Carboxy-terminal Extension Research Articles

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Atypical Evolution of the F1Fo Adenosine Triphosphate Synthase Regulatory ATP6 subunit in Glacier Ice Worms (Annelida: Clitellata: Mesenchytraeus).

The glacier ice worm, Mesenchytraeus solifugus, is among a few animals that reside permanently in glacier ice. Their adaptation to cold temperature has been linked to relatively high intracellular adenosine triphosphate (ATP) levels, which compensate for reductions in molecular motion at low physiological temperatures. Here, we show that ATP6—the critical regulatory subunit of the F1Fo-ATP synthase and primary target of mitochondrial disease—acquired an unprecedented histidine-rich, 18-amino acid carboxy-terminal extension, which counters the strong evolutionary trend of mitochondrial genome compaction. Furthermore, sequence analysis suggests that this insertion is not of metazoan origin, but rather is a product of horizontal gene transfer from a microbial dietary source, and may act as a proton shuttle to accelerate the rate of ATP synthesis.

Backbone 1H, 13C and 15N resonance assignments of the OB domain of the single stranded DNA-binding protein hSSB2 (NABP1/OBFC2A) and chemical shift mapping of the DNA-binding interface.

Single stranded DNA-binding proteins (SSBs) are essential for the maintenance of genome integrity and are required in in all known cellular organisms. Over the last 10 years, the role of two new human SSBs, hSSB1 (NABP2/OBFC2B) and hSSB2 (NABP1/OBFC2A), has been described and characterised in various important DNA repair processes. Both these proteins are made up of a conserved oligonucleotide-binding (OB) fold that is responsible for ssDNA recognition as well a unique flexible carboxy-terminal extension involved in protein-protein interactions. Due to their similar domain organisation, hSSB1 and hSSB2 have been found to display some overlapping functions. However, several studies have also revealed cell- and tissue-specific roles for these two proteins, most likely due to small but significant differences in the protein sequence of the OB domains. While the molecular details of ssDNA binding by hSSB1 has been studied extensively, comparatively little is known about hSSB2. In this study, we use NMR solution-state backbone resonance assignments of the OB domain of hSSB2 to map the ssDNA interaction interface. Our data reveal that ssDNA binding by hSSB2 is driven by four key aromatic residues in analogy to hSSB1, however, some significant differences in the chemical shift perturbations are observed, reflecting differences in ssDNA recognition. Future studies will aim at determining the structural basis of these differences and thus help to gain a more comprehensive understanding of the functional divergences that these novel hSSBs display in the context of genome maintenance.

The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch

Heat shock protein 27 (HSP27), functioning as a stress induced protective protein, has been reported to participate in various biological processes, including apoptosis, thermal protection, and virus infection. In this study, a HSP27-like gene from the seawater fish sea perch, designated as LjHSP27, was characterized. The 1361 bp full-length cDNA of LjHSP27 encoded a 221 amino acid protein containing a conserved α-crystallin domain, two variable amino- and carboxy-terminal extensions, a WD/EPF motif, two serine phosphorylation sites, and two putative actin binding regions. Phylogenetic analysis showed that LjHSP27 shared the closest genetic relationship with HSP27 of the Asian seabass Lates calcarifer. LjHSP27 mRNA was ubiquitously expressed in all tissues examined, but significantly up-regulated in spleen and kidney and down-regulated in brain post red spotted grouper nervous necrosis virus (RGNNV) infection. In vitro, LjHSP27 transcript was remarkably reduced post RGNNV infection, but rapidly increased after polyinosinic-polycytidylic acid treatment. Up-regulation and down-regulation of LjHSP27 inhibited and promoted RGNNV replication in cultured LJB cells, respectively. Luciferase assay indicated that LjHSP27 could enhance the promoter activities of zebrafish interferon (IFN)1 and IFN3, suggesting its potential role in innate immune responses. Moreover, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, as indicated by the up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes, while KNK437 caused down-regulation of LjHSP27 dramatically led to opposite results, suggesting that LjHSP27 might exert its anti-RGNNV activities by regulating the apoptosis signaling pathway. Our results would provide a new insight into the underlying molecular mechanism of HSP and RGNNV interaction.

