The Telomere-Binding Protein Taz1p as a Target for Modification by a SUMO-1 Homologue in Fission Yeast

In fission yeast (Schizosaccharomyces pombe) the homologue of the mammalian SUMO-1 ubiquitin-like modifier is encoded by the pmt3 gene. A two-hybrid screen using the telomere-binding protein Taz1p as bait identified Pmt3p as an interacting factor. In vitro experiments using purified components of the fission yeast Pmt3p modification system demonstrated that Taz1p could be modified directly by Pmt3p. The amino acid sequence of Taz1p contains a close match to the consensus modification site for SUMO-1, and a PEST sequence similar to those found in established SUMO-1 targets. Although previous experiments have identified an increase in telomere length as one consequence of the pmt3– genotype, we could not detect Pmt3p modification of Taz1p in protein extracts made from exponentially growing haploid cells or any effect of Pmt3p on the localization of GFP-Taz1p at discrete foci in the haploid cell nucleus.     

To the 30-nm chromatin fiber and beyond

Chromatin fibers are intrinsically dynamic macromolecular complexes whose biological functions are intimately linked with their structure and interactions with chromatin-associated proteins (CAPs). Three-dimensional architectural transitions between or within the two co-existing chromatin types referred to as euchromatin and heterochromatin have been associated with activation or repression of nuclear functions. The presence of specific subsets of chromosomal proteins co-existing with the different chromatin conformations suggests a functional significance for their co-localization. The major points of emphasis of this review will assess the structure, function and recently documented exchanges amongst various members of the CAP family.     

Identifying anisotropic constraints in multiply labeled bacteriorhodopsin by 15N MAOSS NMR: a general approach to structural studies of membrane proteins

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically (15)N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the (15)N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results.     

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.     

Differentiation-induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2L dopamine receptor markedly potentiated both forskolin- and A2-adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D2L dopamine receptor-mediated inhibition of Galphas-stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.     

Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis

Despite their unparalleled catalytic prowess and environmental compatibility, enzymes have yet to see widespread application in synthetic chemistry. This lack of application and the resulting underuse of their enormous potential stems not only from a wariness about aqueous biological catalysis on the part of the typical synthetic chemist but also from limitations on enzyme applicability that arise from the high degree of substrate specificity possessed by most enzymes. This latter perceived limitation is being successfully challenged through rational protein engineering and directed evolution efforts to alter substrate specificity. However, such programs require considerable effort to establish. Here we report an alternative strategy for expanding the substrate specificity, and therefore the synthetic utility, of a given enzyme through a process of "substrate engineering". The attachment of a readily removable functional group to an alternative glycosyltransferase substrate induces a productive binding mode, facilitating rational control of substrate specificity and regioselectivity using wild-type enzymes.     

Sumoylation of PCNA: Wrestling with recombination at stalled replication forks

Post-replication repair encompassses error-prone and error-free processes for bypassing lesions encountered during DNA replication. In Saccharomyces cerevisiae, proteins acting in the Rad6-dependent pathway are required to channel lesions into these pathways. Until recently there was little information as to how this channelling was regulated. However, several recent papers, and in particular from the Jentsch and Ulrich groups have provided striking insights into the role of modified forms of PCNA in these events [C. Hoege, B. Pfander, G.L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO, Nature 419 (2002) 135-141; P. Stelter, H.D. Ulrich, Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation, Nature 425 (2003) 188-191; B. Pfander, G.L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase, Nature 436 (2005) 428-433; E. Papouli, S. Chen, A.A. Davies, D. Huttner, L. Krejci, P. Sung, H.D. Ulrich, Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p, Mol. Cell. 19 (2005) 123-133]. In particular they have shown that mono-ubiquitinated PCNA directs translesion synthesis via DNA polymerases with low stringency, and that polyubiquitinated PCNA is associated with error-free avoidance of lesions. Recent data have shown that the role of small ubiquitin-like modifier (SUMO) modification of PCNA is not an event that occurs merely in the absence of ubiquitination, rather it serves to recruit Srs2 to replication forks in order to inhibit recombination. The implications of these findings for post-replication repair in S. cerevisiae and other eukaryotes are discussed.     

Assessing the trypanocidal potential of natural and semi-synthetic diketopiperazines from two deep water marine-derived fungi

Human African trypanosomiasis (HAT, commonly known as African sleeping sickness) is categorized as a neglected disease, as it afflicts >50,000 people annually in sub-saharan Africa, and there are few formal programs in the world focused on drug discovery approaches for this disease. In this study, we examined the crude extracts of two fungal strains (Aspergillus fumigatus and Nectria inventa) isolated from deep water sediment which provided >99% growth inhibition at 1 μg/mL of Trypanosoma brucei, the causative parasite of HAT. A collection of fifteen natural products was supplemented with six semi-synthetic derivatives and one commercially available compound. Twelve of the compounds, each containing a diketopiperazine core, showed excellent activity against T. brucei (IC₅₀ = 0.002–40 μM), with selectivity over mammalian cells as great as 20-fold. The trypanocidal diketopiperazines were also tested against two cysteine protease targets Rhodesain and TbCatB, where five compounds showed inhibition activity at concentrations less than 20 μM. A preliminary activity pattern is described and analyzed.     

A description of larval and early juvenile development in Paralomis spinosissima (Decapoda: Anomura: Paguroidea: Lithodidae) from South Georgia waters (Southern Ocean)

The early ontogenetic stages of Paralomis spinosissima Birstein and Vinogradow, 1972, are described in detail and illustrated, with notes on morphological variability observed. Larval and early juvenile development was described to the crab I instar reared under controlled conditions of temperature and food supply. The abbreviated larval development invariably passed through two zoeal stages and the benthic megalopa stage. The larval development was completed without food supply, and food Artemia nauplii were first given after moult to the crab-I stage. Simplification and retarded development of the mouthparts are discussed as a function of lecithotrophy of these larvae and based on morphology no facultative feeding mode is suggested. Lecithotrophy in the Southern Ocean Lithodidae is discussed to be an adaptation allowing independence from seasonal food availability at high latitudes.