Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Docking studies on novel analogues of 8 methoxy fluoroquinolones against GyrA mutants of Mycobacterium tuberculosis

Background

Modeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues.

Results

Using sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rβ and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus.

Conclusions

We succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.     

Signal Transduction Protocols (R. C. Dickson and M. D. Mendenhall (eds.), in <Emphasis Type="Italic">Methods in Molecular Biology</Emphasis>, Vol. 284, J. M. Walker (Series Editor), Humana Press,Totowa-New Jersey, 2004, 327 p., $ 99.50)

Book Review

Signal Transduction Protocols (R. C. Dickson and M. D. Mendenhall (eds.), in Methods in Molecular Biology, Vol. 284, J. M. Walker (Series Editor), Humana Press, Totowa-New Jersey, 2004, 327 p., $ 99.50)     

Silencing human NKG2D, DAP10, and DAP12 reduces cytotoxicity of activated CD8+ T cells and NK cells

Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.     

Marine Heterotrophic Amoebae, Flagellates and Heliozoa From Belize (Central America) and Tenerife (Canary Islands), With Descriptions of New Species, Luffisphaera Bulbochaete N. Sp., L. Longihastis N. Sp., L. Turriformis N. Sp. and Paulinella Intermedia N. Sp.

ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids, the bicosoecids, the chromulinids, the pedinellids and a variety of laxa of uncertain affinities (Protista incertae sedis). the identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described: one new species of the amoeba genus Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Luffisphaera Belcher & Swale, 1975 ( Luffisphaera bulbochaete n. sp.; L. longihastis n. sp.; L. turriformis n. sp.).     

Taxol from fungal endophytes and the issue of biodiversity

Fungi represent one of the most understudied and diverse group of organisms. Commonly, these organisms make associations with higher life forms and may proceed to biochemically mimic the host organism. An excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in the plant genusTaxus (yew). However, taxol has been reported in a novel endophytic fungus—Taxomyces andreanae, but also has been demonstrated to occur in a number of unrelated fungal endophytes includingPestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia and others. Thus, this report presents information on the presence of taxol among disparate fungal genera, and uses these observations as an additional argument to support efforts to study fungal endophytes and preserve their associated host plants.     

Evolution and phylogenetic utility of the period gene in Lepidoptera

Evolution and phylogenetic utility of the period gene are explored through sequence analysis of a relatively conserved 909-bp fragment in 26 lepidopteran species. Taxa range from tribes to superfamilies, primarily within the putative clade Macrolepidotera plus near outgroups, and include both strongly established and problematic groupings. Their divergence dates probably range from the late Cretaceous through much of the Tertiary. Comparisons within the same set of closely related species show that amino acid substitutions in period occur 4.9 and 44 times as frequently as they do in two other nuclear genes--dopa decarboxylase and elongation factor-1 alpha, respectively. In contrast, rates of observed synonymous substitution are within 60% of each other for these three genes. Synonymous changes in period approach saturation by the family level, whereas nonsynonymous and amino acid divergences across the Macrolepidoptera are less than half the maximal values reported for this gene. Phylogenetic analyses of period strongly supported groupings at the family level and below. In contrast to previous analyses at this level with other nuclear genes, much of the information lies in nonsynonymous change. Relationships up to the superfamily level were recovered with decreasing effectiveness, and little, if any, signal was apparent regarding relationships among superfamilies. This could reflect rapid radiation of the superfamilies, however, rather than saturation in the period locus; thus, period, in combination with other genes, remains a plausible candidate for approaching the difficult problems of lepidopteran family and superfamily relationships.      

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

New Thioureas and Related Substances Intended for Melanoma Targeting

Thiouracil and a few related drugs are known to be melanoma-seeking agents owing to specific incorporation into nascent melanin. The melanin-affinic properties are apparently due to binding to intermediates, preferably dopaquinone, produced in the melanin synthetic pathway by tyrosinase-catalysed oxidation of tyrosine. In the present paper, in vitro screening methods have been used for the identification of possible melanoma seekers according to the above principle. The binding of test substance to dopaquinone suppresses dopachrome formation by the withdrawal of dopaquinone from the reaction mixture, and the decrease in dopachrome concentration was monitored spectrophotometrically at 475 nm. In order to eliminate false results caused by tyrosinase inhibition, which also will decrease the dopachrome concentration, the oxygen consumption was followed potentiometrically. To avoid the effect of tyrosinase inhibition on dopachrome formation, additional experiments with autoxidation of L-dopa in the presence of test substance were performed. Of the 22 substances (mainly thioureylenes and thioamides) studied, 4,5,6-triamino-2(H)-pyrimidinehtionsulfate, trithiocyanuric acid, 2-thiouracil, 6-methyl-2-thiouracil, and 4-amino-2-mercaptopyrimidine most effectively decreased the dopachrome formation with no or little inhibition of tyrosinase activity. They should therefore be regarded as potential melanoma seekers. In a complementary autoradiographic study on the uptake of the potent tyrosinase inhibitor mercaptobenzothiazole (MBT) in B 16 melanoma, transplanted to mice, it was found that strong tyrosinase inhibition seems to decrease incorporation into melanin in vivo. MBT was partly accumulated in restricted areas of the tumor, which may be explained by the small molar dose injected.