The Interaction of Polyglutamine Peptides with Lipid Membranes Is Regulated by Flanking Sequences Associated with Huntingtin

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q₃₅-KK, KK-Q₃₅-P₁₀-KK, Nt17-Q₃₅-KK, and Nt17-Q₃₅-P₁₀-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q₃₅-P₁₀-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.     

Psammaplysin F: A unique inhibitor of bacterial chromosomal partitioning

Described is the antibiotic activity of a marine natural product. Psammaplysin F (1) inhibited the growth of four Gram-positive strains by >80% at 50 μM, and the amine at position C-20 is responsible for the observed antibacterial activity. When tested against two strains of methicillin resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentrations (MICs) for psammaplysin F (40–80 μM) were similar to the structurally-related alkaloid psammaplysin H (2). Psammaplysin F (1) increased membrane permeability by two to four-fold compared to psammaplysin H (2) or control-treated bacteria, respectively. Unlike psammaplysin H (2), we show that psammaplysin F (1) inhibits equal partitioning of DNA into each daughter cell, suggesting that this natural product is a unique prokaryotic cell division inhibitor.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

A small molecule that preferentially binds the closed conformation of Hsp90

Described is the synthesis of three different fluorescein-tagged derivatives of a macrocycle, and their binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-dependent closed state. We show that the compounds demonstrate a conformation-dependent preference for binding to the closed state.     

Design and synthesis of Hsp90 inhibitors: Exploring the SAR of Sansalvamide A derivatives

Utilizing the structure–activity relationship we have developed during the synthesis of the first two generations and mechanism of action studies that point to the interaction of these molecules with the key oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their synthesis. Our new structures, designed from previously reported potent compounds, were tested for cytotoxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed using computational studies involving blind docking of derivatives using Autodock. Further, we show new evidence that our molecules bind directly to Hsp90 and modulate Hsp90’s binding with client proteins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.     

Parental Control over Mate Choice to Prevent Marriages with Out-group Members

The present research examined how a preference for influencing the mate choice of one's offspring is associated with opposition to out-group mating among parents from three ethnic groups in the Mexican state of Oaxaca: mestizos (people of mixed descent, n?=?103), indigenous Mixtecs (n?=?65), and blacks (n?=?35). Nearly all of the men in this study were farmworkers or fishermen. Overall, the level of preferred parental influence on mate choice was higher than in Western populations, but lower than in Asian populations. Only among the Mixtecs were fathers more in favor of parental influence on the mate choice of children than mothers were. As predicted, opposition to out-group mating was an important predictor of preferred parental influence on mate choice, more so among fathers than among mothers, especially in the mestizo group-the group with the highest status. In addition, women, and especially mestizo women, expressed more opposition to out-group mating than men did.     

Condensing and fluidizing effects of ganglioside GM1 on phospholipid films

Mixed monolayers of the ganglioside G_M1 and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by G_M1 on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for G_M1 concentrations ranging from 5 to 70 mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of G_M1 (<25 mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher G_M1 concentrations (>25 mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that G_M1 and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/G_M1 binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of G_M1 to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the G_M1 molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the G_M1 molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the G_M1 molecule.     

X-Ray Diffraction and Reflectivity Validation of the Depletion Attraction in the Competitive Adsorption of Lung Surfactant and Albumin

Lung surfactant (LS) and albumin compete for the air-water interface when both are present in solution. Equilibrium favors LS because it has a lower equilibrium surface pressure, but the smaller albumin is kinetically favored by faster diffusion. Albumin at the interface creates an energy barrier to subsequent LS adsorption that can be overcome by the depletion attraction induced by polyethylene glycol (PEG) in solution. A combination of grazing incidence x-ray diffraction (GIXD), x-ray reflectivity (XR), and pressure-area isotherms provides molecular-resolution information on the location and configuration of LS, albumin, and polymer. XR shows an average electron density similar to that of albumin at low surface pressures, whereas GIXD shows a heterogeneous interface with coexisting LS and albumin domains at higher surface pressures. Albumin induces a slightly larger lattice spacing and greater molecular tilt, similar in effect to a small decrease in the surface pressure. XR shows that adding PEG to the LS-albumin subphase restores the characteristic LS electron density profile at the interface, and confirms that PEG is depleted near the interface. GIXD shows the same LS Bragg peaks and Bragg rods as on a pristine interface, but with a more compact lattice corresponding to a small increase in the surface pressure. These results confirm that albumin adsorption creates a physical barrier that inhibits LS adsorption, and that PEG in the subphase generates a depletion attraction between the LS aggregates and the interface that enhances LS adsorption without substantially altering the structure or properties of the LS monolayer.     

Synthesis of Sansalvamide A derivatives and their cytotoxicity in the MSS colon cancer cell line HT-29

We report the synthesis of thirty-six Sansalvamide A derivatives, and their biological activity against colon cancer HT-29 cell line, a microsatellite stable (MSS) colon cancer cell-line. The thirty-six compounds can be divided into three subsets, where the first subset of compounds contains L-amino acids, the second subset contains D-amino acids, and the third subset contains both D- and L-amino acids. Five compounds exhibited excellent inhibitory activity (>75% inhibition). The structure-activity relationship (SAR) of the compounds established that a single D-amino acid in position 2 or 3 gave up to a 10-fold improved cytotoxicity over Sansalvamide A peptide. This work highlights the importance of residues 2 and 3 and the role of D-amino acids in the extraordinary SAR for this compound class.