Different point mutations in the met oncogene elicit distinct biological properties.

The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties.     

Role of rusticyanin in the electron transport process in Thiobacillus ferrooxidans.

Effect of diethyl dithiocarbamate (DEDC), an antimicrobial agent, on growth of Thiobacillus ferrooxidans, possibly by inhibiting rusticyanin present in the periplasmic space of the microorganism, has been studied to gain more insight into the electron transport chain in the bioleaching process. DEDC is found to form a stable complex with rusticyanin in solution and also in polyacrylamide gel. The spectrum of the complex is identical to that of Cu-DEDC complex, suggesting binding of DEDC with copper moiety of rusticyanin and resulting in inhibition of growth. In vitro reduction of purified rusticyanin by Fe(II) in absence of acid-stable cytochrome c is very slow, indicating the importance of cytochrome c in electron transport. Thus, in the iron oxidation process, acid-stable cytochrome c is the primary acceptor of electron, transferring the electron to rusticyanin at pH 2.0, which, in turn, affects electron transfer to iron-cytochrome c reductase around pH 5.5.     

Conserved water molecular dynamics of the different X-ray structures of rusticyanin: an unique aquation potentiality of the ligand bonded Cu++ center

The invariant water molecular interaction involving in the Rusticyanin of Thiobacillus ferrooxidans is thought to be important for its molecular complexation with other proteins at differential acidophilic situation. The comparative analysis of the different x-ray, energy minimized, and auto solvated structures of Rusticyanin revealed the presence of five specific invariant bound water molecules (among the approximately 150 water molecules per monomer) in the crystals. The five W 205, W 206, W 112, W 214, and W 221 water molecules (in Rusticyanin PDB code: 1RCY) were seem to be invariant in all the seven structures (PDB codes: 1RCY, 1A3Z, 1A8Z, 1E3O, 1GY1, 1GY2, 2CAL). Among the five conserved water molecules the W 221 (of 1 RCY or the equivalent water molecules in the other oxidized form of Rusticyanin structures) had endowed an interesting coordination potentiality to Cu(+2) ion during the energy minimization. The W 221 was observed to approach toward the tetrahedrally bonded Cu(+2) ion through the opposite (or trans) route of metal-bonded Met 148. This direct water molecular coordination affected the tetrahedral geometry of Cu(+2) to trigonal bipyramidal. Presumably this structural dynamics at the Cu(+2) center could involve in the electron transport process during protein-protein complexation.     

Antibody neutralization escape mediated by point mutations in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.     

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.     

Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids.

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro alpha 1(I) and pro alpha 2(I) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for alpha 1(I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A+)-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes. We have used this strategy for the detection and sequencing of a point mutation in alpha 2(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.     

Functional analysis of the neurofibromatosis type 2 protein by means of disease-causing point mutations

Despite intense study of the neurofibromatosis type 2 (NF2) tumor-suppressor protein merlin, the biological properties and tumor-suppressor functions of merlin are still largely unknown. In this study, we examined the molecular activities of NF2-causing mutant merlin proteins in transfected mammalian cells, to elucidate the merlin properties that are critical for tumor-suppressor function. Most important, we found that 80% of the merlin mutants studied significantly altered cell adhesion, causing cells to detach from the substratum. This finding implies a function for merlin in regulating cell-matrix attachment, and changes in cell adhesion caused by mutant protein expression may be an initial step in the pathogenesis of NF2. In addition, five different mutations in merlin caused a significant increase in detergent solubility of merlin compared to wild type, indicating a decreased ability to interact with the cytoskeleton. Although not correlated to the cell-adhesion phenotype, four missense mutations decreased the binding of merlin to the ERM-interacting protein EBP-50, implicating this interaction in merlin inhibition of cell growth. Last, we found that some NF2 point mutations in merlin most closely resembled gain-of-function alleles in their cellular phenotype, which suggests that mutant NF2 alleles may not always act in a loss-of-function manner, as had been assumed, but may include a spectrum of allelic types with different phenotypic effects on the function of the protein. In aggregate, these cellular phenotypes provide a useful assay for identifying the functional domains and molecular partners necessary for merlin tumor-suppressor activity.     

An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea-induced Kitl(Steel) alleles

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced Kitl(Sl) alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved alpha-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.     

Water exchange in plant tissue studied by proton NMR in the presence of paramagnetic centers.

