Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies

The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.     

Hsp70/Hsc70 regulates the effect phosphorylation has on stabilizing ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an inherited neurodegenerative disorder. The mutation causing SCA1 is an expansion in the polyglutamine tract of the ATXN1 protein. Previous work demonstrated that phosphorylation of mutant ATXN1 at serine 776 (S776), a putative Akt phosphorylation site, is critical for pathogenesis. To examine this pathway further, we utilized a cell-transfection system that allowed the targeting of Akt to either the cytoplasm or the nucleus. In contrast to HeLa cells, we found that Akt targeted to the cytoplasm increased the degradation of ATXN1 in Chinese hamster ovary cells. However, Akt targeted to the cytoplasm failed to destabilize ATXN1 if Hsp70/Hsc70 was present. Thus, Hsp70/Hsc70 can regulate ATXN1 levels in concert with phosphorylation of ATXN1 at S776.     

Increased expression of glial cell line-derived neurotrophic factor protects against oxidative damage-induced retinal degeneration

Oxidative damage contributes to retinal cell death in patients with age-related macular degeneration or retinitis pigmentosa. One approach to treatment is to identify and eliminate the sources of oxidative damage. Another approach is to identify treatments that protect cells from multiple sources of oxidative damage. In this study, we investigated the effect of increased expression of glial cell line-derived neurotrophic factor (GDNF) in three models of oxidative damage-induced retinal degeneration. Double transgenic mice with doxycycline-inducible expression of GDNF in the retina were exposed to paraquat, FeSO(4), or hyperoxia, all sources of oxidative damage and retinal cell death. Compared to controls, mice with increased expression of GDNF in the retina showed significant preservation of retinal function measured by electroretinograms, reduced thinning of retinal cell layers, and fewer TUNEL-positive cells indicating less retinal cell death. Mice over-expressing GDNF also showed less staining for acrolein, nitrotyrosine, and 8-hydroxydeoxyguanosine, indicating less oxidative damage to lipids, proteins, and DNA. This suggests that GDNF did not act solely to allow cells to tolerate higher levels of oxidative damage before initiation of apoptosis, but also reduced damage from oxidative stress to critical macromolecules. These data suggest that gene transfer of Gdnf should be considered as a component of therapy for retinal degenerations in which oxidative damage plays a role.     

Addition of a non-photic component to a light-based mathematical model of the human circadian pacemaker

Mathematical models have become vital to the study of many biological processes in humans due to the complexity of the physiological mechanisms underlying these processes and systems. While our current mathematical representation of the human circadian pacemaker has proven useful in many experimental situations, it uses as input only a direct effect of light on the circadian pacemaker. Although light (a photic stimulus) has been shown to be the primary synchronizer of the circadian pacemaker across a number of species, studies in both animals and humans have confirmed the existence of non-photic effects that also contribute to phase shifting and entrainment. We modified our light-based circadian mathematical model to reflect evidence from these studies that the sleep-wake cycle and/or associated behaviors have a non-photic effect on the circadian pacemaker. In our representation, the sleep-wake cycle and its associated behaviors provides a non-photic drive on the circadian pacemaker that acts both independently and concomitantly with light stimuli. Further experiments are required to validate fully our model and to understand the exact effect of the sleep-wake cycle as a non-photic stimulus for the human circadian pacemaker.     

Enterovirus 70 receptor utilization is controlled by capsid residues that also regulate host range and cytopathogenicity

Enterovirus type 70, an etiologic agent of acute hemorrhagic conjunctivitis, may bind different cellular receptors depending on cell type. To understand how EV70-receptor interaction is controlled, we studied two variants of the virus with distinct receptor utilization. EV70-Rmk, derived by passage in rhesus monkey kidney cells, replicates poorly in HeLa cells and does not cause cytopathic effects. Decay accelerating factor (DAF) is not a cell receptor for EV70-Rmk. Passage of EV70-Rmk in HeLa cells lead to isolation of EV70-Dne, which does not replicate in rhesus monkey kidney cells but grows to high titers in HeLa cells and causes cytopathic effects. DAF is sufficient for cell entry of EV70-Dne. EV70-Rmk replicates in human eye and brain-derived cell lines, whereas the Dne strain replicates only in HeLa cells and in conjunctiva-derived 15C4 cells. The two EV70 strains differ by five amino acid changes in the viral capsid. Single substitution of four of the five EV70-Rmk amino acids with the residue from EV70-Dne leads to lytic replication in HeLa cells. Conversely, substitution of any of the five EV70-Dne amino acids with the EV70-Rmk amino acid does not alter replication in HeLa cells. Three of these capsid amino acids are predicted to be located in the canyon encircling the fivefold axis of symmetry, one amino acid is found at the fivefold axis of symmetry, and one is located the interior of the capsid. The five EV70 residues define a region of the capsid that controls viral host range, DAF utilization, and cytopathogenicity.     

