HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells

The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells (BMECs). The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia despite the lack of productive HIV infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. In this study we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in vitro on primary human BMECs (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites compared with control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat Abs or with the specific inhibitor SU-1498, which interferes with vascular endothelial growth factor receptor type 2 (Flk-1/KDR) phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and was found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant interfering mutant form, FAK-related nonkinase, significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time-dependent manner. Tat also impaired BBB permeability, as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs via activation of FAK, leading to cytoskeletal reorganization and permeability changes.     

An evolutionarily conserved network of amino acids mediates gating in voltage-dependent potassium channels

A novel sequence-analysis technique for detecting correlated amino acid positions in intermediate-size protein families (50-100 sequences) was developed, and applied to study voltage-dependent gating of potassium channels. Most contemporary methods for detecting amino acid correlations within proteins use very large sets of data, typically comprising hundreds or thousands of evolutionarily related sequences, to overcome the relatively low signal-to-noise ratio in the analysis of co-variations between pairs of amino acid positions. Such methods are impractical for voltage-gated potassium (Kv) channels and for many other protein families that have not yet been sequenced to that extent. Here, we used a phylogenetic reconstruction of paralogous Kv channels to follow the evolutionary history of every pair of amino acid positions within this family, thus increasing detection accuracy of correlated amino acids relative to contemporary methods. In addition, we used a bootstrapping procedure to eliminate correlations that were statistically insignificant. These and other measures allowed us to increase the method's sensitivity, and opened the way to reliable identification of correlated positions even in intermediate-size protein families. Principal-component analysis applied to the set of correlated amino acid positions in Kv channels detected a network of inter-correlated residues, a large fraction of which were identified as gating-sensitive upon mutation. Mapping the network of correlated residues onto the 3D structure of the Kv channel from Aeropyrum pernix disclosed correlations between residues in the voltage-sensor paddle and the pore region, including regions that are involved in the gating transition. We discuss these findings with respect to the evolutionary constraints acting on the channel's various domains. The software is available on our website     

A medium-density genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project. Received: 15 August 1996 / Accepted: 15 September 1996     

Structural divergence among cannabinoids influences membrane dynamics: a 2H solid-state NMR analysis

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB(1) receptor. We used (2)H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Delta(9)-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S(CD) ( approximately 0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S(CD) ( approximately 0.14) at the top of the phospholipid upper chain. The CB(1) antagonist AM281 had average S(CD) values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R(1Z) versus |S(CD)|(2) plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.     

High‐accuracy modeling of antibody structures by a search for minimum‐energy recombination of backbone fragments

Current methods for antibody structure prediction rely on sequence homology to known structures. Although this strategy often yields accurate predictions, models can be stereo‐chemically strained. Here, we present a fully automated algorithm, called AbPredict, that disregards sequence homology, and instead uses a Monte Carlo search for low‐energy conformations built from backbone segments and rigid‐body orientations that appear in antibody molecular structures. We find cases where AbPredict selects accurate loop templates with sequence identity as low as 10%, whereas the template of highest sequence identity diverges substantially from the query's conformation. Accordingly, in several cases reported in the recent Antibody Modeling Assessment benchmark, AbPredict models were more accurate than those from any participant, and the models' stereo‐chemical quality was consistently high. Furthermore, in two blind cases provided to us by crystallographers prior to structure determination, the method achieved <1.5 Ångstrom overall backbone accuracy. Accurate modeling of unstrained antibody structures will enable design and engineering of improved binders for biomedical research directly from sequence. Proteins 2016; 85:30–38. © 2016 Wiley Periodicals, Inc.     

Multifunctional Manganese Ions Trapping and Hydrofluoric Acid Scavenging Separator for Lithium Ion Batteries Based on Poly(ethylene‐alternate‐maleic acid) Dilithium Salt

Manganese dissolution from positive electrodes seriously reduces the life of Li‐ion batteries, due to its detrimental impact on the passivation of negative electrodes. A novel multifunctional separator incorporating inexpensive mass‐produced polymeric materials may dramatically increases the durability of Li‐ion batteries. The separator is made by embedding the poly(ethylene‐alternate‐maleic acid) dilithium salt polymer into a poly(vinylidene fluoride‐hexafluoropropylene) copolymer matrix. LiMn₂O₄‐graphite cells comprising a 1 m LiPF₆ solution in ethylene carbonate plus dimethyl carbonate (1:1 v/v) and the functional separator retain 31% and 100% more capacity than baseline cells with plain commercial separators after 100 cycles at C/5 rate, respectively, at 30 and 55 °C. Analyses of cycled cells indicate greatly reduced Mn contamination of the graphite negative electrodes and almost no irreversible structural change in the LiMn₂O₄ positive electrodes from cells containing the functional separator. The Mn amount in the graphite electrodes from cycled cells with functional separators is ≈80% lower than in the graphite electrodes from cycled baseline cells. Mn ions are found in the functional separators but not in baseline (plain) separators from cycled cells. Finally, it is shown that the reported performance improvements stem from the ability of the novel separator to chelate Mn ions and to scavenge trace HF.     

Ocular neuroprotection by siRNA targeting caspase-2

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.     

Assessing and enhancing foldability in designed proteins

Recent advances in protein‐design methodology have led to a dramatic increase in reliability and scale. With these advances, dozens and even thousands of designed proteins are automatically generated and screened. Nevertheless, the success rate, particularly in design of functional proteins, is low and fundamental goals such as reliable de novo design of efficient enzymes remain beyond reach. Experimental analyses have consistently indicated that a major reason for design failure is inaccuracy and misfolding relative to the design conception. To address this challenge, we describe complementary methods to diagnose and ameliorate suboptimal regions in designed proteins: first, we develop a Rosetta atomistic computational mutation scanning approach to detect energetically suboptimal positions in designs (available on a web server https://pSUFER.weizmann.ac.il); second, we demonstrate that AlphaFold2 ab initio structure prediction flags regions that may misfold in designed enzymes and binders; and third, we focus FuncLib design calculations on suboptimal positions in a previously designed low‐efficiency enzyme, improving its catalytic efficiency by 330‐fold. Furthermore, applied to a de novo designed protein that exhibited limited stability, the same approach markedly improved stability and expressibility. Thus, foldability analysis and enhancement may dramatically increase the success rate in design of functional proteins.