Glutamine metabolism ofCorynebacterium glutamicum: role of the glutaminase GlsK

Corynebacterium glutamicum is able to metabolize different nitrogen and carbon sources. In standard minimal media, ammonium and urea typically serve as nitrogen source and glucose or sucrose as carbon and energy source; however, amino acids might also play a role as nitrogen, carbon and energy source. In this study, the function of the putative glutaminase GlsK was investigated. A glsK deletion strain showed impaired growth with L-glutamine as carbon and energy source, while growth was improved upon glsK overexpression. GlsK possesses a carboxy-terminal domain that seems to be restricted to Corynebacterium species. A truncated GlsK lacking this extension led to faster growth, indicating a regulatory function of this domain. In fact, GlsK activity is regulated in a pH-dependent manner depending on the carboxy-terminal extension, and is positively influenced by cAMP. Furthermore, the C-terminal extension seems to be important for oligomerization of GlsK.

Role of the yeast ribosomal protein L16 in ribosome biogenesis.

Most ribosomal proteins play essential roles in ribosome synthesis and function. In this study, we have analysed the contribution of yeast ribosomal protein L16 to ribosome biogenesis. We show that in vivo depletion of the essential L16 protein results in a deficit in 60S subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability and rapid turnover of early and intermediate pre-60S particles, as evidenced by the reduced steady-state levels of 27SBS and 7SL /S pre-rRNA, and the low amounts of de novo synthesized 27S pre-rRNA and 25S rRNA. Additionally, depletion of L16 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we show that L16 assembles in the nucleolus and binds to early 90S preribosomal particles. Many evolutionarily conserved ribosomal proteins possess extra eukaryote-specific amino- or carboxy-terminal extensions and/or internal loops. Here, we have also investigated the role of the eukaryote-specific carboxy-terminal extension of L16. Progressive truncation of this extension recapitulates, albeit to a lesser extent, the growth and ribosome biogenesis defects of the L16 depletion. We conclude that L16 assembly is a prerequisite to properly stabilize rRNA structures within early pre-60S particles, thereby favouring efficient 27S pre-rRNA processing within the internal transcribed spacer 1 at sites A3 and B1 . Upon depletion of L16, the lack of this stabilization aborts early pre-60S particle assembly and subjects these intermediates to turnover.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.

The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1′ protein and the secreted NS1′ can be used for diagnostic biomarker for virulent virus infection

Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1′ protein and to investigate the function of NS1′ protein during virus infection. NS1′ is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1′. The NS1′ protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1′ protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1′-expressing virus (rA66G) could induce a higher humoral immune response than the NS1′-non-expressing virus (rSA14-14-2). NS1′ protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1′ can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1′ was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1′, most of the virulent JEV strains produce the NS1′ protein. And the secreted NS1′ may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the −1 programmed ribosomal frameshift to produce the NS1′ protein and demonstrated the secreted NS1′ could be used for diagnostic biomarker during JEV infection.

Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

Truncated Human ATP-Specific Succinyl-CoA Synthetase

Two isoforms of the heterodimeric enzyme succinyl-CoA synthetase (SCS) exist in the mitochondria of humans. One is specific for ATP, while the other is specific for GTP. Both catalyze the reversible reaction: succinate + CoA + NTP ⇌ succinyl-CoA + NDP + Pi, where N denotes adenosine or guanosine. SCS is best known as an enzyme of the citric acid cycle where the reaction generates NTP. In the reverse direction, SCS replenishes succinyl-CoA required for the catabolism of ketone bodies and for heme synthesis. Nucleotide-specific forms are thought to be required for SCS to serve its different metabolic roles. The nucleotide specificity lies in the β-subunit [1], and the β-subunit of human ATP-specific SCS has been shown to interact with the C-terminus of erythroid-specific aminolevulinic acid synthase (ALAS2) [2]. ALAS2 catalyzes the committed step in heme synthesis: succinyl-CoA + Gly ⇌ 5-aminolevulinate + CoA + CO2. An interaction between SCS and ALAS2 makes biological sense, since this could provide channeling of succinyl-CoA from SCS to ALAS2. We hypothesize that the interaction is with the carboxy-terminus of the β-subunit of ATP-specific SCS because sequence comparisons show that the β-subunit of ATP-specific SCS has a carboxy-terminal extension when compared to other SCSs' β-subunits. To test this hypothesis, we added a carboxy-terminal His8-tag to the α-subunit of human ATP-specific SCS and mutated the codon for Thr 396β to a stop codon. This truncated version of human ATP-specific SCS has been produced in E. coli and purified. As well as testing to see if truncated human ATP-specific SCS interacts with ALAS2, we are using the truncated version in crystallization trials. Crystals of full-length human ATP-specific SCS diffract to only 3.2 Å and our goal is to obtain better-diffracting crystals of the complex of ATP with truncated human ATP-specific SCS.

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling.

Discrimination between two rice metallothionein isoforms belonging to type 1 and type 4 in metal‐binding ability

Metallothioneins (MTs) are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. Plants have several MT isoforms, which are classified into four types based on the arrangement of Cys residues. In this study, two rice (Oryza sativa) MT isoforms, OsMTI-1b and OsMTII-1a from type 1 and type 4, respectively, were heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). Transformed cells expressing GST-OsMTI-1b showed increased tolerance to Ni(2+) , Cd(2+) , and Zn(2+) and accumulated more metal ions compared with cells expressing GST alone. However, heterologous expression of GST-OsMTII-1a had no significant effects on metal tolerance or ion accumulation. The UV absorption spectra and competitive reactions of in vitro Cd-incubated proteins with 5-5'-dithiobis(2-nitrobenzoic) acid revealed that GST-OsMTI-1b, but not GST-OsMTII-1a, is able to form Cd-thiolate clusters. Furthermore, heterologous expression of both GST-OsMTI-1b and GST-OsMTII-1a conferred H2 O2 tolerance to E. coli cells. Taken together, the results presented here show that two different rice MT isoforms belonging to type 1 and type 4 differ in Ni(2+) , Cd(2+) , and Zn(2+) binding abilities, but they may have overlapping function in protection of cells against oxidative stress.

Molecular cloning, expression analysis, and potential food intake attenuation effect of peptide YY in grass carp (Ctenopharyngodon idellus)

The peptide YY (PYY) is a 36 amino acid peptide involved in the food intake control in vertebrates. We have cloned and characterized a PYY gene from grass carp Ctenopharyngodon idellus. The full-length cDNA encodes a precursor protein of grass carp PYY (gcPYY) that consists of a putative 28-amino acid signal peptide, a 36-amino acid mature peptide, an amidation-proteolytic site, and a 30-amino acid carboxy-terminal extension. The gcPYY gene is comprised of 4 exons interspaced by 3 introns as seen in PYYs from other species. Amino acid alignment and gene structure comparison indicate that the structure of PYY is well preserved throughout vertebrate phylogeny. The tissue distribution and postprandial changes in gcPYY mRNA expression were evaluated by real-time PCR, which showed that the gcPYY is expressed abundantly in the central nervous system, with significantly increased expression following a single meal. During embryogenesis, the presence of gcPYY mRNA was detected in early developing embryos, and high expression levels were observed when most larvae completed their switch from endogenous nourishment to exogenous feeding. Reduced food intake by juveniles during a single meal after giving perpheral injection of gcPYY1-36 suggests a potentially important role of PYY in the food intake attenuation in grass carp.

