Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition

Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction. Dug1p exists as dimer and the stoichiometry of peptide-Dug1p complex is 2:1 indicating each monomer in the dimer binds to one peptide. Thermodynamic studies indicate that the free energy change for Dug1p-peptide complex formation is similar (?G_bind ∼ −7.0 kcal/mol) for a variety of peptides of different composition and length (22 peptides). Three-dimensional model of Dug1p is constructed and docking of peptides to the modeled structure suggests that hydrogen bonding to active site residues (E172, E171, and D137) lock the N-terminal of the peptide into the binding site. Dug1p recognizes peptides in a metal independent manner and peptide binding is not sensitive to salts (dlogK/dlog[salt] ∼ 0) over a range of [NaCl] (0.02-0.5 M), [ZnCl₂], and [MnCl₂] (0-0.5 mM). Our results indicate that promiscuity in peptide binding results from the locking of peptide N-terminus into the active site. These observations were supported by our competitive inhibition activity assays. Dug1p activity towards Cys-Gly peptide is significantly reduced (∼ 70%) in the presence of Glu-Cys-Gly. Therefore, Dug1p can recognize a variety of oligopeptides, but has evolved with post-binding screening potential to hydrolyze Cys-Gly peptides selectively.     

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Egg chambers from apterous⁴ (ap⁴), a female sterile mutant of Drosophila melanogaster, show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oöcyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oöcytes of ap⁴ flies. Within 12 hr after topical application of ZR515 to homozygous ap⁴ females the oöcyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oöcyte also shows some changes. In vitro studies in which ap⁴ ovaries were incubated in Schneider's Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oöcyte membrane.     

Extraction of Protein Interaction Data: A Comparative Analysis of Methods in Use

Several natural language processing tools, both commercial and freely available, are used to extract protein interactions from publications. Methods used by these tools include pattern matching to dynamic programming with individual recall and precision rates. A methodical survey of these tools, keeping in mind the minimum interaction information a researcher would need, in comparison to manual analysis has not been carried out. We compared data generated using some of the selected NLP tools with manually curated protein interaction data (PathArt and IMaps) to comparatively determine the recall and precision rate. The rates were found to be lower than the published scores when a normalized definition for interaction is considered. Each data point captured wrongly or not picked up by the tool was analyzed. Our evaluation brings forth critical failures of NLP tools and provides pointers for the development of an ideal NLP tool.     

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.     

Identification of function for CD44 intracytoplasmic domain (CD44-ICD): modulation of matrix metalloproteinase 9 (MMP-9) transcription via novel promoter response element

CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.