any antibody tested. For a third M-CLL mAb (CLL 169, IGHV3-33), two peptides of considerable specificity were identified that did not bind any other mAb tested. These peptides were also synthesized in MAP configuration, with a biotin tag, in order to mimic multivalent antigens; their reactivity in ELISA was comparable to the highly specific binding of the linear peptide. Tetramers were produced using streptavidin for the CLL169 peptide having the best ELISA binding as well as its MAP peptide counterpart. Binding was examined by FACS to the original CLL cells from which the mAb was derived, as well as to cells from another case with the same stereotypic IGHV, and to cells from a distinct IGHV subgroup. These studies indicate that binding to the cells depends not only on the VH, but also on the D and J segments as well as on the configuration of the peptide (linear, MAP, tetramer linear or tetramer MAP). Binding to the cells above background could be detected for the tetramer MAP peptide but not the linear peptide. The original cells bound best to the tetramer MAP peptide. The CLL cells with the same VH subgroup exhibited similar binding. Cells from a distinct VH sharing the same D and J segments as the original cells also showed some binding to the tetramer MAP peptide. Effects on CLL cell fate after in vitro incubation with the peptides were examined using 7AAD/Annexin V. Adding the tetramer MAP peptide to the CLL cells with the BCR from which they were derived led to 63% increase in cell death after 24 hours over that seen for control cultures. Thus, ligands specific for CLL BCRs can be identified by phage display technologies. For one peptide which displayed high specificity for the selecting mAb in ELISA, binding to CLL cells as well as increased cell death in vitro have been demonstrated. In future, this may lead to the identification of BCR-targeted therapeutic agents.

Repression of motility during fimbrial expression: identification of 14 mrpJ gene paralogues in Proteus mirabilis

Proteus mirabilis alternates between motile and adherent forms. MrpJ, a transcriptional regulator previously reported to repress motility, is encoded at the 3' end of the mrp fimbrial operon in P. mirabilis. Sequencing of the P. mirabilis genome revealed 14 additional paralogues of mrpJ, 10 of which are associated with fimbrial operons. Twelve of these genes, when overexpressed, repressed motility; several distinct patterns of swarming motility were noted. Expression of 10 of the 14 mrpJ paralogues repressed flagellin (FlaA) synthesis. Alignment of the predicted amino acid sequences of MrpJ and its 14 paralogues revealed a conserved consensus motif (SQQQFSRYE) within the helix-turn-helix domain. Site-directed mutagenesis of these residues coupled with linker insertion mutagenesis of MrpJ confirmed the importance of this domain for repression of motility. Gel shift assays demonstrated that MrpJ and another paralogue UcaJ bind directly to the promoter region of the flagellar master regulator flhDC. Thus, P. mirabilis appears to use a related mechanism to inhibit motility during the production of at least 10 of its predicted fimbriae.

Foxp2 Inhibits Nkx2.1-Mediated Transcription of SP-C via Interactions with the Nkx2.1 Homeodomain

The transcription factor (TF) Foxp2 has been shown to partially repress surfactant protein C (SP-C) transcription, presumably through interaction of an independent repressor domain with a conserved Foxp2 consensus site in the SP-C promoter. We explored the role of interactions between Foxp2 and the homeodomain TF Nkx2.1 that may contribute to the marked reduction in SP-C expression accompanying phenotypic transition of alveolar epithelial type II (AT2) to type I (AT1) cells. Foxp2 dose-dependently inhibited Nkx2.1-mediated activation of SP-C in MLE-15 cells. While electrophoretic mobility shift assays and chromatin immunoprecipitations revealed an interaction between Foxp2 and the conserved consensus motif in the SP-C promoter, Nkx2.1-mediated activation of the 318-bp proximal SP-C promoter (which lacks a Foxp2 consensus) was attenuated by increasing amounts of Foxp2. Co-immunoprecipitation and mammalian two-hybrid assays confirmed a physical interaction between Nkx2.1 and Foxp2 mediated through the Nkx2.1 homeodomain. Formation of an Nkx2.1 complex with an SP-C oligonucleotide was inhibited dose-dependently by recombinant Foxp2. These findings demonstrate that direct interaction between Foxp2 and Nkx2.1 inhibits Nkx2.1 DNA-binding and transcriptional activity and suggest a mechanism for down-regulation of SP-C (and probably other AT2 cell genes) during transition of AT2 cells to an AT1 cell phenotype.

BCRs of Mutated and Unmutated CLL Bind Peptides with Distinct Sequence Features: Evidence from Phage Display Libraries.