The proton NMR relaxation of water in maize roots in the presence of paramagnetic centers, Mn2+, Mn- EDTA2 -, and dextran-magnetite was measured. It was shown that the NMR method of Conlon and Outhred (1972, Biochem. Biophys. Acta. 288:354-361) can be applied to a heterogenous multicellular system, and the water exchange time between cortical cells and the extracellular space can be calculated. The water exchange is presumably controlled by the intracellular unstirred layers. The Mn- EDTA2 - complex is a suitable paramagnetic compound for complex tissue, while the application of dextran-magnetite is probably restricted to studies of water exchange in cell suspensions. The water free space of the root and viscosity of the cells cytoplasm was estimated with the use of Mn- EDTA2 -. The convenience of proton NMR for studying the multiphase uptake of paramagnetic ions by plant root as well as their transport to leaves is demonstrated. A simple and rapid NMR technique (spin-echo recovery) for continuous measurement of the uptake process is presented.     

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure

Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.     

Identification of point mutations in mixtures by capillary electrophoresis hybridization.

We have developed a rapid method for unambiguous identification and mutant fraction determination of individual mutants in mixtures of DNA sequence variants each differing by one or a few nucleotides. This method has applications to such diverse areas as interpretation of mutational spectra, screening of populations for polymorphisms and identification of species in environmental mixtures. In our approach, a mixture of unknown sequences labeled with a fluorescent dye is combined with a set of predetermined sequences (standards) representing the variants to be assayed. Labeling the standards with another dye allows the two sets of variants to be measured independently. Using constant denaturing capillary electrophoresis, the sequence variants are separated as individual peaks on the basis of differential melting equilibria. The unknown sequence variants are initially identified based on co-migration with particular standards. This preliminary identification is verified by hybridization of the unknown variants with the co-migrating standards within the capillary. We demonstrate the use of capillary electrophoresis hybridization to dissect complex mutational spectra of human cells in culture.     

Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 Å. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases. Proteins 32:523–531, 1998. © 1998 Wiley-Liss, Inc.     

On the role of the axial ligand in heme-based catalysis of the peroxidase and P450 type

 The present commentary focusses on the role of the axial ligand in peroxidase- and P450-type catalysis. Based on molecular orbital calculations and the experimental evidence available, it is argued that the ligand of a heme-containing enzyme may be a factor in setting the relative chance, although not the intrinsic capability, of the enzyme to catalyse a specific type of heme-based reaction chemistry. The ligand can do so by influencing the electrophilicity, i.e. the redox potential of the high-valency iron-oxo complex, and also by influencing the energy barrier for a reaction pathway through delocalization of valence electrons along the axial ligands, thereby, in the case of a cysteinate but not a histidine axial ligand, stabilizing oxygen transfer pathways. Received and accepted: 7 May 1996     

Perturbation of tryptophan residues by point mutations in bacteriophage T4 lysozyme studied by optical detection of triplet-state magnetic resonance spectroscopy

We have investigated perturbations of the triplet-state properties of Trp residues in bacteriophage T4 lysozyme caused by point mutations using low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Five temperature-sensitive mutants have been studied in detail. These include lysozymes with the point mutations Gln-105----Ala, Gln-105----Gly, Gln-105----Glu, Ala-146----Thr, and Trp-126----Gln. Changes in phosphorescence 0,0 band wavelength, intensity, the triplet-state zero-field splitting (ZFS), and the wavelength dependence of the ZFS were detected only from Trp-138 in each mutant. In the case of the Q105A mutation, the perturbations on Trp-138 have been ascribed to the combination of an increase in the polarizability of the environment and to the loss of hydrogen bonding of the enamine nitrogen of indole. For the Q105G mutation, we believe that Q is replaced by a solvent molecule in H bonding, leading to relatively small changes. In the Q105E mutation, the perturbation results largely from the introduction of a charged residue. In the case of the mutation A146T, the perturbation is associated with a local conformational change in which Trp-138 is shifted to a more solvent-exposed location. On the other hand, no significant spectroscopic changes in Trp-126 and Trp-158 were found in any of the mutants, suggesting that the perturbations are probably localized near Trp-138 for the mutations of positions 105 and 146. However, in the mutation W126Q, which occurs approximately 16 A away from Trp-138, significant changes of Trp-138 are detected, suggesting that the effects of this mutation are propagated over large distances.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.     

Analysis of point mutations of the BRCA1 gene by hybridization with hydrogel microarrays

BRCA1 mutations are associated with a higher risk of breast (BC) and ovarian cancer in women. Testing for such mutations allows BC prognosis, selection of an individual treatment strategy, and prevention of disease recurrence. Hybridization on a hydrogel microarray was developed for identifying point mutations in BRCA1. The microarray was designed to detect five-point mutations: 185delAG, 300T→G, 4153delA, 4158A→G, and 5382insC. The microarray was tested with 36 control specimens with known genotypes and used to examine 65 BC patients. The results demonstrated the advantage of employing the microarray in analyzing BRCA1 mutations.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

High-throughput profiling of point mutations across the HIV-1 genome

Background

The HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance.

Results

Here we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions – representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket.

Conclusion

The approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.