Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors.

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.

Mutational analysis of cell cycle inhibition by integrin beta1C.

Integrin beta1C is an alternatively spliced cytoplasmic variant of the beta1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta1A, beta1B, beta1D, beta3, and beta5 cytoplasmic domains had weak and variable effects. The beta1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta1C. The beta1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta1C as a growth inhibitor in vivo.

An activated Rac mutant functions as a dominant negative for membrane ruffling.

Previous studies have shown that point mutations in the effector domain of Rac1 block specific downstream pathways such as PAK, JNK/SAPK kinases and membrane ruffling. Specifically, the F37A mutation, made in a constitutively activated Q61L background, activates PAK but fails to induce membrane ruffles. We now show that Q61L/F37A Rac not only fails to induce ruffling but potently blocks membrane ruffling induced by serum or PDGF. In the presence of serum, cells do extend filopodia, suggesting that this mutant only blocks a subset of the effectors that induce cytoskeletal reorganization. At later times, this rac mutant induces membrane blebbing, but not apoptosis. These results show that Q61L/F37A Rac, is constitutively activated with respect to PAK activation but functions as a dominant negative for another pathway, membrane ruffling. That an effector domain point mutant can simultaneously function as a dominant negative and dominant positive for different pathways implies that effects of these variants on cell functions must be interpreted with caution.

Integrins, adhesion and apoptosis.

Integrin-mediated adhesion to extracellular matrix proteins is required for survival of many cell types. This phenomenon appears to be a mechanism of tumour suppression and to participate in embryogenesis. Here, our current understanding of how integrin-dependent signals prevent apoptosis and implications of anchorage-dependent survival for development, physiology and pathology are discussed.

Transcriptional and post-transcriptional regulation of maternal and zygotic cytoskeletal tropomyosin mRNA during Drosophila development correlates with specific morphogenic events.

We have characterized maternal and zygotic cytoskeletal tropomyosin mRNA expression during Drosophila embryogenesis using in situ hybridization to endogenous cytoskeletal tropomyosin mRNA and a cytoskeletal tropomyosin promoter/beta-galactosidase-encoding fusion gene mRNA in transgenic flies. A 2.0-kb maternal cytoskeletal tropomyosin mRNA is synthesized in the nurse cells and transported into the oocyte during oogenesis. During early embryogenesis, this mRNA becomes localized to the pole cell region and then to the cortex during the cellular blastoderm stage. In later embryos it is localized to the ventral and cephalic furrows and extending germ band. The major zygotic mRNA is 2.4 kb and is first detected at gastrulation. In early embryos, this mRNA is expressed in the invaginating anterior and posterior midgut, the transverse furrows, and the amnioserosa, all regions of the embryo undergoing intense cellular movement, and in later embryos, predominantly in the gut, brain, and epidermis. A transgene construct containing 1.2 kb of 5' cytoskeletal tropomyosin promoter sequences driving expression of Escherichia coli beta-galactosidase and the cytoskeletal tropomyosin 3' untranslated region in transgenic flies has the same distribution of maternal and zygotic transcripts throughout oogenesis and embryonic development as the endogenous transcripts. A transgene containing the 3' untranslated region from either the hsp70 gene or the SV40 early genes, on the other hand, does not express maternal RNA. Furthermore, none of the transgenes expressed in the follicle cells, suggesting that expression in these cells is under different transcriptional control. Our results indicate that maternal and zygotic cytoskeletal tropomyosin mRNAs are localized to specific regions of the developing embryo, particularly in regions of the embryo undergoing cell movement and invagination. Furthermore, the synthesis, transport, and accumulation of this RNA is under transcriptional and post-transcriptional control.

The extracellular matrix as a cell survival factor.

Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. In many cases, PCD is induced by the withdrawal of specific hormones or growth factors that function as survival factors. In this study, we have investigated the potential role of the extracellular matrix (ECM) as a cell survival factor. Our results indicate that in the absence of any ECM interactions, human endothelial cells rapidly undergo PCD, as determined by cell morphology, nuclei fragmentation, DNA degradation, protein cross-linking, and the expression of the PCD-specific gene TRPM-2. PCD was blocked by plating cells on an immobilized integrin beta 1 antibody but not by antibodies to either the class I histocompatibility antigen (HLA) or vascular cell adhesion molecule-1 (VCAM-1), suggesting that integrin-mediated signals were required for maintaining cell viability. Treatment of the cells in suspension with the tyrosine phosphatase inhibitor sodium orthovanadate also blocked PCD. When other cell types were examined, some, but not all, underwent rapid cell death when deprived of adhesion to the ECM. These results suggest that in addition to regulating cell growth and differentiation, the ECM also functions as a survival factor for many cell types.