In CLL, the use of specific IgV genes to code the clone's BCR is non-random and there is an apparent selection for particular genetic and amino acid structures that can be shared by different patients, supporting the hypothesis that antigenic stimulation influences the development and course of CLL. As the binding specificities of the BCR are largely unknown, a vast variety of antigens may affect the BCRs and defined antigens have yet to be identified. Therefore, we used peptide phage display technology to identify ligands for CLL BCRs. BCRs from 2 IgVH unmutated (U-CLL) and 3 mutated (M-CLL) patients were expressed as IgG1 mAbs and used to probe a 12-mer peptide phage display library. In each case, after 3 rounds of selection, we isolated ligands reactive with the CLL mAbs. For the 3 M-CLL mAbs, phage clones carrying peptide inserts with conserved consensus motifs were found. Specificity of the BCR-ligand interactions was demonstrated in direct and indirect ELISA, since selected phage clones and synthetic peptides bound to their respective M-CLL mAb but not to other M-CLL mAbs. Variation of the amino acid sequence of the synthetic peptides significantly altered their reactivity with the corresponding M-CLL mAb. Furthermore, synthetic peptides were bound only by the proper mAb/BCR, but not by mAbs of other M-CLL or U-CLL patients with BCRs comprised of different IgVH genes, supporting the hypothesis that BCRs of M-CLL recognize a defined epitope. In contrast, the mAbs from 2 U-CLL cases did not select phages bearing a consensus motif. Rather these U-CLL mAbs bound multiple phages expressing the same 12-mer peptides, although these differed in sequence between the two U-CLL cases tested. Furthermore, 2 separate selection procedures using 1 U-CLL mAb isolated multiple phage bearing the same 12-mer sequence on each occasion as well as another set of phages with a completely distinct sequence in 1 of 2 selections. ELISAs demonstrated specific binding of all phage clones and of the synthetic peptides by the U-CLL mAbs. Despite this level of specificity, the 2 U-CLL mAbs also reacted with peptides isolated from panning with other CLL mAbs, thereby displaying considerable polyreactivity. Rather than binding only one distinct epitope, mAbs from U-CLL appear capable of interacting with multiple, unrelated structures. Finally, one of the peptides isolated with an U-CLL mAb was bound by all of the CLL mAbs tested, including those from M-CLL cases; therefore this target is antigenically “polyreactive”. Thus, phage display is a feasible approach to identify specific ligands for CLL BCRs. The two classes of BCRs in M-CLL and U-CLL show substantially different binding properties - the former binding shared amino acid motifs and the latter binding multiple ligands of distinct and identical 12-mer amino acid sequences. These peptides can be used to analyze more precisely the binding sites of CLL BCRs as well as the consequences that ensue after BCR crosslinking, and they might help develop BCR-specific therapeutic agents.

Identification of a Hypothetical Protein of Plant Pathogenic Xanthomonas campestris as a Novel β-Galactosidase

Xc17L, a lactose-utilizing mutant of Xanthomonas campestris pv. campestris previously isolated by mutagenesis with nitrous acid, displays a level of β-galactosidase 3.5-fold higher than that in the parental Xc17. In this study, the gene encoding the enzyme displaying a higher specific activity in Xc17L was inactivated by mini-Tn5 transposition.Sequencing revealed that the product (579 aa, 63.5 kDa) of this gene, designated galD, was previously annotated to encode a hypothetical protein on the genome. Mutation of the gene by marker exchange, complementation test and Western blot analysis together confirmed that galD is indeed the gene involved in β-galactosidase elevation in Xc17L. With only the N-terminal region possessing similarity to the known β-galactosidases and partially conserved consensus motif, GalD is recognized as a member of the glycosyl hydrolase family 35. Insertion with GmΩ, which causes polar effects, into the upstream genes followed by Western blotting showed that galD is cotranscribed with the upstream genes and expressed constitutively. Mutation in galD causes no significant changes including pathogenicity in the bacterium.

Alternative Oxidase (AOX) Genes of African Trypanosomes: Phylogeny and Evolution of AOX and Plastid Terminal Oxidase Families

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.

Characterization of a Drosophilamelanogaster orthologue of muskelin

Muskelin was identified in vertebrates as a novel, intracellular, kelch repeat protein that is needed in cell-spreading responses to the matrix adhesion molecule, thrombospondin-1. The identification and characterization of an orthologue of muskelin in Drosophila melanogaster is now reported. The Drosophila muskelin gene, located on chromosome 2R, is encoded in ten exons. Drosophila muskelin is expressed in embryos, larvae and adult flies. The protein has 45% sequence identity to vertebrate muskelins, with highest sequence identity in an amino-terminal domain and the six kelch repeats that form a β-propeller structure. Multiple sequence alignment of human, mouse, rat and Drosophila muskelins and protein database searches revealed a novel highly conserved motif within the amino-terminal domain, lissencephaly homology motif (LisH) and C-terminal to LisH motifs in the central region of the molecule, and several conserved consensus motifs for phosphorylation by protein kinase C and casein kinase II. These findings provide new information on the modular structure of muskelin and indicate potential for conserved mechanisms of function.

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins.

This study describes the development of 3PO, a nonviral, protein-based gene delivery vector which utilizes the highly evolved cell-binding, cell-entry and intracellular transport functions of the adenovirus serotype 5 (Ad5) capsid penton protein. A penton fusion protein containing a polylysine sequence was produced by recombinant methods and tested for gene delivery capability. As the protein itself is known to bind integrins through a conserved consensus motif, the penton inherently possesses the ability to bind and enter cells through receptor-mediated internalization. The ability to lyse the cellular endosome encapsulating internalized receptors is also attributed to the penton. The recombinant protein gains the additional function of DNA binding and transport with the appendage of a polylysine motif. This protein retains the ability to form pentamers and mediates delivery of a reporter gene to cultured cells. Interference by oligopeptides bearing the integrin binding motif suggests that delivery is mediated specifically through integrin receptor binding and internalization. The addition of protamine to penton-DNA complexes allows gene delivery in the presence of serum.

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

The Arabidopsis thaliana K+ channel AKT1 was expressed in a yeast strain defective for K+ uptake at low K+ concentrations (&lt;3 mM). Besides restoring K+ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K+ concentration used (50 μM, 5 mM, or 50 mM). We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so‐called P domain) of animal K+ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4. Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na+ content, decreased K+ content, and lower growth rate, as compared to control cells expressing the wild‐type channel) were selected. By co‐expressing them with the wild‐type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pore), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N‐terminal flanking region.

Analysis of the Site Occupancy Constraints of Primary Amino Acid Sequences in the Motif Directing Palmitylation of the Vaccinia Virus 37-kDa Envelope Protein

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A(Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1–12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines;A,aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)Amotif is required for efficient palmitylation of p37.