Protein Elongation Research Articles

Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii.

Proper protein localization is essential for critical cellular processes, including vesicle-mediated transport and protein translocation. Tail-anchored (TA) proteins are integrated into organellar membranes via the C-terminus, orienting the N-terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C-terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early-branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow-up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.

Identification of a novel LRRK1 mutation in a family with osteosclerotic metaphyseal dysplasia.

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare skeletal dysplasia characterized by osteosclerotic metaphyses with osteopenic diaphyses of the long tubular bones. Our previous study identified a homozygous elongation mutation in leucine-rich repeat kinase 1 gene (LRRK1) in a patient with OSMD and showed that Lrrk1 knockout mice exhibited phenotypic similarity with OSMD. Here we report a second LRRK1 mutation in Indian sibs with OSMD. They had homozygous mutation (c.5971_5972insG) that produces an elongated mutant protein (p.A1991Gfs*31) similar to the first case. The sibs had normal stature, normal intelligence and recurrent fractures. The common radiographic feature was asymmetric and variable sclerosis of vertebral end plates, pelvic margin and metaphyses of tubular bones. One of the sibs had facial dysmorphisms, dentine abnormalities and acro-osteolysis. A comparison between the three OSMD cases with LRRK1 mutations with different ages suggested that the sclerotic lesions resolved with age. Our findings further support that LRRK1 would cause a subset of OSMD cases.

The β-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection.

The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β‐1,3‐glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate‐binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72 + GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3‐glucans have a higher degree of polymerization and are less branched than the wild‐type strain. The mutant showed significant differences in global patterns of gene expression, a hyper‐branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium‐mediated plant infection.

Validation of reference genes for RT-qPCR analysis of growth hormone receptor and growth hormone expression in the tench (Tinca tinca) fed substituting poultry meal for fish meal

The tench (Tinca tinca L., 1758) is a promising freshwater species in which numerous studies have been conducted recently to develop a dedicated feed for optimized growth rate. The aim of this study was to assess the stability of seven candidate reference genes, including 18S ribosomal RNA (18S rRNA), β-actin (actb), α-tubulin (tuba), ribosomal protein l8 (rpl8), 40S ribosomal protein s11 (rps11), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and elongation factor 1 alpha (ef1a), to select the best calibrator for nutrigenomic studies that would improve our understanding and illustrate the molecular basis of nutrient-related gene expression in response to feed mixes in the tench. An additional aim was to verify the hypothesis that the functioning of the somatotropic axis in the tench (growth hormone receptor, ghr, and growth hormone, gh) depends on dietary treatment. The analysis was based on five tissue samples (liver, spleen, gut, heart and muscle) collected from adult tench females fed with 0% and 100% replacement of fish meal (FM) with poultry by-product meal (PBM). The study demonstrated that the most stable genes in most tissues were rpl8 and rps11, with gapdh showing the greatest stability, along with rpl8, in the liver. Moreover, a comparison of relative transcript abundance between the tissues revealed that ghr demonstrated the highest expression in the liver, while gh demonstrated the highest expression in the spleen. Based on the selected reference genes, it was determined that the feed did not affect the level of expression of ghr and gh in most tissues except the spleen (ghr). The study also involved a phylogenetic analysis based on the amino acid sequences of Rpl8, Rps11 and Ghr, and it was confirmed that the tench is the most closely related with Asian cyprinids (i.e. Megalobrama amblycephala). The study identified suitable reference genes for RT-qPCR in the tench, and the effect of two feed variants on the somatotropic axis was determined. The study opens the way for further nutrigenomic studies aimed to develop a feed best matched for the needs of the tench and optimizing its growth. Statement of relevanceThe effect of feed intake on gene expression in fish species is of particular interest of aquaculture sector which seeks for alternative protein sources, but also new species to adapt for intensive culture.The study demonstrated that the most stable genes in most tissues were rpl8 and rps11, with gapdh showing the greatest stability, along with rpl8, in the liver.Comparison of relative transcript abundance between tissues revealed that ghr demonstrated the highest expression in the liver, while gh demonstrated the highest expression in the spleen.Presented work characterizes genes with stable expression across various tench tissue types and identifies suitable reference genes for RTq-PCR for nutrient-related gene expression studies.Presented study was performed for tench for the first time and provides information on influence of poultry by-product meal on the tench organism.Information on tench genetic studies, especially combined with dietary treatments are sparse, therefore presented results address this gap and will bring attention of Aquaculture readers.

