Notch-independent RBPJ controls angiogenesis in the adult heart.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.

The Fas-FADD death domain complex structure unravels signalling by receptor clustering.

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.

Maspin alters the carcinoma proteome.

Maspin, a member of the serine protease inhibitor (serpin) family, is a tumor suppressor in breast and prostate cancer. To address molecular mechanisms underlying maspin's activity, we restored its expression in invasive carcinoma cells and analyzed the resulting changes by shotgun proteomics. Using a mass spectrometry-based multidimensional proteomic method, we observed changes to the expression of approximately 27% of the detectable proteome. In particular, we noted changes to the expression of proteins that regulate cytoskeletal architecture, cell death, and protein turnover. In each case, changes in protein expression were accompanied by measurable changes in tumor cell phenotype. Thus, maspin-expressing cells exhibit a more prominent actin cytoskeleton, a reduced invasive capacity, an increased rate of spontaneous apoptosis, and an altered proteasome function. These observations reveal for the first time the far reaching effects of maspin on multiple protein networks and a new hypothesis of maspin function based on the regulation of proteasome function.

Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X(L).

Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.

Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of membrane type-1 matrix metalloproteinase in malignant cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in cell locomotion and tissue remodeling. Trafficking to the plasma membrane and internalization into the transient storage compartment both regulate the cell surface presentation of MT1-MMP. Our data indicate that mutant MT1-MMP lacking the cytoplasmic tail is recruited to the caveolae-enriched lipid raft membrane microdomains in breast carcinoma MCF7 cells. In contrast, the wild-type protease is not permanently associated with lipid rafts. Trafficking to lipid rafts correlated with poor internalization and the persistent presentation of MT1-MMP at the cell surface. The tail mutant efficiently functioned in inducing the activation of the latent proMMP-2 zymogen, matrix remodeling, and contraction of three-dimensional collagen lattices. Recruitment of the tail mutant to lipid raft antagonized, however, the cleavage of the plasma membrane-associated E-cadherin. These events limited the contribution of the tail mutant to cell locomotion and malignant growth. It is conceivable that the tail peptide sequence plays a crucial role in the translocations of MT1-MMP across the cell and contributes to coordinated cellular functions. It is tempting to hypothesize that the mechanisms involved in trafficking of MT1-MMP to caveolin-enriched lipid rafts may be targeted in a clinically advantageous manner.

The cytoplasmic tail peptide sequence of membrane type-1 matrix metalloproteinase (MT1-MMP) directly binds to gC1qR, a compartment-specific chaperone-like regulatory protein.

Membrane type-1 matrix metalloproteinase (MT1-MMP), a key enzyme in cell locomotion, is known to be primarily recruited to the leading edge of migrating cells. This raises a possibility that the C-terminal cytoplasmic tail of MT1-MMP interacts with intracellular regulatory proteins, which modulate translocations of the protease across the cell. Here, we demonstrated that MT1-MMP via its cytoplasmic tail directly associates with a chaperone-like compartment-specific regulator gC1qR. Although a direct functional link between these two proteins remains uncertain, our observations suggest that the transient associations of gC1qR with the cytoplasmic tail of MT1-MMP are likely to be involved in the mechanisms regulating presentation of the protease at the tumor cell surface.