P-selectin,and not E-selectin,negatively regulates murine megakaryocytopoiesis

To assess the role of P-selectin and E-selectin in megakaryocytopoiesis, in vitro assays were performed in animal models deficient in both adhesion receptors. There was a significantly greater number of IL-3-responsive megakaryocyte progenitors CFU (CFU-MK) and an increase in immature megakaryoblasts in response to IL-6 in the P-selectin-null mice compared with the wild-type controls. Furthermore, P-selectin-null mice showed a greater number of CFU-MK colonies derived from CD34(+) cells in response to IL-3 or IL-3 plus stem cell factor. A significant shift in baseline ploidy with a reduction in 8N cells and an increase in 32N cells was also observed in the P-selectin-null mice. Secretion of the inhibitory growth factor TGF-beta1 and not TGF-beta2 was significantly lower in the supernatants of cultures containing bone marrow cells from P-selectin-deficient mice as compared with those from the wild-type control bone marrow cells. No differences in the responsiveness of murine CFU-MK, immature megakaryocytes, or 5-fluorouracil-selected stem cells to cytokines were observed in E-selectin-null mice as compared with the control mice. These studies indicate that the absence of P-selectin, and not E-selectin, resulted in an altered adhesion environment with subsequent expansion of megakaryocyte progenitors and immature megakaryoblasts, enhanced secretion of TGF-beta1, and apparent increased responsiveness to inflammatory cytokines.     

A novel scoring function for predicting the conformations of tightly packed pairs of transmembrane alpha-helices

Pairs of helices in transmembrane (TM) proteins are often tightly packed. We present a scoring function and a computational methodology for predicting the tertiary fold of a pair of alpha-helices such that its chances of being tightly packed are maximized. Since the number of TM protein structures solved to date is small, it seems unlikely that a reliable scoring function derived statistically from the known set of TM protein structures will be available in the near future. We therefore constructed a scoring function based on the qualitative insights gained in the past two decades from the solved structures of TM and soluble proteins. In brief, we reward the formation of contacts between small amino acid residues such as Gly, Cys, and Ser, that are known to promote dimerization of helices, and penalize the burial of large amino acid residues such as Arg and Trp. As a case study, we show that our method predicts the native structure of the TM homodimer glycophorin A (GpA) to be, in essence, at the global score optimum. In addition, by correlating our results with empirical point mutations on this homodimer, we demonstrate that our method can be a helpful adjunct to mutation analysis. We present a data set of canonical alpha-helices from the solved structures of TM proteins and provide a set of programs for analyzing it (http://ashtoret.tau.ac.il/~sarel). From this data set we derived 11 helix pairs, and conducted searches around their native states as a further test of our method. Approximately 73% of our predictions showed a reasonable fit (RMS deviation <2A) with the native structures compared to the success rate of 8% expected by chance. The search method we employ is less effective for helix pairs that are connected via short loops (<20 amino acid residues), indicating that short loops may play an important role in determining the conformation of alpha-helices in TM proteins.     

Synthesis and biological evaluation of pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane derivatives as potential therapeutic agents in Parkinson's disease

In previous studies, the polycyclic cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane (NGP1-01) and a number of its derivatives showed positive effects in neuroprotection studies with MPTP, in vivo. In view of these findings, we examined these compounds for their effects on [(3)H]dopamine ([(3)H]DA) release and uptake inhibition in murine striatal synaptosomes, as well as for inhibition of baboon liver monoamine oxidase (MAO) B. In order to assess specificity, initial experiments focused on compounds that blocked dopamine uptake without causing appreciable release (<40% at 100 microM) of the transmitter. NGP1-01 blocked the uptake of [(3)H]DA with an IC(50) of 57 microM, while another compound, 8-phenylethyl-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane, blocked uptake at an IC(50) value of 23 microM. These values were comparable to that of another polycyclic cage amine, amantadine (IC(50); 82 micro), that is used in parkinsonian therapy. Structure-activity relationships of this series of compounds support the importance of geometric and steric, rather than electronic effects, in determining biological activity. MAO-B inhibition for this group was weak, with less than 50% inhibition at 300 microM for any of the compounds in the series. The present study suggests that blockage of the dopamine transporter may underlie, at least in part, their neuroprotective effects against MPTP-induced parkinsonism. These compounds may be considered as potential lead compounds for Parkinson's Disease therapy.     

