Crystal structures of the Dab homology domains of mouse disabled 1 and 2

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.     

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin-14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary-shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary-shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2-2.4 microm min(-1), 2-5-fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.     

Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.Few of the hundreds of known neutralizing anti-HIV monoclonal antibodies (MAbs) display broad cross-reactive activities (4). Of those derived from clade B-infected patients, b12 binds to the gp120 subunit of the HIV envelope protein (Env), to an epitope that overlaps the CD4 binding site, and neutralizes approximately 50% of virus isolates tested, including non-clade B viruses (27). 2G12 binds to N-linked carbohydrates on gp120 (32, 34) and neutralizes 41% of isolates tested, although not clade C or E isolates. 447-52D also binds to the gp120 subunit, to an epitope within the V3 loop, and potently neutralizes up to 45% of clade B isolates but rarely non-clade B isolates. 4E10 and 2F5 recognize adjacent epitopes located at the membrane-proximal external region (MPER) of the gp41 Env subunit (9, 22, 24, 28, 42). Two neutralizing antibodies (NAbs) isolated from a clade A-infected patient (PG9 and PG16) show broad and potent neutralizing activity by recognizing epitopes consisting of conserved regions of the V2 and V3 loops of gp120, preferentially on native trimers (40).4E10 is capable of neutralizing all isolates tested at some level (4), although there is evidence for the existence of rare viruses that are resistant to 4E10 neutralization (30). The exact structure of the epitope recognized by 4E10 within the trimeric, functional HIV Env is unknown, but structural studies have shown that an isolated peptide spanning the epitope adopts a helical conformation, a short 3₁₀ segment followed by a 4₁₃ (or true α-helical) segment, with an extended structure at the N terminus when bound to 4E10 (9). It has also been reported that 4E10 interacts with a variety of lipids and membrane components, particularly the phospholipid cardiolipin (15), suggesting that difficulties in eliciting 4E10-like broadly neutralizing antibodies by immunization and the apparent rarity of 4E10-like antibody responses in HIV-1-infected subjects (19, 33) are linked to this polyspecificity to autoantigens, contributing to their elimination through tolerance mechanisms. However, subsequent studies have shown that the measurable, but quite weak, affinity of 4E10 for certain lipids is comparable to that of some antiphospholipid antibodies elicited during many infections, suggesting that 4E10 is not remarkably autoreactive (35). Therefore, it is still unclear whether lipid binding properties are linked to the rarity of 4E10-like specificities. It has also been proposed that the neutralizing activity of 4E10 may partly depend on lipid binding, either through interactions with viral membrane lipids that disturb the membrane-bound structure of the MPER on the trimeric, virion-associated Env spike (39) or through an encounter model. In the latter, initial interactions with membrane components align 4E10 with its protein epitope or allow 4E10 to gain proximity to its epitope (1), perhaps partially alleviating steric occlusion effects (for example, see reference 17). We sought to determine whether specific interactions exist between 4E10 and membrane lipid components and whether such interactions meaningfully contribute to neutralization by any mechanism.     

Thiourea compound AW00178 sensitizes human H1299 lung carcinoma cells to TRAIL-mediated apoptosis

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a wide variety of cancer cells. Recently, cancer cell resistance to TRAIL-mediated apoptosis has become a challenging issue in the development of TRAIL-based anti-cancer therapies. In this study, we found that 1-(5-chloro-2-methyl-phenyl)-3-[4-(5-trifluoromethyl-pyrazol-1-yl)-phenyl]-thiourea (AW00178) was able to sensitize TRAIL-resistant human lung cancer H1299 cells to TRAIL-mediated apoptosis. Treatment with AW00178, either alone or in combination with TRAIL, induced the expression of CHOP, a protein related to TRAIL sensitivity, and reduced the expression of survivin, an anti-apoptotic protein involved in TRAIL resistance. Additionally, AW00178, alone or in combination with TRAIL, induced the activation of c-Jun and inactivation of Akt. A pharmacologic inhibition study revealed that c-Jun activation and Akt inactivation were strongly related to CHOP induction and survivin down-regulation, respectively. In summary, these results suggested that AW00178 mediated sensitization to TRAIL-mediated apoptosis in H1299 cells by increasing sensitivity and decreasing resistance to TRAIL via the induction of c-Jun-dependent CHOP expression and the reduction of Akt-dependent survivin expression, respectively.     

Characterization of Antibiotic Peptide Pores Using Cryo-EM and Comparison to Neutron Scattering

Magainin, a 23-residue antibiotic peptide, interacts directly with the lipid bilayer leading to cell lysis in a strongly concentration-dependent fashion. Utilizing cryo-electron microscopy, we have directly observed magainin interacting with synthetic DMPC/DMPG membranes. Visual examination shows that visibly unperturbed vesicles are often found adjacent to vesicles that are lysed or porous, demonstrating that magainin disruption is a highly stochastic process. Quantitatively, power spectra of large numbers of porous vesicles can be averaged together to produce the equivalent of an electron scattering curve, which can be related to theory, simulation, and published neutron scattering experiments. We demonstrate that magainin-induced pores in lipid vesicles have a mean diameter of ∼80 Å, compatible with earlier reported results in multilayer stacks. In addition to establishing a connection between experiments in multilayer stacks and vesicles, this also demonstrates that computed power spectra from windowed-out regions of cryo-EM images can be compared to neutron scattering data in a meaningful way, even though the pores of interest cannot yet be individually identified in images. Cryo-EM offers direct imaging of systems in configurations closely related to in vivo conditions, whereas neutron scattering has a greater variety of mechanisms for specific contrast variation via D₂O and deuterated lipids. Combined, the two mechanisms support each other, and provide a clearer picture of such ‘soft’ systems than either could provide alone.     

CHESS (CgHExpreSS): A comprehensive analysis tool for the analysis of genomic alterations and their effects on the expression profile of the genome

Background  

Genomic alterations frequently occur in many cancer patients and play important mechanistic roles in the pathogenesis of cancer. Furthermore, they can modify the expression level of genes due to altered copy number in the corresponding region of the chromosome. An accumulating body of evidence supports the possibility that strong genome-wide correlation exists between DNA content and gene expression. Therefore, more comprehensive analysis is needed to quantify the relationship between genomic alteration and gene expression. A well-designed bioinformatics tool is essential to perform this kind of integrative analysis. A few programs have already been introduced for integrative analysis. However, there are many limitations in their performance of comprehensive integrated analysis using published software because of limitations in implemented algorithms and visualization modules.     

Demonstration of motoneuron-12 sparing in cultured Manduca sexta ventral nerve cords

The emergence of the adult Manduca sexta moth is accompained by the death of half of the neurons present in the pupal abdominal nervous system (Truman, 1983). This developmental neuronal death is highly selective, so that the same neurons die at the same time relative to emergence in every moth. In the case of the MN-12 motoneurons, this cell death is regulated both by hemolymph concentrations of a steroid hormone, 20-hydroxyecdysone, and by actions exerted by adjacent ganglia (Truman and Schwartz, 1984; Fahrbach and Truman, 1987). This latter effect, which has been previously described in isolated abdomens and in moths with transected ventral nerve cords, has now been reproduced under controlled culture conditions in which the selectivity and extent of postemergence neuronal death is comparable to that seen in vivo. With respect to the MN-12 neurons found in the most anterior unfused abdominal ganglion, A₃, the pterothoracic ganglion appears to be the source of a factor that permits these neurons to die according to their usual developmental schedule. © 1992 John Wiley & Sons, Inc.