Specificity of dimer formation in tropomyosins: influence of alternatively spliced exons on homodimer and heterodimer assembly.

Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.

Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.

Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos.

The dorsoventral axis is established early in Xenopus development and may involve signaling by Wnts, a family of Wnt1-protooncogene-related proteins. The protein kinase shaggy functions in the wingless/Wnt signaling pathway, which operates during Drosophila development. To assess the role of a closely related kinase, glycogen synthase kinase 3 beta (GSK-3 beta), in vertebrate embryogenesis, we cloned a cDNA encoding a Xenopus homolog of GSK-3 beta (XGSK-3 beta). XGSK-3 beta-specific transcripts were detected by Northern analysis in Xenopus eggs and early embryos. Microinjection of the mRNA encoding a catalytically inactive form of rat GSK-3 beta into a ventrovegetal blastomere of eight-cell embryos caused ectopic formation of a secondary body axis containing a complete set of dorsal and anterior structures. Furthermore, in isolated ectodermal explants, the mutant GSK-3 beta mRNA activated the expression of neural tissue markers. Wild-type XGSK-3 beta mRNA suppressed the dorsalizing effects of both the mutated GSK-3 beta and Xenopus dishevelled, a proposed upstream signaling component of the same pathway. These results strongly suggest that XGSK-3 beta functions to inhibit dorsoventral axis formation in the embryo and provide evidence for conservation of the Wnt signaling pathway in Drosophila and vertebrates.

Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell–collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd ≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α1(I)87–92, KGHRGF, with intermediate affinities (Kd ≈ 2 μM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd ≫ 10 μM). Thus, heparin–type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen–heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

Inhibition of advanced glycation endproduct formation by acetaldehyde: Role in the cardioprotective effect of ethanol

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called “French paradox,” (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation

Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86). In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining [V(D)J] recombination. Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination. It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints. The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation. An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation. To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice. We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation. Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant. These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids of Marsilea

During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while β-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea β-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither β-tubulin- nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita.

Biological impact on mineral dissolution: Application of the lichen model to understanding mineral weathering in the rhizosphere

Microorganisms modify rates and mechanisms of chemical and physical weathering and clay growth, thus playing fundamental roles in soil and sediment formation. Because processes in soils are inherently complex and difficult to study, we employ a model based on the lichen–mineral system to identify the fundamental interactions. Fixed carbon released by the photosynthetic symbiont stimulates growth of fungi and other microorganisms. These microorganisms directly or indirectly induce mineral disaggregation, hydration, dissolution, and secondary mineral formation. Model polysaccharides were used to investigate direct mediation of mineral surface reactions by extracellular polymers. Polysaccharides can suppress or enhance rates of chemical weathering by up to three orders of magnitude, depending on the pH, mineral surface structure and composition, and organic functional groups. Mg, Mn, Fe, Al, and Si are redistributed into clays that strongly adsorb ions. Microbes contribute to dissolution of insoluble secondary phosphates, possibly via release of organic acids. These reactions significantly impact soil fertility. Below fungi–mineral interfaces, mineral surfaces are exposed to dissolved metabolic byproducts. Through this indirect process, microorganisms can accelerate mineral dissolution, leading to enhanced porosity and permeability and colonization by microbial communities.

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.