Class-specific correlations between protein folding rate, structure-derived, and sequence-derived descriptors

Small single-domain proteins that fold by simple two-state kinetics have been shown to exhibit a wide variation in their folding rates. It has been proposed that folding mechanisms in these proteins are largely determined by the native-state topology, and a significant correlation between folding rate and measures of the average topological complexity, such as relative contact order (RCO), has been reported. We perform a statistical analysis of folding rate and RCO in all three major structural classes (alpha, beta, and alpha/beta) of small two-state proteins and of RCO in groups of analogous and homologous small single-domain proteins with the same topology. We also study correlation between folding rate and the average physicochemical properties of amino acid sequences in two-state proteins. Our results indicate that 1) helical proteins have statistically distinguishable, class-specific folding rates; 2) RCO accounts for essentially all the variation of folding rate in helical proteins, but for only a part of the variation in beta-sheet-containing proteins; and 3) only a small fraction of the protein topologies studied show a topology-specific RCO. We also report a highly significant correlation between the folding rate and average intrinsic structural propensities of protein sequences. These results suggest that intrinsic structural propensities may be an important determinant of the rate of folding in small two-state proteins.     

CDC91L1 (PIG-U) is a newly discovered oncogene in human bladder cancer

Genomic amplification at 20q11-13 is a common event in human cancers. We isolated a germline translocation breakpoint at 20q11 from a bladder cancer patient. We identified CDC91L1, the gene encoding CDC91L1 (also called phosphatidylinositol glycan class U (PIG-U), a transamidase complex unit in the glycosylphosphatidylinositol (GPI) anchoring pathway), as the only gene whose expression was affected by the translocation. CDC91L1 was amplified and overexpressed in about one-third of bladder cancer cell lines and primary tumors, as well as in oncogenic uroepithelial cells transformed with human papillomavirus (HPV) E7. Forced overexpression of CDC91L1 malignantly transformed NIH3T3 cells in vitro and in vivo. Overexpression of CDC91L1 also resulted in upregulation of the urokinase receptor (uPAR), a GPI-anchored protein, and in turn increased STAT-3 phosphorylation in bladder cancer cells. Our findings suggest that CDC91L1 is an oncogene in bladder cancer, and implicate the GPI anchoring system as a potential oncogenic pathway and therapeutic target in human cancers.     

A Calpha model for the transmembrane alpha helices of gap junction intercellular channels

Gap junction channels connect the cytoplasms of apposed cells via an intercellular conduit formed by the end-to-end docking of two hexameric hemichannels called connexons. We used electron cryomicroscopy to derive a three-dimensional density map at 5.7 angstroms in-plane and 19.8 angstroms vertical resolution, allowing us to identify the positions and tilt angles for the 24 alpha helices within each hemichannel. The four hydrophobic segments in connexin sequences were assigned to the alpha helices in the map based on biochemical and phylogenetic data. Analyses of evolutionary conservation and compensatory mutations in connexin evolution identified the packing interfaces between the helices. The final model, which specifies the coordinates of Calpha atoms in the transmembrane domain, provides a structural basis for understanding the different physiological effects of almost 30 mutations and polymorphisms in terms of structural deformations at the interfaces between helices, revealing an intimate connection between molecular structure and disease.     

AbDesign: An algorithm for combinatorial backbone design guided by natural conformations and sequences

Computational design of protein function has made substantial progress, generating new enzymes, binders, inhibitors, and nanomaterials not previously seen in nature. However, the ability to design new protein backbones for function—essential to exert control over all polypeptide degrees of freedom—remains a critical challenge. Most previous attempts to design new backbones computed the mainchain from scratch. Here, instead, we describe a combinatorial backbone and sequence optimization algorithm called AbDesign, which leverages the large number of sequences and experimentally determined molecular structures of antibodies to construct new antibody models, dock them against target surfaces and optimize their sequence and backbone conformation for high stability and binding affinity. We used the algorithm to produce antibody designs that target the same molecular surfaces as nine natural, high‐affinity antibodies; in five cases interface sequence identity is above 30%, and in four of those the backbone conformation at the core of the antibody binding surface is within 1 Å root‐mean square deviation from the natural antibodies. Designs recapitulate polar interaction networks observed in natural complexes, and amino acid sidechain rigidity at the designed binding surface, which is likely important for affinity and specificity, is high compared to previous design studies. In designed anti‐lysozyme antibodies, complementarity‐determining regions (CDRs) at the periphery of the interface, such as L1 and H2, show greater backbone conformation diversity than the CDRs at the core of the interface, and increase the binding surface area compared to the natural antibody, potentially enhancing affinity and specificity. Proteins 2015; 83:1385–1406. © 2015 Wiley Periodicals, Inc.