Sanjay S. Khandekar

Temperature-sensitive Differential Affinity of TRAIL for Its Receptors DR5 IS THE HIGHEST AFFINITY RECEPTOR

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 “decoy” receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the “decoy” receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 °C, their rank-ordered affinities are substantially different at 37 °C, with DR5 having the highest affinity (K D ≤ 2 nm) and OPG having the weakest (K D = 400 nm). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.

Reciprocal Expression of the TNF Family Receptor Herpes Virus Entry Mediator and Its Ligand LIGHT on Activated T Cells: LIGHT Down-Regulates Its Own Receptor

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8+ than CD4+ T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.

Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis.

β-Ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys112 proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His244 and Asn274. The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.

Mechanism of De Novo Initiation by the Hepatitis C Virus RNA-Dependent RNA Polymerase: Role of Divalent Metals

ABSTRACT We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg2+ and Mn2+ gave rise to significant amounts of synthesis. Mg2+ and Mn2+ affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn2+ significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn2+ reduced the Km for the initiation nucleotide, a GTP, from 103 to 3 μM. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn2+-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.

Potent, Selective and Orally Bioavailable Dihydropyrimidine Inhibitors of Rho Kinase (ROCK1) as Potential Therapeutic Agents for Cardiovascular Diseases

Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.

Bacterial β-ketoacyl-Acyl Carrier Protein Synthases as Targets for Antibacterial Agents

As a result of increasing drug resistance in pathogenic bacteria, there is a critical need for novel broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria is an essential process for cell survival, the enzymes involved in the FAS pathway have emerged as promising targets for antimicrobial agents. Several lines of evidence have indicated that bacterial condensing enzymes are central to the initiation and elongation steps in bacterial fatty acid synthesis and play a pivotal role in the regulation of the entire fatty acid synthesis pathway. beta-ketoacyl-acyl carrier protein (ACP) synthases (KAS) from various bacterial species have been cloned, expressed and purified in large quantities for detailed enzymological, structural and screening studies. Availability of purified KAS from a variety of bacteria, along with a combination of techniques, including combinatorial chemistry, high-throughput screening, and rational drug design based on crystal structures, will undoubtedly aid in the discovery and development of much needed potent and broad-spectrum antibacterial agents. In this review we summarize the biochemical, biophysical and inhibition properties of beta-ketoacyl-ACP synthases from a variety of bacterial species.

Affinity and kinetics of the interactions between an αβ T-cell receptor and its superantigen and class II-MHC/peptide ligands

Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alphabeta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Valpha2, Vbeta8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69+/-0.12 x 10(4)M(-1) sec(-1) and a dissociation rate of 1.9+/-0.47 x 10(-2) sec(-1), giving a dissociation constant of 1.1 microM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I A(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07+/-0.19 x 10(4)M(-1)sec(-1) and a dissociation rate of 2.2+/-0.65 x 10(-2) sec(-1), giving a dissociation constant of 2.1 microM.

Conformational Integrity and Ligand Binding Properties of a Single Chain T-cell Receptor Expressed in Escherichia coli

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Vα2Cα, Vβ8.2Cβ) expressed in insect cells binds both Vβ8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak·conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Vα and Vβ domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak·conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- orO-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mm. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by β-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.