A selective serotonin 5-HT1B receptor inhibition suppresses cells proliferation and induces apoptosis in human uterine leiomyoma cells

ObjectiveUterine leiomyomas (fibroids or myomas) are the most common pelvic tumor in women with about 70% lifetime incidence rate. Currently, the only definitive treatment is surgery, causing undesirable side effects and negative impact on women’s quality of life, reproductive ability, and a substantial impact on healthcare costs. Therefore, curative medical treatments are needed to be developed. In this study, we investigated the impact of serotonin receptor 5-HT1B on cell proliferation and survival in human uterine leiomyoma cells (huLM). Study designThe impact of 5-HT1B receptor on cell proliferation, survival and apoptosis was investigated using a selective 5-HTR1B antagonist SB216641 in huLM cells, utilizing MTS, colony formation assay and Annexin V staining, respectively. Mechanisms of inhibition of cell proliferation, survival and induction of apoptosis were investigated by Western blot analysis after treatment with various doses of HT1B antagonist. Results5-HT1B receptor inhibition leads to a significant decrease in proliferation and colony formation in huLM cells, reduction of cyclin D1 and alpha-smooth muscle actin (α-SMA) expressions and the activity of Mitogen Activated Protein Kinase (MAPK) ERK and Elongation Factor 2 kinase (EF2K) pathways. 5-HT1B receptor blockage also induces apoptotic cell death by inducing cleavage of caspase-8, -9, and -3 and PARP. ConclusionOur findings show for the first time that 5-HT1B receptor promotes uterine leiomyoma cell survival and proliferation and its inhibition may be a potential therapeutic approach for human uterine leiomyomas. Thus, 5-HT1B expression and antagonists should be further investigated in leiomyoma tumors.

Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies.

The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.

Content of oenothein B in clonally propagated Chamaenerion angustifolium (L.) Scop. genotypes

Caenorhabditis elegans-Pseudomonas aeruginosa infection model is commonly used for pathogenesis studies over the decades. In the present study, upon exposure to the Pseudomonas aeruginosa PAO1, the 2D-PAGE was performed to examine the total proteins differences of C. elegans during the PAO1 infection at different time durations (12–48 h). Also, the 2D-DIGE using the cyanine dyes were performed (48 h) to identify the differentially regulated proteins against the PAO1 infection. Among the 19 short-listed proteins, 5 proteins were down-regulated and 14 proteins were up-regulated. Eukaryotic elongation factor-2 (EEF-2), a GTP binding protein involves in protein elongation process was down regulated during the pathogen infection. The 2D-PAGE analysis and MS data for the 12 and 24 h infections identified the NDK-1 and other essential protein includes, ACS-18, ACT-1, GPD-3, GDH-1 and LBP-6 which are involved in important cellular homeostasis were down regulated. Validation studies using qPCR analysis for eef-2 and other selected genes, western blot analysis for EEF-2 and effect of host translational inhibition studies using Cycloheximide during PAO1 infection suggests that P. aeruginosa systematically restrains the function of host by arresting the expression of EEF-2 and thereby inhibiting protein translational events. Further, in silico analysis revealed the Exotoxin A could directly bind with the host EEF-2 and NDK-1 during the C. elegans- PAO1 interactions.Model system, C. elegans facilitates the identification of virulence mechanisms during bacterial pathogenesis. Upon infection by the fungal and bacterial pathogens, the C. elegans system induces an array of transcriptional responses, including differential expression of effector/modulator genes that provide safeguard and fight against infection. However, the in-depth knowledge of host response by the pathogen at protein level remains unclear. Much of the studies were carried out only at the transcripts level and scarce reports are available at the protein level for the host-pathogen interaction studies. In order to provide few interesting clues at the protein level, the nematode, C. elegans was infected with the human pathogen P. aeruginosa and the response(s) of host was investigated at the protein level by 2D-DIGE analysis and further validation studies using qPCR and western blotting techniques. Our differential proteomics data suggest that translational inhibition as one of the patterns of pathogenesis in C. elegans during P. aeruginosa infection. Since many of the effectors identified through C. elegans are conserved in other systems including human, our data pave the way for understanding important regulatory pathways involved during bacterial pathogenesis that can be translated into higher eukaryotic organisms.

Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus.

Elongator protein 3 (Elp3) is the enzymatic unit of the elongator protein complex, a histone acetyltransferase complex involved in transcriptional elongation. It has long been shown to play an important role in cell migration; however, the underlying mechanism is unknown. Here, we showed that Elp3 is expressed in pre-migratory and migrating neural crest cells in Xenopus embryos, and knockdown of Elp3 inhibited neural crest cell migration. Interestingly, Elp3 binds Snail1 through its zinc-finger domain and inhibits its ubiquitination by β-Trcp without interfering with the Snail1/Trcp interaction. We showed evidence that Elp3-mediated stabilization of Snail1 was likely involved in the activation of N-cadherin in neural crest cells to regulate their migratory ability. Our findings provide a new mechanism for the function of Elp3 in cell migration through stabilizing Snail1, a master regulator of cell motility.

Proteome analysis reveals translational inhibition of Caenorhabditis elegans enhances susceptibility to Pseudomonas aeruginosa PAO1 pathogenesis

Caenorhabditis elegans-Pseudomonas aeruginosa infection model is commonly used for pathogenesis studies over the decades. In the present study, upon exposure to the Pseudomonas aeruginosa PAO1, the 2D-PAGE was performed to examine the total proteins differences of C. elegans during the PAO1 infection at different time durations (12–48h). Also, the 2D-DIGE using the cyanine dyes were performed (48h) to identify the differentially regulated proteins against the PAO1 infection. Among the 19 short-listed proteins, 5 proteins were down-regulated and 14 proteins were up-regulated. Eukaryotic elongation factor-2 (EEF-2), a GTP binding protein involves in protein elongation process was down regulated during the pathogen infection. The 2D-PAGE analysis and MS data for the 12 and 24h infections identified the NDK-1 and other essential protein includes, ACS-18, ACT-1, GPD-3, GDH-1 and LBP-6 which are involved in important cellular homeostasis were down regulated. Validation studies using qPCR analysis for eef-2 and other selected genes, western blot analysis for EEF-2 and effect of host translational inhibition studies using Cycloheximide during PAO1 infection suggests that P. aeruginosa systematically restrains the function of host by arresting the expression of EEF-2 and thereby inhibiting protein translational events. Further, in silico analysis revealed the Exotoxin A could directly bind with the host EEF-2 and NDK-1 during the C. elegans- PAO1 interactions. Biological significanceModel system, C. elegans facilitates the identification of virulence mechanisms during bacterial pathogenesis. Upon infection by the fungal and bacterial pathogens, the C. elegans system induces an array of transcriptional responses, including differential expression of effector/modulator genes that provide safeguard and fight against infection. However, the in-depth knowledge of host response by the pathogen at protein level remains unclear. Much of the studies were carried out only at the transcripts level and scarce reports are available at the protein level for the host-pathogen interaction studies. In order to provide few interesting clues at the protein level, the nematode, C. elegans was infected with the human pathogen P. aeruginosa and the response(s) of host was investigated at the protein level by 2D-DIGE analysis and further validation studies using qPCR and western blotting techniques. Our differential proteomics data suggest that translational inhibition as one of the patterns of pathogenesis in C. elegans during P. aeruginosa infection. Since many of the effectors identified through C. elegans are conserved in other systems including human, our data pave the way for understanding important regulatory pathways involved during bacterial pathogenesis that can be translated into higher eukaryotic organisms.

Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis

Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHopTPR2AB) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved.

