Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent ␥-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimer's disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog ␥-secretase inhibitor. We propose that ␥-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

From an investigation of how transfected DNA navigates abrogation of endosome-lysosome fusion or translocation from the cell surface to the nucleus, we have developed with microfilament or microtubule toxins, respectively, a transfection method for primary human fibroblasts that inhibits the nuclear accumulation of transfected DNA, but approaches the efficiency of viruses. We have visually interruption of lysosomal function with protease inhibitors tracked the subcellular routing of exogenous DNA and find promotes it. Second, in normal human fibroblasts, which that all cells in an asynchronous population are surprisingly are refractory to transfection, the exogenous DNA is rapidly competent in the nuclear uptake of DNA, but two steps excluded from the nucleus, but in HeLa cells, which are practically limit efficient transfection to a minority of cells.readily transfected, there is prolonged nuclear stability of First, regardless of the method used to traverse the cell the DNA, indicating the failure in HeLa cells of a mechanism membrane -CaPO 4 precipitation, lipofection or electroporfor the elimination of foreign DNA. These observations ation -it appears that nuclear transport of DNA requires imply strategies for optimizing gene transfer efficiency in routing through endosomes and lysosomes. Apparent virus-independent approaches to gene therapy.

Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.Satellite cells are a lineage derived from somites that reside under the basement membrane of the myofiber and are responsible for replenishing skeletal muscle during growth in the postnatal period and in response to exercise and injury in the adult animal (24). As muscles hypertrophy, the satellite cells divide and fuse in order to increase the complement of myonuclei in myofibers. Transplant studies in chick embryos indicate that satellite cells are unable to take part in muscle embryogenesis, suggesting that they serve a specialized function as a presumptive skeletal muscle stem cell (3). Satellite cells are required to both proliferate and terminally differentiate, but invariably the proportion of satellite cells in a muscle remains constant, independent of both the age of the animal and the size of the muscle, and returns to this fixed value at the conclusion of muscle regeneration following injury (23).Understanding the molecular mechanisms responsible for the switch between proliferation and differentiation in satellite cells could be a key to understanding skeletal muscle regeneration in response to disease and trauma. However, relatively little is known about the mechanisms at work to ensure maintenance of the satellite cell population. Most of this knowledge comes from embryological studies of mesoderm differentiation in somites or the developing limb bud that have been extrapolated to tissue culture models of satellite myoblast differentiation. As with embryonic myogenesis, expression of MyoD or t...

Abstract. We report on a general strategy for engineering dominant negative mutations that, in principle, requires neither extensive structural or functional knowledge of the targeted protein. The approach consists of fusing the lysosomal protease cathepsin B (CB) to a subunit of a multimeric protein. The CB fusion polypeptide can proteolytically digest the multimer and/or detour the multimer from its usual subcellular destination to the lysosome. We first demonstrate the general validity of the approach with CB fusion to E. coli lacZ, encoding tetrameric 13-galactosidase. Cotransfection of NIH 3T3 cells with a vector expressing a CBlacZ fusion inhibits the 13-galactosidase activity produced by transfection of lacZ alone. We infer that the dominant negative inhibition results from both direct proteolysis of the 13-galactosidase tetramer by the fusion subunit and detour of the tetramer to the lysosome. In a specific application of this strategy, we have fused CB to the dimeric bHLH skeletal muscle transcription factor MyoD. The CB-MyoD fusion protein localizes to the cytoplasm, presumably the lysosome, demonstrating the dominance of lysosomal localization to nuclear localization. The CB-MyoD fusion appears to divert homodimerizing native MyoD from its usual nuclear destination, consequently inhibiting MyoDmediated transactivation and in vitro differentiation of C2C12 myoblasts. Surprisingly, the CB-MyoD fusion fails to interact with the bHLH heterodimerization partners, El2 and E47, suggesting preferential MyoD homodimer formation, at least in the prenuclear cellular compartments.