The x-ray structure of dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase

The repeating unit of the glycan chain in the S-layer of the bacterium Aneurinibacillus thermoaerophilus L420-91(T) is composed of four alpha-d-rhamnose molecules and two 3-acetamido-3,6-dideoxy-alpha-d-galactose moieties (abbreviated as Fucp3NAc). Formation of the glycan layer requires nucleotide-activated sugars as the donor molecules. Whereas the enzymes involved in the synthesis of GDP-rhamnose have been well characterized, less is known regarding the structures and enzymatic mechanisms of the enzymes required for the production of dTDP-Fucp3NAc. One of the enzymes involved in the biosynthesis of dTDP-Fucp3NAc is a 3,4-ketoisomerase, hereafter referred to as FdtA. Here we describe the first three-dimensional structure of this sugar isomerase complexed with dTDP and solved to 1.5 A resolution. The FdtA dimer assumes an almost jellyfish-like appearance with the sole alpha-helices representing the tentacles. Formation of the FdtA dimer represents a classical example of domain swapping whereby beta-strands 2 and 3 from one subunit form part of a beta-sheet in the second subunit. The active site architecture of FdtA is characterized by a cluster of three histidine residues, two of which, His(49) and His(51), appear to be strictly conserved in the amino acid sequences deposited to date. Site-directed mutagenesis experiments, enzymatic assays, and x-ray crystallographic analyses suggest that His(49) functions as an active site base.     

Pro-apoptotic activity of novel Isatin-Schiff base copper(II) complexes depends on oxidative stress induction and organelle-selective damage

We characterized the pro-apoptotic activity of two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine: (Cu(isapn)) and (Cu(isaepy)(2)), respectively. We demonstrated that these compounds trigger apoptosis via the mitochondrial pathway. The early induction of the p53/p21 system indicates a role for p53 in cell death, however, experiments carried out with small interfering RNA against p53, or with cells lacking p53, support that a p53-independent mechanism can also occur. The extent of apoptosis mirrors the kinetics of intracellular copper uptake. Particularly, Cu(isaepy)(2) enters the cells more efficiently and specifically damages nuclei and mitochondria, as evidenced by atomic absorption analysis of copper content and by the extent of nuclear and mitochondrial integrity. Conversely, Cu(isapn), although less permeable, induces a wide-spread oxidative stress, as demonstrated by analyses of reactive oxygen species concentration, and oxidation of proteins and lipids. The increase of the antioxidant defense, through the overexpression of Cu,Zn-SOD, partially counteracts cell death; whereas retinoic acid-mediated differentiation completely rescues cells from apoptosis induced by both compounds. The activation of JNK- and Akt-mediated phosphorylative pathways has been found to be not functional for apoptosis induction. On the contrary, apoptosis significantly decreased when the analogous zinc complex was used or when Cu(isaepy)(2) was incubated in the presence of a copper chelator. Altogether, our data provide evidence for a dual role of these copper(II) complexes: they are able to vehicle copper into the cell, thus producing reactive oxygen species, and could behave as delocalized lipophilic cation-like molecules, thus specifically targeting organelles.     

Structural basis of enantioselective inhibition of cyclooxygenase-1 by S-alpha-substituted indomethacin ethanolamides

The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of alpha-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the alpha-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85 A. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-alpha-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.     

p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway

Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75^NTR), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75^NTR to enhance cAMP degradation. The p75^NTR-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75^NTR-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75^NTR regulates degradation of cAMP and perpetuates scar formation after injury.