What an Intron May Tell: Several Sexual Biospecies Coexist in Meriderma spp. (Myxomycetes)

Specimens of the snowbank myxomycete Meriderma atrosporum agg. from five European mountain ranges were sequenced for parts of the nuclear small subunit ribosomal RNA gene (SSU) and the protein elongation factor 1 alpha gene (EF1A). A phylogeny of the EF1A gene, including a very variable spliceosomal intron, resulted in seven phylogroups, and this topology was confirmed by SSU sequences. Two thirds of all specimens were heterozygous for the EF1A gene, and the two haplotypes of these specimens occurred always in the same phylogroup. Except for two cases in closely related phylogroups all ribotypes were as well limited to one phylogroup. This pattern is consistent with the assumption of reproductively isolated sexual biospecies. Numbers of EF1A-haplotypes shared between mountain ranges correlate with geographical distance, suggesting relative isolation but occasional long-distance dispersal by spores. Most subpopulations (divided by putative biospecies and mountain ranges) were in Hardy-Weinberg equilibrium. A simulation assuming panmixis within but not in between subpopulations suggested that similar numbers of shared genotypes can be created by chance through sexual reproduction alone. Our results support the biospecies concept, derived from experiments with cultivable members of the Physarales. We discuss the results on the background of possible reproductive options in myxomycetes.

Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association.

Throughout the four phases of protein biosynthesis-initiation, elongation, termination, and recycling-the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAi (Met) in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

Proteomic Profiling of Bifidobacterium bifidum S17 Cultivated Under In Vitro Conditions

Bifidobacteria are frequently used in probiotic food and dairy products. Bifidobacterium bifidum S17 is a promising probiotic candidate strain that displays strong adhesion to intestinal epithelial cells and elicits potent anti-inflammatory capacity both in vitro and in murine models of colitis. The recently sequenced genome of B. bifidum S17 has a size of about 2.2 Mb and encodes 1,782 predicted protein-coding genes. In the present study, a comprehensive proteomic profiling was carried out to identify and characterize proteins expressed by B. bifidum S17. A total of 1148 proteins entries were identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), representing 64.4% of the predicted proteome. 719 proteins could be assigned to functional categories according to cluster of orthologous groups of proteins (COGs). The COG distribution of the detected proteins highly correlates with that of the complete predicted proteome suggesting a good coverage and representation of the genomic content of B. bifidum S17 by the proteome. COGs that were highly present in the proteome of B. bifidum S17 were Translation, Amino Acid Transport and Metabolism, and Carbohydrate Transport and Metabolism. Complete sets of enzymes for both the bifidus shunt and the Embden-Meyerh of pathway were identified. Further bioinformatic analysis yielded 28 proteins with a predicted extracellular localization including 14 proteins with an LPxTG-motif for cell wall anchoring and two proteins (elongation factor Tu and enolase) with a potential moonlighting function in adhesion. Amongst the predicted extracellular proteins were five of six pilin proteins encoded in the B. bifidum S17 genome as well as several other proteins with a potential role in interaction with host structures. The presented results are the first compilation of a proteomic reference profile for a B. bifidum strain and will facilitate analysis of the molecular mechanisms of physiology, host-interactions and beneficial effects of a potential probiotic strain.

Brain-derived neurotrophic factor promotes vesicular glutamate transporter 3 expression and neurite outgrowth of dorsal root ganglion neurons through the activation of the transcription factors Etv4 and Etv5.

