Non-canonical amino acids in protein engineering

Methods for engineering proteins that contain non-canonical amino acids have advanced rapidly in the past few years. Novel amino acids can be introduced into recombinant proteins in either a residue-specific or site-specific fashion. The methods are complementary: residue-specific incorporation allows engineering of the overall physical and chemical behavior of proteins and protein-like macromolecules, whereas site-specific methods allow mechanistic questions to be probed in atomistic detail. Challenges remain in the engineering of the translational apparatus and in the design of schemes that can be used to encode both canonical and non-canonical amino acids.     

Solid-phase oligosaccharide synthesis and related technologies

Research efforts directed at the development of methodologies effective for solid-phase synthesis of oligosaccharides have resulted in a number of impressive achievements. In addition, closely related technologies, such as soluble polymer-supported synthesis and fluorous synthesis of the same class of molecules, have proved to be quite promising.     

Metazoan phylogenies: falling into place or falling to pieces? A palaeontological perspective

Metazoan phylogeny is in a state of ferment, stirred by the addition of new molecular trees as well as controversial interpretations of molecular ‘clocks’. Concerning the latter topic, the clocks recurrently point to divergence times substantially older than the known fossil record. Some attempt reconciliation by appealing to a conveniently cryptic interval prior to the first fossils. This effectively reduces the fossil record to an erratic search-light giving only glimpses into the true evolutionary history. Other options, however, remain open. Molecular clocks may themselves run erratically and what happens in molecular history may not coincide with the emergence of body plans.     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).     

RGSZ1 and Ret RGS: Two of Several Splice Variants from the Gene RGS20

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.     

Helicobacter pylori chromosomal DNA replication: current status and future perspectives

Helicobacter pylori causes gastritis, gastric ulcer and gastric cancer. Though DNA replication and its control are central to bacterial proliferation, pathogenesis, virulence and/or dormancy, our knowledge of DNA synthesis in slow growing pathogenic bacteria like H. pylori is still preliminary. Here, we review the current understanding of DNA replication, replication restart and recombinational repair in H. pylori. Several differences have been identified between the H. pylori and Escherichia coli replication machineries including the absence of DnaC, the helicase loader usually conserved in gram-negative bacteria. These differences suggest different mechanisms of DNA replication at initiation and restart of stalled forks in H. pylori.     

The signal-anchor sequence of CYP2C1 inserts into the membrane as a hairpin structure

Microsomal cytochrome P450s (CYPs) are anchored to the endoplasmic reticulum membrane by the N-terminal signal-anchor sequence which is predicted to insert into the membrane as a type 1 transmembrane helix with a luminally located N-terminus. We have mapped amino acids of the CYP2C1 signal-anchor, fused to Cys-free glutathione S-transferase, within the membrane by Cys-specific labeling with membrane-impermeant maleimide polyethylene glycol. At the C-terminal end of the signal-anchor, Trp-20 was mapped to the membrane–cytosol interface and Leu-19 was within the membrane. Unexpectedly, at the N-terminal end, Glu-2 and Pro-3 were mapped to the cytoplasmic side of the membrane rather than the luminal side as expected of a type 1 transmembrane helix. Similar results were observed for the N-terminal amino acids of the signal-anchor sequences of CYP3A4 and CYP2E1. These observations indicate that contrary to the current model of the signal-anchor of CYPs as a type 1 transmembrane helix, CYP2C1, CYP2E1, and CYP3A4 are monotopic membrane proteins with N-terminal signal-anchors that have a hairpin or wedge orientation in the membrane.     

Spatial complexity of mechanisms controlling a bacterial cell cycle

Cell cycle progression in Caulobacter is governed by a multilayered regulatory network linking chromosome replication with polar morphogenesis and cell division. Temporal and spatial regulation have emerged as the central themes, with the abundance, activity and subcellular location of key structural and regulatory proteins changing over the course of the cell cycle. An additional layer of complexity was recently uncovered, showing that each segment of the chromosome is located at a specific cellular position both during and after the completion of DNA replication, raising the possibility that this positioning contributes to temporal and spatial control of gene expression.     

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the over-expression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.     

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: implication in multinucleation and chemosensitization

The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC−/−p53−/− mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.