Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

A protein residing at the subunit interface of the bacterial ribosome

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two beta-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-alpha-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Discovery and fine mapping of serum protein loci through transethnic meta-analysis.

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10(-8)) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.

Pathogenesis of cell toxicity by plant toxins

Ribosome inactivating proteins (RIPs) are a family of plant proteins that depurinate the major rRNA, inhibiting the protein synthesis. RIPs are divided into type 1, single chain proteins with enzymatic activity, and type 2 RIPs (toxic and non-toxic), with the enzymatic chain linked to a binding chain. RIPs have been used alone or as toxic component of immunotoxins for experimental therapy of many diseases. The knowledge of cell death pathway(s) induced by RIPs could be useful for clarifying the mechanisms induced by RIPs and for designing specific immunotherapy. The topic of the current study was (i) the determination of the amino acid sequence of the type 2 RIP stenodactylin. The comparison with other RIPs showed that the A chain is related to other toxic type 2 RIPs. whereas the B chain is more related to the non-toxic type 2 RIPs. This latter result is surprising because stenodactylin is actually the most toxic type 2 RIP known; (ii) the study of the cell death mechanisms induced by stenodactylin in human neuroblastoma cells (NB100). High doses of stenodactylin can activate the effector caspases (perhaps through the DNA damage and/or intrinsic/extrinsic pathways) and also cause ROS generation. Low doses cause a caspase-dependent apoptosis, mainly via extrinsic pathway. Moreover, the activation of caspases precedes the inhibition of protein synthesis; (iii) the investigation of the cell death pathway induced by the non-toxic type 2 RIPs ebulin l and nigrin b. These RIPs demonstrated high enzymatic activity in a cell-free system, but they lack high cytotoxicity. These preliminary studies demonstrate that the cell death mechanism induced by the two non-toxic RIPs is partially caspase-dependent apoptosis, but other mechanisms seem to be involved

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of prokaryotic (H. volcanii, S. aureus) and unicellular eukaryotic model organisms. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs. For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. In the archaeon H. volcanii a tRNA-derived fragment was identified to target the small ribosomal subunit upon alkaline stress in vitro and in vivo. As a consequence of ribosome binding, this tRNA-fragment reduces protein synthesis by interfering with the peptidyl transferase activity. Our data reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory sRNAs.

The ribosome as novel target for small regulatory RNAs

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. Furthermore it is strongly assumed that the primordial translation system in the ‘RNA world’ most likely received direct regulatory input from ncRNA-like cofactors. The fundamental question that we would like to ask is: Does the ‘RNA world still communicate’ with the ribosome? To address this question, we have analyzed the small ncRNA interactomes of ribosomes of organisms from all three domains of life. Deep-sequencing and subsequent bioinformatic analyses revealed thousands of putative ribosome-associated ncRNAs.1,2 For a subset of these ncRNA candidates we have gathered experimental evidence that they are expressed in a stress-dependent manner and indeed directly target the ribosome. We show that some of these ribosome-bound small ncRNAs are capable of fine tuning protein synthesis in vitro and in vivo. Our data therefore reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life and suggest the existence of a so far largely unexplored mechanism of translation regulation.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules are key players in controlling gene expression at multiple steps in all domains of life. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily (such as micro RNAs and small-interfering RNAs), not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by directly targeting the ribosome [3]. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator under these stress conditions. Our data reveal the ribosome as a target for small regulatory ncRNAs and unveil the existence of a novel mechanism of translation regulation. Analogous genomic screens in organisms spanning all three domains of life demonstrate the existence of thousands of ncRNA candidates putatively regulating the ribosome. We therefore anticipate that ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

核糖体失活蛋白抗HIV-1作用的研究进展

核糖体失活蛋白(RIPs)抗HIV-1活性研究已有十几年的历史。RIPs类化合物代表了抗HIV/AIDS天然产物或先导化合物发展的一个重要方向。本文从介绍RIPs的酶活性及其抗HIV-1活性入手,对RIPs抗HIV-1的可能机制,从与RIPs酶活性的关系、诱导HIV-1感染细胞的凋亡及相应的信号转导、诱发活性氧的产生,以及对HIV-1整合酶的抑制作用等几个方面做了较详尽的阐述,并对RIPs的结构修饰和抗HIV-1构效关系进行了综述。对RIPs类化合物在抗病毒领域进行深入而系统地研究,能拓宽其在抗HIV/AIDS临床上的进一步应用。

Immunological response in mice bearing LM3 breast tumor undergoing Pulchellin treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Is there a role for eIF5A in translation?

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archaebacteria to mammals. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. This review focuses on the functional characterization of eIF5A. Although this protein was originally identified as a translation initiation factor, subsequent studies did not support a role for eIF5A in general translation initiation. eIF5A has also been implicated in nuclear export of HIV-1 Rev and mRNA decay, but these findings are controversial in the literature and may reflect secondary effects of eIF-5A function. Next, the involvement of eIF5A and hypusination in the control of the cell cycle and proliferation in various organisms is reviewed. Finally, recent evidence in favor of reconsidering the role of eIF5A as a translation factor is discussed. Future studies may reveal the specific mechanism by which eIF5A affects protein synthesis.

Estudo comparativo da atividade antiinflamatória e anti-tumoral de proteínas inativadoras de ribossomos extraídas de plantas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Regulating the expression of eEF1A to decipher its non canonical functions in eukaryotic cells

The canonical function of eEF1A is delivery of the aminoacylated tRNA to the A site of the ribosome during protein translation, however, it is also known to be an actin binding protein. As well as this actin binding function, eEF1A has been shown to be involved in other cellular processes such as cell proliferation and apoptosis. It has long been thought that the actin cytoskeleton and protein synthesis are linked and eEF1A has been suggested to be a candidate protein to form this link, though very little is understood about the relationship between its two functions. Overexpression of eEF1A has also been shown to be implicated in many different types of cancers, especially cancers that are metastatic, therefore it is important to further understand how eEF1A can affect both translation and the organisation of the actin cytoskeleton. To this end, we aimed to determine the effects of reduced expression of eEF1A on both translation and its non canonical functions in CHO cells. We have shown that reduced expression of eEF1A in this cell system results in no change in protein synthesis, however results in an increased number of actin stress fibres and other proteins associated with these fibres such as myosin IIA, paxillin and vinculin. Cell motility and attachment are also affected by this reduction in eEF1A protein expression. The organisational and motility phenotypes were found to be specific to eEF1A by transforming the cells with plasmids containing either human eEF1A1 or eEF1A2. Though the mechanisms by which these effects are regulated have not yet been established, this data provides evidence to show that the translation and actin binding functions of eEF1A are independent of each other as well as being suggestive of a role for eEF1A in cell motility as supported by the observation that overexpression of eEF1A protein tends to be associated with the cancer cells that are metastatic.