Brain-derived neurotrophic factor (BDNF) is critical for sensory neuron survival and is necessary for vesicular glutamate transporter 3 (VGLUT3) expression. Whether the transcription factors Etv4 and Etv5 are involved in these BDNF-induced effects remains unclear. In the present study, primary cultured dorsal root ganglion (DRG) neurons were used to test the link between BDNF and transcription factors Etv4 and Etv5 on VGLUT3 expression and neurite outgrowth. BDNF promoted the mRNA and protein expression of Etv4 and Etv5 in DRG neurons. These effects were blocked by extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor PD98059 but not phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or phospholipase C-γ (PLC-γ) inhibitor U73122. Etv4 siRNA and Etv5 siRNA effectively blocked the VGLUT3 expression and neurite elongation induced by BNDF. The overexpression of Etv4 or Etv5 potentiated the effects of BNDF-induced neurite elongation and growth-associated protein 43 (GAP-43), medium neurofilament (NF-M), and light neurofilament (NF-L) expression while these effects could be inhibited by Etv4 and Etv5 siRNA. These data imply that Etv4 and Etv5 are essential transcription factors in modulating BDNF/TrkB signaling-mediated VGLUT3 expression and neurite outgrowth. BDNF, through the ERK1/2 signaling pathway, activates Etv4 and Etv5 to initiate GAP-43 expression, promote neurofilament (NF) protein expression, induce neurite outgrowth, and mediate VGLUT3 expression for neuronal function improvement. The biological effects initiated by BDNF/TrkB signaling linked to E26 transformation-specific (ETS) transcription factors are important to elucidate neuronal differentiation, axonal regeneration, and repair in various pathological states.

Hydrophobic Interactions Elicit Cooperative Response in Dystrophin

The primary role of dystrophin, a 427 kDa elongated protein, is to stabilize the membrane of muscle cells against the mechanical forces deriving from muscle contraction and relaxation; its absence or mutation lead to various forms of muscular dystrophy. Dystrophin binds to actin through its N-terminus actin binding domain (ABD1), and connects to the cell membrane through its C-terminus; in between, 24 spectrin repeats (SR) with interspersed hinges provide structural flexibility and have been proposed to actively dissipate the mechanical stress. Yet, dystrophin's mechanism of function is largely unknown, preventing effective drug development. Our working hypothesis is that dystrophin's domains are allosterically coupled through specific hydrophobic interactions, and that the response to mechanical stress is propagated across domains through order-to-disorder transitions at their interface. We test this hypothesis with all-atom molecular dynamics simulations of dystrophin's SR 1 monomer, SR 17-18 dimer, and the ABD1 domain. Our results support the hypothesis by showing that (a) forced unfolding by pulling initiates at the interface between SR repeats; (b) the linker between SR 17-18 is flexible and allows for distinct conformations at the interface between domains, characterized by different hydrophobic interaction patterns; (c) mutual information analyses show that conformational changes at the dimer's interface and termini are coupled; and (d) hydrophobic interactions between the two CH domains and the connecting linker stabilize a closed conformation of ABD1 that is alternative to the open crystal structure and explains experimental fluorescence results. Our results suggest that the disease may originate from altered hydrophobic interactions, and pave the way for targeted drug development.

Codon influence on protein expression in E. coli correlates with mRNA levels.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

A requirement for cell elongation protein RodZ and cell division proteins FtsN and DedD to maintain the small rod morphology of Escherichia coli at growth temperatures near 8°C.

As similarly observed in nutrient-poor media at 37°C, Escherichia coli forms small rods in nutrient-rich media at temperatures near 8°C, the minimum temperature of growth. A study was initiated to identify proteins required to facilitate the small rod morphology at low temperature. E. coli contains three nonessential SPOR domain proteins (DamX, RlpA, and DedD) that have been demonstrated to bind to the septal ring. In contrast to the normal growth and small rod morphology of damX and rlpA null mutants at 10°C, the dedD null mutant exhibited reduced growth and formed filamentous cells. The presence of plasmid-encoded DedD restored growth and small rods. Plasmid-encoded FtsN, an essential SPOR domain protein that functions to stabilize the septal ring and to initiate septation, in the dedD null mutant resulted in increased growth and the formation of shorter chained cells. However, plasmid-encoded DedD failed to restore growth and cell division of cells lacking FtsN at 10°C. In contrast to cell division protein DedD, RodZ is a cell elongation protein particularly required for growth at 30°C. However, the rodZ null mutant grew similarly as the wild type strain and produced cocci in LB broth at 10°C. Moreover at 10°C, the concerted deletion of dedD and rodZ resulted in severe inhibition of growth accompanied with the formation of swollen prolate ellipsoids due to a block in septal ring assembly and cell elongation. The data indicate the cellular requirement of both FtsN and DedD for septation as well as RodZ for cell elongation to maintain the small rod morphology at temperatures near 8°C. In comparison to the growth and small rods of the wild type in M9-glucose minimal media at 37°C, the dedD null mutant grew at the same rate and produced elongated cells while the rodZ null mutant grew at a slightly slower rate and produced cocci. The data indicate that DedD and RodZ are also required to maintain the small rod morphology in nutrient-poor media, but there is a higher cellular requirement of DedD for growth and cell division in nutrient-rich media at low temperature.

