A comparative study of metabolic engineering anti-metabolite tolerance in Escherichia coli

A problem in strain engineering is that mutations that benefit the expression of a phenotype in one environment may impose a cost to biological fitness in a new environment. The overall objective of this study was to improve understanding of this phenomenon within the context of a classic anti-metabolite selection strategy. We have engineered Escherichia coli using three mutagenesis techniques (chemical mutagenesis, insertional mutagenesis, and plasmid-based overexpression) and assessed the relative costs and benefits to biological fitness of mutants selected for tolerance to five amino acid analogs whose target amino acids (glutamatic acid, aspartic acid, tryptophan, glycine, and serine) differ in metabolic connectivity and biosynthetic energy requirements. Our major findings include (i) the fold increase in anti-metabolite tolerance, independent of mutagenesis strategy, was much greater for aspartic acid beta-hydroxamate (AAH) compared to all other tested hydroxamates, (ii) increased tolerance to glutamic acid gamma-hydroxamate (GAH) was not achieved using any of the mutagenesis strategies, and (iii) characteristics of the anti-metabolite, rather than those of the corresponding metabolite, were more important in determining the ability to increase tolerance.     

Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.     

Discovery of potent,selective, and orally bioavailable 3H-spiro[isobenzofuran-1,4′-piperidine] based melanocortin subtype-4 receptor agonists

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4′-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.     

Multifocal signal modulation therapy of cancer: ancient weapon, modern targets

Although safe in most cases, ancient treatments are ignored because neither their active components nor their molecular targets are well defined. This is not the case, however, with curcumin, a yellow-pigment substance and component of turmeric (Curcuma longa), which was identified more than a century ago. Recently, extensive research has addressed the chemotherapeutic potential of this relatively nontoxic-plant-derived polyphenol. Because most cancers are caused by deregulation of as many as 500 different genes, agents that target multiple gene products are needed for prevention and treatment of cancer. In this regard, curcumin has been reported to have immense potentiality for being used in cancer chemotherapy because of its control over the machineries of cell survival, proliferation, invasion, and angiogenesis. The mechanisms implicated are diverse and appear to involve a combination of cell signaling pathways at multiple levels. This review seeks to summarize the unique multifocal signal modulatory properties of the “ancient weapon,” curcumin, which may be exploited for successful clinical cancer prevention.     

Characterization of the Yeast Telomere Nucleoprotein Core: Rap1 BINDS INDEPENDENTLY TO EACH RECOGNITION SITE

At the core of Saccharomyces cerevisiae telomeres is an array of tandem telomeric DNA repeats bound site-specifically by multiple Rap1 molecules. There, Rap1 orchestrates the binding of additional telomere-associated proteins and negatively regulates both telomere fusion and length homeostasis. Using electron microscopy, viscosity, and light scattering measurements, we show that purified Rap1 is a monomer in solution that adopts a ringlike or C shape with a central cavity. Rap1 could orchestrate telomere function by binding multiple telomere array sites through either cooperative or independent mechanisms. To determine the mechanism, we analyze the distribution of Rap1 monomers on defined telomeric DNA arrays. This analysis clearly indicates that Rap1 binds independently to each nonoverlapping site in an array, regardless of the spacing between sites, the total number of sites, the affinity of the sites for Rap1, and over a large concentration range. Previous experiments have not clearly separated the effects of affinity from repeat spacing on telomere function. We clarify these results by testing in vivo the function of defined telomere arrays containing the same Rap1 binding site separated by spacings that were previously defined as low or high activity. We find that Rap1 binding affinity in vitro correlates with the ability of telomeric repeat arrays to regulate telomere length in vivo. We suggest that Rap1 binding to multiple sites in a telomere array does not, by itself, promote formation of a more energetically stabile complex.     

Ancient and recent adaptive evolution of primate non-homologous end joining genes

In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes.     

Functional characterization of the chicken peptide transporter 1 (pept1, slc15a1) gene

Dietary amino acids can be transported into intestinal epithelial cells as di- and tripeptides by the action of the peptide transporter, PepT1 (SLC15A1). Expression of the chicken PepT1 (cPepT1) gene changes in response to dietary crude protein level; however, the molecular mechanism governing this regulation is unknown. This study analyzed the promoter region of the cPepT1 gene. Using deletion analysis, positive-acting (-314 to -261, -169 to -155, and -120 to -60) and negative-acting (-419 to -386 and -214 to -169) regions were mapped in transfected chick embryo fibroblasts (CEF). The addition of neither amino acids Phe, Arg, or Val, nor the dipeptides Gly-Sar (glycyl-sarcosine), Gly-Pro, Gly-Phe, Met-Pro, Met-Lys or Lys-Lys, had an effect on cPepT1 promoter activity in transfected CEF. The cPepT1 promoter was more active in CEF and primary chicken intestinal cells than in chicken liver cells. This study represents a functional characterization of the molecular regulation of the chicken PepT1 gene.