Distinct functions of tapasin revealed by polymorphism in MHC class I peptide loading

Peptide assembly with class I molecules is orchestrated by multiple chaperones including tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the tapasin-deficient human cell line 721.220. These findings indicate distinct roles for tapasin in class I peptide loading. First, tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.     

Mechanisms of DNA cleavage by copper complexes of 3-clip-phen and of its conjugate with a distamycin analogue

Mechanisms of DNA oxidation by copper complexes of 3-Clip-Phen and its conjugate with a distamycin analogue, in the presence of a reductant and air, were studied. Characterisation of the production of 5-methylenefuranone (5-MF) and furfural, associated with the release of nucleobases, indicated that these copper complexes oxidised the C1′ and C5′ positions of 2-deoxyribose, respectively, which are accessible from the DNA minor groove. Oxidation at C1′ was the major degradation route. Digestion of DNA oxidation products by P1 nuclease and bacterial alkaline phosphatase allowed characterisation of glycolic acid residues, indicating that these copper complexes also induced C4′ oxidation. However, this pathway was not associated with base propenal release. The ability of the copper complex of the 3-Clip-Phen conjugate with the distamycin analogue to produce sequence-selective DNA cleavage allowed confirmation of these mechanisms of DNA oxidation by PAGE. Comparison of DNA cleavage activity showed that conjugation of 3-Clip-Phen with a DNA minor groove binder, like the distamycin analogue, decreased both its ability to perform C1′ oxidation as well as the initial rate of the reaction, but this conjugate is still active after 5 h at 37°C, making it an efficient DNA cleaver.     

The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Temperature and Electrolyte Optimization of the α-Hemolysin Latch Sensing Zone for Detection of Base Modification in Double-Stranded DNA

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol⁻¹ decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.     

The RNA binding protein Larp1 regulates cell division,apoptosis and cell migration

The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.     

Biometrical genetic analysis of luteovirus transmission in the aphid Schizaphis graminum

The aphid Schizaphis graminum is an important vector of the viruses that cause barley yellow dwarf disease. We studied the genetic architecture of virus transmission by crossing a vector and a non-vector genotype of S. graminum. F1 and F2 hybrids were generated, and a modified line-cross biometrical analysis was performed on transmission phenotype of two of the viruses that cause barley yellow dwarf: Cereal yellow dwarf virus (CYDV)-RPV and Barley yellow dwarf virus (BYDV)-SGV. Our aims were to (1) determine to what extent differences in transmission ability between vectors and non-vectors is due to net additive or non-additive gene action, (2) estimate the number of loci that determine transmission ability and (3) examine the nature of genetic correlations between transmission of CYDV-RPV and BYDV-SGV. Only additive effects contributed significantly to divergence in transmission of both CYDV-RPV and BYDV-SGV. For each luteovirus, Castle-Wright's estimator for the number of effective factors segregating for transmission phenotype was less than one. Transmission of CYDV-RPV and BYDV-SGV was significantly correlated in the F2 generation, suggesting that there is a partial genetic overlap for transmission of these luteoviruses. Yet, 63% of the F2 genotypes transmitted CYDV-RPV and BYDV-SGV at significantly different rates. Our data suggest that in S. graminum, the transmission efficiency of both CYDV-RPV and BYDV-SGV is regulated by a major gene or set of tightly linked genes, and the transmission efficiency of each virus is influenced by a unique set of minor genes.