Comparative mRNA Expression of eEF1A Isoforms and a PI3K/Akt/mTOR Pathway in a Cellular Model of Parkinson's Disease.

The PI3K/Akt/mTOR pathway is one of dysregulated pathways in Parkinson's disease (PD). Previous studies in nonneuronal cells showed that Akt regulation can be increased by eukaryotic protein elongation factor 1 alpha 2 (eEF1A2). eEF1A2 is proposed to contribute protection against apoptotic death, likely through activation of the PI3K/Akt pathway. Whether eEF1A2 plays a role in the prevention of cell death in PD has not been investigated. Recently, gene profiling on dopaminergic neurons from postmortem PD patients showed both upregulation and downregulation of some PI3K and mTOR genes. In this paper, the expression of all gene members of the PI3K/Akt/mTOR pathway in relation to those of the eEF1A isoforms in a cellular model of PD was investigated at the mRNA level. The results showed a similar trend of upregulation of genes of the eEF1A isoforms (eEF1A1 and eEF1A2) and of the PI3K (classes I–III)/Akt (Akt1, Akt2, and Akt3)/mTOR (mTORC1 and mTORC2) pathway in both nondifferentiated and differentiated SH-SY5Y dopaminergic cells treated with 1-methyl-4-phenylpyridinium (MPP+). Upregulation of eEF1A2, Akt1, and mTORC1 was consistent with the relative increase of eEF1A2, Akt, phospho-Akt, and mTORC1 proteins. The possible role of eEF1A isoforms in the regulation of the PI3K/Akt/mTOR pathway in PD is discussed.

Characterization of inner membrane protein YciB in Escherichia coli: YciB interacts with cell elongation and division proteins.

The function of inner membrane protein YciB in Escherichia coli has not been identified. In this study, the membrane topology of the protein that contains five transmembrane domains was clarified. YciB was found to interact with various proteins involved in cell elongation and cell division using a bacterial two-hybrid system. It was also found that the deletion mutant of yciB is susceptible to the low osmolarity. These observations together with previous reports indicate that YciB is involved in synthesis of the cell envelope by interacting with cell elongation and cell division complexes.

Noncanonical roles for Tropomyosin during myogenesis

For skeletal muscle to produce movement, individual myofibers must form stable contacts with tendon cells and then assemble sarcomeres. The myofiber precursor is the nascent myotube, and during myogenesis the myotube completes guided elongation to reach its target tendons. Unlike the well-studied events of myogenesis, such as myoblast specification and myoblast fusion, the molecules that regulate myotube elongation are largely unknown. In Drosophila, hoi polloi (hoip) encodes a highly conserved RNA-binding protein and hoip mutant embryos are largely paralytic due to defects in myotube elongation and sarcomeric protein expression. We used the hoip mutant background as a platform to identify novel regulators of myogenesis, and uncovered surprising developmental functions for the sarcomeric protein Tropomyosin 2 (Tm2). We have identified Hoip-responsive sequences in the coding region of the Tm2 mRNA that are essential for Tm2 protein expression in developing myotubes. Tm2 overexpression rescued the hoip myogenic phenotype by promoting F-actin assembly at the myotube leading edge, by restoring the expression of additional sarcomeric RNAs, and by promoting myoblast fusion. Embryos that lack Tm2 also showed reduced sarcomeric protein expression, and embryos that expressed a gain-of-function Tm2 allele showed both fusion and elongation defects. Tropomyosin therefore dictates fundamental steps of myogenesis prior to regulating contraction in the sarcomere.