Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.     

Extended disordered proteins: targeting function with less scaffold

It has been estimated that a large fraction of cellular proteins are natively disordered. Current opinion largely holds that natively disordered proteins are more 'adaptive', leading to advantages in regulation and in binding diverse ligands. Here, we argue for another, simple, physically based reason. Disordered proteins often have large intermolecular interfaces, the size of which is dictated by protein function. For proteins to be stable as monomers with extensive interfaces, protein size would need to be 2-3 times larger. This would either increase cellular crowding or enlarge the size of the cell by 15-30%, owing to the increase in the sequence length. Smaller sizes of cells, proteins, DNA and RNA conserve energy. Thus, disordered proteins provide a simple yet elegant solution to having large intermolecular interfaces, but with smaller protein, genome and cell sizes.     

8-quinolinamines and their pro prodrug conjugates as potent blood-schizontocidal antimalarial agents

Synthesis and antimalarial activities of N8-(4-amino-1-methylbutyl)-5-alkoxy-4-ethyl-6-methoxy-8-quinolinamines (5) and their pro prodrug analogues (6-7) prepared by covalently linking 5 to the redox-sensitive (8) and esterase-sensitive (9) linkers through the amide linkage are reported. The most effective 8-quinolinamines [5c (R=C5H11) and 5f (R=C8H17)] have exhibited in vitro and in vivo biological efficacy superior to that of the standard drug chloroquine against both drug-sensitive and drug-resistant malaria strains. Analogues 6-7 were evaluated for in vivo blood-schizontocidal activity as potential pro prodrug models for the primary amino group containing 8-quinolinamines (5). The most effective pro prodrug analogue (6c) has displayed promising activities against drug-sensitive and drug-resistant strains of Plasmodia in vivo.     

Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

Growth and yield functions for irrigated plantations of Eucalyptus camaldulensis in the hot desert of India

Eucalyptus camaldulensis forms a major portion of irrigated plantations in the hot desert of India and for effective management of these plantations, growth and yield functions are necessary. This paper describes the development of height, diameter and volume growth functions based on the analysis of the data collected from the permanent sample plots laid out in the study area. The Chapman-Richards equation could be used for the prediction of diameter growth while the Schumacher model could be considered for predicting volume growth. Some base-age variant and invariant site-index models, already reported in the literature, were compared in terms of relative accuracy and it was found that the Payandeh and Wang model performed the best among the four models tried.     

Cutting edge: activation of the p38 mitogen-activated protein kinase signaling pathway mediates cytokine-induced hemopoietic suppression in aplastic anemia

Myelosuppressive cytokines, in particular IFN-gamma and TNF-alpha, play an important role in the pathogenesis of idiopathic aplastic anemia in humans. It is unknown whether these negative regulators of hemopoiesis suppress stem cells by activating a common signaling cascade or via distinct nonoverlapping pathways. In this study, we provide evidence that a common element in signaling for IFN-gamma and TNF-alpha in human hemopoietic progenitors is the p38/MapKapK-2 signaling cascade. Our studies indicate that pharmacological inhibition of p38 reverses the suppressive effects of IFN-gamma and TNF-alpha on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 strongly enhances hemopoietic progenitor colony formation from aplastic anemia bone marrows in vitro. Thus, p38 appears to play a critical role in the pathogenesis of aplastic anemia, suggesting that selective pharmacological inhibitors of this kinase may prove useful in the treatment of aplastic anemia and other cytokine-mediated bone marrow failure syndromes.