Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers

Complete resolution of the amide resonances in a three-dimensional solid-state NMR correlation spectrum of a uniformly 15N-labeled membrane protein in oriented phospholipid bilayers is demonstrated. The three orientationally dependent frequencies, 1H chemical shift, 1H–15N dipolar coupling, and 15N chemical shift, associated with each amide resonance are responsible for resolution among resonances and provide sufficient angular restrictions for protein structure determination. Because the protein is completely immobilized by the phospholipids on the relevant NMR time scales (10 kHz), the linewidths will not degrade in the spectra of larger proteins. Therefore, these results demonstrate that solid-state NMR experiments can overcome the correlation time problem and extend the range of proteins that can have their structures determined by NMR spectroscopy to include uniformly 15N-labeled membrane proteins in phospholipid bilayers.

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.

The anterior determinant bicoid of Drosophila is a derived Hox class 3 gene

The Drosophila gene bicoid functions as the anterior body pattern organizer of Drosophila. Embryos lacking maternally expressed bicoid fail to develop anterior segments including head and thorax. In wild-type eggs, bicoid mRNA is localized in the anterior pole region and the bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal caudal mRNA in the anterior region of the embryo. caudal genes as well as other homeobox genes or members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila. This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior–posterior polarity. We have cloned bicoid from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza), and show that the gene originated from a recent duplication of the direct homolog of the vertebrate gene Hox3, termed zerknüllt, which specifies extraembryonic tissues in insects.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

The cluA− mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria

The cluA gene of Dictyostelium discoideum encodes a novel 150-kDa protein. Disruption of cluA results in clustering of mitochondria near the cell center. This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm. The mutant cell populations also exhibit an increased frequency of multinucleated cells, suggesting an impairment in cytokinesis. Both phenotypes are reversed by transformation of cluA− cells with a plasmid carrying a constitutively expressed cluA gene. The predicted sequence of the cluA gene product is homologous to sequences encoded by open reading frames in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans, but not to any known protein. The only exception is a short region with some homology to the 42-residue imperfect repeats present in the kinesin light chain, which probably function in protein–protein interaction. These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria.

Different response to Plasmodium falciparum malaria in West African sympatric ethnic groups

The comparison of malaria indicators among populations that have different genetic backgrounds and are uniformly exposed to the same parasite strains is one approach to the study of human heterogeneities in the response to the infection. We report the results of comparative surveys on three sympatric West African ethnic groups, Fulani, Mossi, and Rimaibé, living in the same conditions of hyperendemic transmission in a Sudan savanna area northeast of Ouagadougou, Burkina Faso. The Mossi and Rimaibé are Sudanese negroid populations with a long tradition of sedentary farming, while the Fulani are nomadic pastoralists, partly settled and characterized by non-negroid features of possible caucasoid origin. Parasitological, clinical, and immunological investigations showed consistent interethnic differences in Plasmodium falciparum infection rates, malaria morbidity, and prevalence and levels of antibodies to various P. falciparum antigens. The data point to a remarkably similar response to malaria in the Mossi and Rimaibé, while the Fulani are clearly less parasitized, less affected by the disease, and more responsive to all antigens tested. No difference in the use of malaria protective measures was demonstrated that could account for these findings, and sociocultural or environmental factors do not seem to be involved. Known genetic factors of resistance to malaria did not show higher frequencies in the Fulani. The differences in the immune response were not explained by the entomological observations, which indicated substantially uniform exposure to infective bites. The available data support the existence of unknown genetic factors, possibly related to humoral immune responses, determining interethnic differences in the susceptibility to malaria.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Identification and classification of p53-regulated genes

Sequence-specific transactivation by p53 is essential to its role as a tumor suppressor. A modified tetracycline-inducible system was established to search for transcripts that were activated soon after p53 induction. Among 9,954 unique transcripts identified by serial analysis of gene expression, 34 were increased more than 10-fold; 31 of these had not previously been known to be regulated by p53. The transcription patterns of these genes, as well as previously described p53-regulated genes, were evaluated and classified in a panel of widely studied colorectal cancer cell lines. “Class I” genes were uniformly induced by p53 in all cell lines; “class II” genes were induced in a subset of the lines; and “class III” genes were not induced in any of the lines. These genes were also distinguished by the timing of their induction, their induction by clinically relevant chemotherapeutic agents, the absolute requirement for p53 in this induction, and their inducibility by p73, a p53 homolog. The results revealed substantial heterogeneity in the transcriptional responses to p53, even in cells derived from a single epithelial cell type, and pave the way to a deeper understanding of p53 tumor suppressor action.

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA

Genetic analysis of limiting quantities of genomic DNA play an important role in DNA forensics, paleoarcheology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity studies. We have tested the ability of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify picogram quantities of human genomic DNA for the purpose of increasing the amount of template for genotyping with microsatellite repeat markers. DNA was uniformly amplified at a large number of typable loci throughout the human genome with starting template DNAs from as little as 15 pg to as much as 400 ng. A much greater-fold enrichment was seen for the smaller genomic DOP-PCRs. All markers tested were amplified from starting genomic DNAs in the range of 0.6–40 ng with amplifications of 200- to 600-fold. The DOP-PCR-amplified genomic DNA was an excellent and reliable template for genotyping with microsatellites, which give distinct bands with no increase in stutter artifact on di-, tri-, and tetranucleotide repeats. There appears to be equal amplification of genomic DNA from 55 of 55 tested discrete microsatellites implying near complete coverage of the human genome. Thus, DOP-PCR appears to allow unbiased, hundreds-fold whole genome amplification of human genomic DNA for genotypic analysis.

Agrin in Alzheimer’s disease: Altered solubility and abnormal distribution within microvasculature and brain parenchyma

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer’s disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.

Role of the J-domain in the cooperation of Hsp40 with Hsp70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2–75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2–75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2–388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2–75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.