The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

Cells preconditioned with mild,transient UVA irradiation acquire resistance to oxidative stress and UVA-induced apoptosis: role of 4-hydroxynonenal in UVA-mediated signaling for apoptosis

Because 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213-41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis.     

Exploring CYP1A1 as anticancer target: homology modeling and in silico inhibitor design

Microsomal cytochrome P450 family 1 enzymes has great importance in the bioactivation of mutagens. P450 catalyzed reactions involve a wide range of substrates, and this versatality is reflected in a structural diversity, evident in the active sites of available P450 structures. This structure offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. In this paper, we document a homology model of CYP P450 1A1 from Homo sapiens, developed on the basis of template crystal structure of human microsomal P450 1a2 in complex with inhibitor (PDB Id: 2HI4). Homology modeling is performed at the programs, both in the commercial and public realms. We tried to explore CYP1A1 as a potential target for anticancer chemotherapy. To gain an insight into the binding of ligands with enzyme, protein-ligand complex was developed by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. Active site characterization and the study for involvement of specific aminoacids in binding with ligand, facilitates structure based inhibitor design. This study should prove useful in the design and development of potential novel anticancer agents. Figure The refined structure of homology model of CYP1A1     

Characterization of the adaptive response and growth upon hyperosmotic shock in Saccharomyces cerevisiae

Molecular and physiological details of osmoadaptation in yeast Saccharomyces cerevisiae are well characterized. It is well known that a cell, upon osmotic shock, delays its growth, produces a compatible solute like glycerol in yeast to maintain the osmotic equilibrium. Many genes are regulated by the hyperosmolarity glycerol (HOG) singling pathway, some of which in turn control the carbon flux in the glycolytic pathway for glycerol synthesis and reduced growth. The whole process of survival of cells under hyperosmotic stress is controlled at multiple levels in signaling and metabolic pathways. To better understand the multi-level regulations in yeast to osmotic shock, a mathematical model is formulated which integrates the growth and the osmoadaptation process. The model included the HOG pathway which consists of Sho1 and Sln1 signaling branches, gene regulation, metabolism and cell growth on glucose and ethanol. Experiments were performed to characterize the effect of various concentrations of salt on the wild-type and mutant strains. The model was able to successfully predict the experimental observations for both the wild-type and mutant strains. Further, the model was used to analyze the effects of various regulatory mechanisms prevalent in the signaling and metabolic pathways which are essential in achieving optimum growth in a saline medium. The analysis demonstrated the relevance of the combined effects of regulation at several points in the signaling and metabolic pathways including activation of GPD1 and GPD2, inhibition of PYK and PDC1, closure of the Fps1 channel, volume effect on the glucose uptake rate, downregulation of ethanol synthesis and upregulation of ALD6 for acetate synthesis. The analysis demonstrated that these combined effects orchestrated the phenomena of adaptation to osmotic stress in yeast.     

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

The GAL regulatory system is highly conserved in yeast species of Saccharomyces cerevisiae and Kluyveromyces lactis. While the GAL system is a well studied system in S. cerevisiae, the dynamic behavior of the KlGAL system in K. lactis has not been characterized. Here, we have characterized the GAL system in yeast K. lactis by developing a dynamic model and comparing its performance to its not-so-distant cousin S. cerevisiae. The present analysis demonstrates the significance of the autoregulatory feedbacks due to KlGal4p, KlGal80p, KlGal1p and Lac12p on the dynamic performance of the KlGAL switch. The model predicts the experimentally observed absence of bistability in the wild type strain of K. lactis, unlike the short term memory of preculturing conditions observed in S. cerevisiae. The performance of the GAL switch is distinct for the two yeast species although they share similarities in the molecular components. The analysis suggests that the whole genome duplication of S. cerevisiae, which resulted in a dedicated inducer protein, Gal3p, may be responsible for the high sensitivity of the system to galactose concentrations. On the other hand, K. lactis uses a bifunctional protein as an inducer in addition to its galactokinase activity, which restricts its regulatory role and hence higher galactose levels in the medium are needed to trigger the GAL system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9082-7) contains supplementary material, which is available to authorized users.     

Mapping DNA-binding domains of the autoimmune regulator protein

The human autoimmune regulator (AIRE) gene encodes a putative DNA-binding protein, which is mutated in patients affected by the autoimmune polyglandular syndrome type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We have recently reported that AIRE can bind to two different DNA sequence motifs, suggesting the existence of at least two DNA-binding domains in the AIRE protein. By expressing a series of recombinant AIRE protein fragments, we demonstrate here that the two well-known plant homeodomains (PHD) domains in AIRE can bind to the ATTGGTTA sequence motif. The first ATTGGTTA-binding domain is mapped to amino acids 299-355 and the second ATTGGTTA-binding domain to amino acids 434-475. Furthermore, the SAND domain of AIRE is shown to bind to TTATTA motif. Results presented herein show that the residues at position 189-196 of AIRE (QRAVAMSS) are required for its binding to the TTATTA motif. The required sequence for DNA binding in the SAND domain of AIRE is remarkably different from other SAND-containing proteins such as Sp-100b and NUDR. Data presented in this paper indicate that the two PHD domains contained in AIRE, in addition to the SAND domain, can bind to specific DNA sequence motifs.     

Na+ and K+ transport in Nitrosomonas europaea and Nitrobacter agilis

Potassium-depleted cells of Nitrosomonas europaea and Nitrobacter agilis were prepared by diethanolamine treatment and contained less than 5 mM intracellular K⁺. The addition of K⁺ to K⁺-depleted cells of N. europaea and N. agilis resulted in a depolarization of membrane potential (ΔΨ) by about 5 and 10 mV, respectively. This depolarization was, however, compensated by an equivalent increase in transmembrane pH gradient (ΔpH), so that the total proton-motive force (Δp) remained constant, indicating that K⁺ transport was electrogenic in both bacteria. Using ²²Na⁺-loaded cells, it is shown that both bacteria lack a respiration-dependent Na⁺ pump; however, antiporters for Na⁺/H⁺, K⁺/Na⁺ and K⁺/H⁺ were detected. Of these, at least the K⁺/Na⁺ antiporter required an electrochemical gradient for its operation. It is also shown that the unprotonated form of NH₄⁺ is transported into these bacteria by a simple diffusion mechanism.     

Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate‐conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnF‐UBP domain in tandem with a linkage‐selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48‐linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48‐linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t‐UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48‐linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain‐selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the ‘ubiquitome’. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 ( http://proteomecentral.proteomexchange.org/dataset/PXD004059 ).     

Peptide vaccines of the HER-2/neu dimerization loop are effective in inhibiting mammary tumor growth in vivo

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20-30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266-333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266-296, 298-333, and 315-333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266-296 constructs being equally effective as compared with Herceptin. The 266-296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF(+/-)Neu2-5(+/-)). The 266-296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.