Behavioral and Transcriptome Profiling of Heterozygous Rab10 Knock-Out Mice

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.A central question in the field of aging research is to identify the cellular and molecular basis of neuroresilience. One potential candidate is the small GTPase, Rab10. Here, we used Rab101/ mice to investigate the molecular mecha-nisms underlying Rab10-mediated neuroresilience. Brain expression analysis of 880 genes involved in neurodegener-ation showed that Rab101/ mice have increased activation of pathways associated with neuronal metabolism, structural integrity, neurotransmission, and neuroplasticity compared with their Rab101/1 littermates. Lower activation was observed for pathways involved in neuroinflammation and aging. We identified and validated several differentially expressed genes (DEGs), including Stx2, Stx1b, Vegfa, and Lrrc25 (downregulated) and Prkaa2, Syt4, and Grin2d (upregulated). Behavioral testing showed that Rab101/ mice perform better in a hippocampal-dependent spatial task (object in place test), while their performance in a classical conditioning task (trace eyeblink classical condition-ing, TECC) was significantly impaired. Therefore, our findings indicate that Rab10 differentially controls the brain cir-cuitry of hippocampal-dependent spatial memory and higher-order behavior that requires intact cortex-hippocampal circuitry. Transcriptome and biochemical characterization of these mice suggest that glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D or GluN2D) is affected by Rab10 signaling. Further work is needed to evaluate whether GRIN2D mediates the behavioral phenotypes of the Rab101/ mice. We conclude that Rab101/ mice de-scribed here can be a valuable tool to study the mechanisms of resilience in Alzheimer’s disease (AD) model mice and to identify novel therapeutical targets to prevent cognitive decline associated with normal and pathologic aging.ECU Open Access Publishing Support Fun

Viruses: incredible nanomachines. New advances with filamentous phages

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account

The Effects of Apolipoprotein F Deficiency on High Density Lipoprotein Cholesterol Metabolism in Mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls

Molecular dynamics simulations of monomeric apolipoprotein A-I from a recent X-ray structure

We have examined the X-ray crystal structure recently refined by Ajees and colleagues (Ajees et al. 2006) for monomeric apolipoprotein A-I (apoA-I). Because the structure, which has been crystallized together with chromium organic compounds, possesses a substantially higher percentage of alpha helicity than is generally estimated experimentally for the lipid-free monomeric apoA-I in solution (~80% vs ~50%), we have performed molecular dynamics (MD) simulations for ~10 ns of the model in order to explore the dynamic behavior of the single apoA-I monomer at a physiological salt concentration and a temperature range of 310-410 K. While 10 ns simulation is only a starting point, a few important observations have been made: i) the percentage of alpha helicity decreased substantially to below 70% (i.e., towards a lower experimental estimate); ii) the structure became more globular in overall appearance; iii) the flexible N-terminal domain (amino acid residues 1 to 43) has lost most of its alpha helicity; iv) the hydrophobic core of the 4-helix bundle is defined by stacking of a cluster of aromatic amino acid residues, outlined by a shell of aliphatic hydrophobic residues; v) The four helix bundle portion of the simulated structure is clustering around a pronounced stacking of aromatic residues derived from all four helixes and the aromatic cluster is overlaid by a shell of aliphatic hydrophobic residues. We conjecture that this aromatic cluster and its surrounding hydrophobic residues are the driving force for creation of a dynamic (molten globular) four helix bundle arrangement in lipid-free monomeric apoA-I in solution. This work was supported by NIH grant

Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer.

The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments

Preliminary models of spheroidal HDL particles through molecular dynamics

In vivo high density lipoproteins (HDL) originate as discoidal prebeta-migrating complexes of the apolipoprotein (apo) A-I and phospholipids which are either secreted from the liver and intestine or are formed extracellularly by the interaction of lipid poor apoA-I with small amounts of phospholipids. These prebeta HDL complexes of apoA-I and phospholipids are highly effective acceptors of unesterified cholesterol (UC) from cell membranes; the accumulation of UC converts them into larger discoidal particles. These latter particles are excellent substrates for the enzyme Lecithin Cholesterol Acyl Transferase (LCAT), which catalyses the transfer of an acyl group from phosphatidylcholine (PC) to cholesterol, generating cholesteryl ester (CE) and lyso-PC. The transition from discoidal to spheroidal HDL (the form of circulating HDL) is driven by phase separation of CE molecules to create a hydrophobic core in the middle of the lipid bilayer. To investigate the conformation of apoA-I in spheroidal HDL particles, we performed all-atom and coarse-grained (CG) molecular dynamics (MD) simulations on a starting model HDL particle produced by a methodology involving incremental removal of palmitoyloleoylphosphatidylcholine (POPC) from a particle with 160 POPC and a belt of two antiparallel amphipathic helical lipid-associating domains of apoA-I. The initial particle, a previously 1 ns MD simulated and a delta40 apoA-I double belt both surrounding a saddle-shaped POPC bilayer with 80 lipid molecules, was subjected to removal of 24 POPC molecules from the center, followed by the insertion of a cluster of 16 cholesteryl oleate molecules (CO). The goal was simulation of spheroidal HDL particle by creating a CO hydrophobic core between the monolayers of the POPC bilayer. In both atomistic and CG MD simulations at 310K the starting model assumes a spheroidal shape. This work was supported by a National Institute of Health grant