Point mutations in the HIV-1 matrix protein turn off the myristyl switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.     

Reduced C(beta) statistical potentials can outperform all-atom potentials in decoy identification

We developed a series of statistical potentials to recognize the native protein from decoys, particularly when using only a reduced representation in which each side chain is treated as a single C(beta) atom. Beginning with a highly successful all-atom statistical potential, the Discrete Optimized Protein Energy function (DOPE), we considered the implications of including additional information in the all-atom statistical potential and subsequently reducing to the C(beta) representation. One of the potentials includes interaction energies conditional on backbone geometries. A second potential separates sequence local from sequence nonlocal interactions and introduces a novel reference state for the sequence local interactions. The resultant potentials perform better than the original DOPE statistical potential in decoy identification. Moreover, even upon passing to a reduced C(beta) representation, these statistical potentials outscore the original (all-atom) DOPE potential in identifying native states for sets of decoys. Interestingly, the backbone-dependent statistical potential is shown to retain nearly all of the information content of the all-atom representation in the C(beta) representation. In addition, these new statistical potentials are combined with existing potentials to model hydrogen bonding, torsion energies, and solvation energies to produce even better performing potentials. The ability of the C(beta) statistical potentials to accurately represent protein interactions bodes well for computational efficiency in protein folding calculations using reduced backbone representations, while the extensions to DOPE illustrate general principles for improving knowledge-based potentials.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Characterization of the Bex gene family in humans, mice, and rats

To better understand the development of ventral mesencephalic dopamine neurons, we performed subtractive hybridization screens to find ventral mesencephalic genes expressed at rat embryonic day 10 when these neurons begin to differentiate. The most commonly identified genes in these screens were members of the Bex (Brain expressed X-linked) gene family, rat Bex1 (Rex3), and a novel gene, rat Bex4. After identifying these genes, we then sought to characterize the Bex gene family. Two additional novel Bex genes (human Bex5 and mouse Bex6) were discovered through genomic databases. Bex5 is present in humans and monkeys, but not rodents, while Bex6 exists in mice, but not humans. Bex4 and Bex5 are localized to the X chromosome, are expressed in brain, and are similar in sequence. Bex4 and Bex5 are 54% and 56% identical to human Bex3 (pHGR74, NADE). Mouse Bex6 is on chromosome 16 and is 67% identical to mouse Bex4. Human Bex gene expression was studied with tissue expression arrays probed with specific oligonucleotides. Human Bex1 and Bex2 have similar expression patterns in the central nervous system with high levels in pituitary, cerebellum, and temporal lobe, and Bex1 is widely expressed outside of the central nervous system with high expression in the liver. Human Bex4 is highly expressed in heart, skeletal muscle, and liver, while Bex3 and Bex5 are more widely expressed. The subcellular localization of the Bex proteins varies from nuclear (rat Bex1) to cytoplasmic (rat Bex3, human Bex5, and mouse Bex6) and to both nuclear and cytoplasmic (rat Bex2 and rat Bex4). Rat Bex3, rat Bex4, human Bex5, and mouse Bex6 are degraded by the proteasome, while rat Bex1 or Bex2 are not. Rat Bex3 protein can likely bind transition metals through a histidine-rich domain. Because this gene family was originally named Bex and because these genes are unified by sequence similarity and gene structure, we believe the Bex nomenclature should prevail over nomenclature based on function (NADE) that has not been extended to the other Bex genes. We conclude that the Bex gene family members are highly homologous but differ in their expression patterns, subcellular localization, and degradation by the proteasome.     

Fractional order viscoelasticity of the aortic valve cusp: an alternative to quasilinear viscoelasticity

BACKGROUND: Quasilinear viscoelasticity (QLV) theory has been widely and successfully used to describe the time-dependent response of connective tissues. Difficulties remain, however, particularly in material parameter estimation and sensitivities. In this study, we introduce a new alternative: the fractional order viscoelasticity (FOV) theory, which uses a fractional order integral to describe the relaxation response. FOV implies a fractal-like tissue structure, reflecting the hierarchical arrangement of collagenous tissues. METHOD OF APPROACH: A one-dimensional (I-D) FOV reduced relaxation function was developed, replacing the QLV "box-spectrum" function with a fractional relaxation function. A direct-fit, global optimization method was used to estimate material parameters from stress relaxation tests on aortic valve tissue. RESULTS: We found that for the aortic heart valve, FOV had similar accuracy and better parameter sensitivity than QLV, particularly for the long time constant (tau2). The mean (n = 5) fractional order was 0.29, indicating that the viscoelastic response of the tissue was strongly fractal-like. RESULTS SUMMARY: mean QLV parameters were C = 0.079, tau1 = 0.004, tau2 = 76, and mean FOV parameters were beta = 0.29, tau = 0.076, and rho = 1.84. CONCLUSIONS: FOV can provide valuable new insights into tissue viscoelastic behavior Determining the fractional order can provide a new and sensitive quantitative measure for tissue comparison.     

Spin-labeled gramicidin a: channel formation and dissociation

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.