Heterologous Expression and Metal-Binding Characterization of a Type 1 Metallothionein Isoform (OsMTI-1b) from Rice (Oryza sativa)

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni(2+), Cd(2+), Zn(2+) and Cu(2+) ions was analyzed. The results demonstrated that this isoform has ability to bind Ni(2+), Cd(2+) and Zn(2+) ions in vitro, whereas it has no substantial ability to bind Cu(2+) ions. From competitive reaction with 5,5'-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni(2+)/Cd(2+) > Zn(2+) > Cu(2+).

Crystal structure of human Intersectin-2L C2 domain

Intersectin-2L (ITSN-2L) is a long isoform of ITSN family, which is a multimodule scaffolding protein functioning in membrane-associated molecular trafficking and signal transduction pathways. ITSN-2L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain, suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. But the role of C2 domain is obscure in this process. Here we report the crystal structure of human ITSN-2L C2 domain at 1.56Å resolution. The sequence and structural alignment of ITSN-2L C2 domain with other members of C2 domain protein family indicate its vital cellular roles in membrane trafficking, the generation of lipid-second messengers and activation of GTPases. Moreover, our data show the possible roles of ITSN-2L C2 domain in regulating the activity of Cdc42.

Insulin-Like Growth Factor-I E-Peptide Activity Is Dependent on the IGF-I Receptor

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity.

Rictor regulates phosphorylation of the novel protein kinase C Apl II in Aplysia sensory neurons

Rapamycin-insensitive companion of TOR (Rictor) is a conserved component of target of rapamycin complex 2 (TORC2), a complex implicated in phosphorylation of a number of signal transduction-related kinases, including protein kinase Cs (PKCs) at their 'hydrophobic' site in the carboxy-terminal extension domain. In the marine mollusk, Aplysia californica, an increase in phosphorylation of the novel PKC, Apl II, at the hydrophobic site is associated with a protein synthesis-dependent increase in synaptic strength seen after continuous application of serotonin. To determine if Rictor plays a role in this increase, we cloned the Aplysia ortholog of Rictor (ApRictor). An siRNA-mediated decrease in ApRictor levels in Aplysia sensory neurons led to a decrease in the phosphorylation of PKC Apl II at the hydrophobic site suggesting a role for ApRictor in hydrophobic site phosphorylation. However, over-expression of ApRictor was not sufficient to increase phosphorylation of PKC Apl II. Continuous application of serotonin increased phosphorylation of PKC Apl II at the hydrophobic site in cultured sensory neurons, and this was blocked by Torin, which inhibits both TORC1 and TORC2. Over-expression of ApRictor did not lead to change in the magnitude of serotonin-mediated phosphorylation, but did lead to a small increase in the membrane localization of phosphorylated PKC Apl II. In conclusion, these studies implicate Rictor in phosphorylation of a novel PKC during synaptic plasticity and suggest an additional role for Rictor in regulating the localization of PKCs.

Analysis of type 2 metallothionein gene from mangrove species (Kandelia candel)

Metallothioneins are Cys-rich, low-molecular weight, metal-binding proteins. In this paper, we aimed to evaluate the roles of type 2 metallothionein in Kandelia candel (KcMT2) by analyzing its mRNA expression in leaves in response to heavy metals, as well as the metal-binding properties of its protein. The transcriptional response of KcMT2 gene to different heavy metals (Zn, Cu, Pb and Cd) was measured using real-time quantitative PCR. The results showed that the gene was regulated by the four metals tested. Significant increase in the expression of KcMT2 mRNA was also found in response to Zn, Cu, Pb and Cd. To analyze further the possible roles of KcMT2 against heavy metals, it was expressed in Escherichia coli BL21 as a carboxy-terminal extension of glutathione-S-transferase (GST). The E. coli cells expressing GST-KcMT2 fusion protein showed higher growth compared to the control cells under Zn, Cu, Pb or Cd exposure, and the GST-KcMT2 fusion protein had a stronger binding affinity for Zn, Cu, Pb and Cd than GST alone. Results presented here reveal that KcMT2 may play important roles in the heavy metal homeostasis or detoxification.