No abstract

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.

Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild-type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F-penetratin variant was larger than for the W48F-penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become a helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild-type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to a helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.

A series of natural peptides and mutants, derived from the Alzheimer ␤-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29 -40 and 29 -42) and a central domain (13-28) of the ␤-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire ␤-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29 -40 and 29 -42 ␤-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29 -42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 ␤-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the ␣-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the ␣-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29 -40 and 29 -42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the ␤-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.The amyloid peptide (A␤), 1 a 39 -43-residue peptide, is a normal 4-kDa derivative of a large transmembrane glycoprotein, the amyloid ␤ precursor protein. The A␤ peptide is found in an aggregated, poorly soluble form in extracellular amyloid deposition in the brains and leptomeniges of patients with Alzheimer's disease (1). In contrast, it occurs in a soluble form in several biological fluids, including the cerebrospinal fluid, where it is produced by glial cells and neurons and where it circulates at nanomolar concentrations (2). The mechanism by which A␤ causes cell death and exerts its cytotoxicity effect remains unclear, and controversies still exist concerning the cytotoxic action of A␤ on neuronal cells. A number of in vitro studies with the synthetic A␤ peptide have shown that this peptide aggregates easily and...

Chylomicrons (CM)1 and very low density lipoproteins (VLDL) are among the largest macromolecular complexes secreted from eukaryotic cells. The assembly of neutral lipids and phospholipids into CM and VLDL is nucleated around a single molecule of apoB and requires a microsomal triglyceride transfer protein (MTP) complexed to the endoplasmic reticulumresident protein, protein disulfide isomerase (PDI). The function of the MTP-PDI complex is to supply apoB with sufficient lipid to form a soluble lipoprotein. Defects of apoB and MTP cause hypobetalipoproteinemia and abetalipoproteinemia, respectively (1, 2).ApoB and MTP have structural homology with lamprey lipovitellin (LV) (3). LV contains an N-terminal ␤-barrel (amino acids 17-296), an ␣-helical structure (amino acids 297-614), and a C-terminal lipid binding cavity (4). The structural relationship between MTP, apoB, and LV is supported by conservation of the gene and protein structure (3,5). Important features of the quaternary structure of the lamprey LV homodimer are adapted in MTP to form a heterodimer with PDI and to associate with apoB during lipoprotein production (3, 5). The defining difference between MTP, apoB, and LV is related to their C-terminal lipid binding structures, which associate with different amounts of lipid (3). LV binds principally phospholipid with a stoichiometry of ϳ35 molecules/subunit (6). MTP binds 1-5 molecules of lipid (7). ApoB has a long C-terminal extension (ϳ3500 amino acids), which incorporates a large neutral lipid core (8).Here, we have addressed the mechanism by which MTP-PDI acquires neutral lipid from phospholipid bilayers for the assembly of VLDL and CMs. In the absence of a crystal structure for MTP, we derived a homology model to guide mutagenesis and biophysical studies. The experimental data substantiate the overall predictions of the model and provide insights into the mechanism of lipid acquisition and binding. EXPERIMENTAL PROCEDURESModeling-Models were developed on the alignment shown in Fig. 1 and the x-ray crystal structure of lamprey LV (Protein Data Bank accession number 1LLV), refined to an R-value of 0.19 at 2.8 Å resolution (4). The C-sheet was modeled using INSIGHT interactive graphics software and the Homology computer program (Biosym Technologies, San Diego) and the A-sheet with the general purpose modeling program O (9). Models were energy minimized and the quality of the coordinates assessed as described (3).Mutagenesis and Expression Studies-Mutagenesis was performed by a polymerase chain reaction-based strategy (3). All constructs were sequenced before use. Transfections, preparation of cell extracts, and triglyceride transfer activities were performed as described (2, 3).Triglyceride Binding and Fusogenic Activity-Wild-type (WT) and mutant MTP-PDI complexes were purified as described (10). Donor small unilamellar vesicles (SUVs) were prepared as described (2), purified to homogeneity (11), and incubated with MTP (w/w 70:1) for 2 h at 37°C. Lipid-protein complexes were separated on a Sepharose CL-4B...