Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

RNA editing in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanosoma brucei, direct the insertion and deletion of uridylate (U) residues. Analyses of gRNAs reveal three functional elements. The 5′-end of the gRNA contains the anchor, which is responsible for selection and binding to the pre-edited mRNA. The second element (the guiding region) provides the information required for editing. At the 3′-end of the gRNA is a non-encoded U-tail, whose function remains unclear. However, the cleavage–ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of editing sites, thereby strengthening the interaction and holding onto the 5′ cleavage product. Our previous studies demonstrated that the U-tail interacts with upstream sequences and may play roles in both stabilization and tethering. These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA–U-tail interaction as editing proceeds in the 3′→5′ direction. We examined gCYb-558 and its U-tail interaction with 5′CYbUT and two partially edited 5′CYb substrates. Our results indicate that the 3′-end of the U-tail interacts with the same sequence in all three mRNAs. Predicted secondary structures using crosslinking data suggest that a similar structure is maintained as editing proceeds. These results indicate that the role of the U-tail may also involve maintenance of important secondary structure motifs.

The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Tissue spreading on implantable substrates is a competitive outcome of cell–cell vs. cell–substratum adhesivity

While the interactions of cells with polymeric substrata are widely studied, the influence of cell–cell cohesivity on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or “tug-of-war” between cell–cell and cell–substratum adhesions. We have generated both a “library” of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesivity. Cell–substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell–cell cohesivity was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell–cell cohesivity on a series of copolymeric substrata having different levels of cell–substratum adhesivity. In these experiments, cell–cell cohesivity was measured by tissue surface tensiometry, and cell–substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell–cell cohesivity dramatically slowed the spreading rate of cell aggregates.

Translocation pathway of protein substrates in ClpAP protease

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPγS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through ≈150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

Using electroactive substrates to pattern the attachment of two different cell populations

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.

Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

Heterocyclic carbene–metal-catalyzed Csp2–Csp2 and Csp–Csp2 couplings using nonmetallic substrates

The use of carbene ligands for transition-metal complexes has been developed in the last decades, being of special interest those carbenes derived from a nitrogen-containing heterocyclic system. An interesting variety of carbene-metal complexes has been tested in the Mizoroki-Heck reaction. In comparison, few examples can be found for the Matsuda-Heck version of this coupling reaction. Additionally, the Sonogashira coupling has been also catalyzed with different carbene-metal catalysts.

Effects of atmospheric deposition on painted substrates : cooperative research and development agreement with Rohm & Haas.

Mode of access: Internet.

Sequential purification and crystal growth for the production of low cost silicon substrates : quarterly technical progress report no. 1, September 15-December 31, 1979 /

"February 1980."