Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution

We have solved and refined the crystal and molecular structures of the calcium-saturated N-terminal domain of troponin C (TnC) to 1.75 A resolution. This has allowed for the first detailed analysis of the calcium binding sites of this molecular switch in the calcium-loaded state. The results provide support for the proposed binding order and qualitatively, for the affinity of calcium in the two regulatory calcium binding sites. Based on a comparison with the high-resolution apo-form of TnC we propose a possible mechanism for the calcium-mediated exposure of a large hydrophobic surface that is central to the initiation of muscle contraction within the cell.     

Mannan-specific immunoglobulin G antibodies in normal human serum accelerate binding of C3 to Candida albicans via the alternative complement pathway

Candida albicans activates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allows C. albicans to initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca2+ with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding to C. albicans incubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab')2 fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system and C. albicans.     

Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow micro-aerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.     

Regression of experimental brain tumors with 6-thioxanthine and Escherichia coli gpt gene therapy

The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 microM, whereas parental C6 cells were not affected at a concentration of 500 microM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This "modified" bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX). In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment. This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005). These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer.     

A discrete three-amino acid segment (LVI) at the C-terminal end of kinase-impaired ErbB3 is required for transactivation of ErbB2

ErbB3 is unique among other members of the receptor tyrosine kinase family of growth factor receptors in that its kinase domain is enzymatically impaired. This renders it incapable of transducing a signal in response to ligand binding. However, in conjunction with ErbB2, ErbB3 is a potent mediator of signaling by the growth factor heregulin. Heregulin binding to ErbB3 induces formation of a heterodimeric complex with ErbB2, and this results in transactivation of the ErbB2 kinase. Although interaction between the extracellular domains of these receptors is an essential part of this process, it was not clear whether interaction between the cytoplasmic domains is also necessary for transactivation. By examining the abilities of a series of cytoplasmic domain mutants of ErbB3 to activate ErbB2, we have found a discrete sequence of three amino acid residues (LVI), located at the carboxyl-terminal end of the impaired ErbB3 kinase region, that is obligatory for transactivation. We conclude that formation of a functional ErbB2-ErbB3 signaling complex requires the presence of a specific structural feature within the ErbB3 cytoplasmic domain and suggest that ErbB2 transactivation results from a physical interaction between the cytoplasmic domains of these receptors.     

Christian's spondylo-digital syndrome: second familial case

We studied a mother and daughter with skeletal dysplasia which was characterized clinically by proximal and distal flexion contractures in the phalanges, and by brachydactyly, clinodactyly and ulnar and radial subdislocations of the fingers. Radiologically, the 2nd metacarpal in the daughter was seen to be longer than the other metacarpals, with bone carpal fusion, and flexion contractures of the fingers in both hands. Thoraco-lumbar xyphorotoscoliosis and malformed vertebrae with dyssegmentation of L2-L3, T12 and L1 with cuneiform shape, asymmetry of the pelvic bones and exostotic lesions in the proximal third of the tibia and the distal third of the femur were also noted. The clinical and radiological characteristics were compatible with the syndrome described by Christian et al. in 1975 and called the second metatarsal syndrome. The purpose of this paper was to present a second corroborative familial case and to propose another name: Christian's spondylo-digital syndrome.     

Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.     

Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.