A new gene-finding tool: using the Caenorhabditis elegans operons for identifying functional partner proteins in human cells

How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.     

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.     

Comparative analysis of inducible expression systems in transient transfection studies

Ectopic protein expression in mammalian cells is a valuable tool to analyze protein functions. Increasingly, inducible promoters are being used for regulated gene expression. Here, we compare expression maxima, induction rates, and "leakiness" of the following promoter systems: (I) two tetracycline-responsive Tet systems (Tet-On, Tet-Off), (II) the glucocorticoid-responsive mouse mammary tumor virus promoter (MMTVprom), (III) the ecdysone-inducible promoter (EcP), and (IV) the T7 promoter/T7 RNA polymerase system (T7P). The systems were analyzed by expressing an enhanced green fluorescent protein (EGFP) luciferase fusion reporter protein in transiently transfected cells. Expression was assessed qualitatively by fluorescence microscopy of the EGFP component and quantitatively by measuring the enzymatic activity of the luciferase component of the fusion protein. Basal expression levels ("leakiness") were ranked Tet-On>Tet-Off>MMTVprom>EcP>T7P. Induction rates were EcP>MMTVprom>T7P>Tet-Off>Tet-On. Expression maxima were ranked. Tet-On>Tet-Off>MMTVprom>EcP>T7P. To increase T7-promoter-mediated expression we inserted an internal ribosomal entry site element into the T7 expression cassette. In presence of T7 RNA polymerase this modified T7 promoter achieved expression levels of 42% of a Rous Sarcoma virus promoter, while keeping basal expression extremely low.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH(2)- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.     

Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D

We characterized two novel members of the RAET1/ULBP gene cluster, RAET1E and RAET1G. The encoded proteins were similar to the ULBP in their class I-like alpha1 and alpha2 domains, but differed in that, instead of being GPI-anchored, their sequences were type 1 membrane-spanning molecules. Both proteins were capable of being expressed at the cell surface. Both proteins bound the activating receptor NKG2D, and RAET1G bound the human CMV protein UL16. The expression of diverse NKG2D-binding molecules in different tissues and with different properties is consistent with multiple modes of infection- or stress-induced activation.     

Inhibition of the growth of Toxoplasma gondii in immature human dendritic cells is dependent on the expression of TNF-alpha receptor 2

An effective immunity to Toxoplasma gondii in humans is dependent on the cellular immune response. Toxoplasma can infect and replicate in almost all nucleated cells, and the most important cytokine regulating the growth in humans is IFN-gamma; however, the role of TNF-alpha has to date been largely described to be synergistic. We show that, compared with mature human dendritic cells (mDC), immature human DC (iDC) demonstrate a reduced parasite proliferation when infected with Toxoplasma. This toxoplasmostasis was only present in iDC after 11 days of culture and was not present in DC that had been matured ex vivo using a cytokine mixture (mDC). Spontaneous toxoplasmostatic activity has previously only been described in fresh human monocytes, and the mechanism involved is as yet unclear. We show that, in comparison with an absence of expression in mDC, TNF-R2 is expressed in both iDC and monocytes infected with Toxoplasma, and furthermore, that blocking the TNF-R2 with Abs abrogates the toxoplasmostasis in the iDC. These findings demonstrate a functional role for TNF-R2 in the newly described spontaneous toxoplasmostasis of iDC.     

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.