Faculty Bibliography

Presenilin 1 (PS1), presenilin 2, and nicastrin form high molecular weight complexes that are necessary for the endoproteolysis of several type 1 transmembrane proteins, including amyloid precursor protein (APP) and the Notch receptor, by apparently similar mechanisms. The cleavage of the Notch receptor at the 'S3-site' releases a C-terminal cytoplasmic fragment (Notch intracellular domain) that acts as the intracellular transduction molecule for Notch activation. Missense mutations in the presenilins cause familial Alzheimer's disease by augmenting the 'gamma-secretase' cleavage of APP and overproducing one of the proteolytic derivatives, the Abeta peptide. Null mutations in PS1 inhibit both gamma-secretase cleavage of APP and S3-site cleavage of the Notch receptor. Mice lacking PS1 function have defective Notch signaling and die perinatally with severe skeletal and brain deformities. We report here that a genetic modifier on mouse distal chromosome 1, coinciding with the locus containing Nicastrin, influences presenilin-mediated Notch S3-site cleavage and the resultant Notch phenotype without affecting presenilin-mediated APP gamma-site cleavage. Two missense substitutions of residues conserved among vertebrates have been identified in nicastrin. These results indicate that Notch S3-site cleavage and APP gamma-site cleavage are distinct presenilin-dependent processes and support a functional interaction between nicastrin and presenilins in vertebrates. The dissociation of Notch S3-site and APP gamma-site cleavage activities will facilitate development of gamma-secretase inhibitors for treatment of Alzheimer's disease

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons

The phosphoprotein (P) of human parainfluenza virus type 3 (HPIV 3) plays a central role in the viral genome RNA transcription and replication. It acts as an essential cofactor of the RNA polymerase (L) by forming a functional L-P complex, binds to the genomic N-RNA template to recruit the L-P complex for RNA synthesis, and interacts with the nucleocapsid protein (N) to form the encapsidation complex (N-P). We have earlier demonstrated that the P protein forms oligomers (B. P. De, M. A. Hoffman, S. Choudhary, C. C. Huntley, and A. K. Banerjee, 2000, J. Virol. 74, 5886-5895) and in this article we identified the putative oligomerization domain of the P protein and studied the role of this domain in transcription. By computer analyses, we have localized a high-score coiled-coil motif characteristic of oligomerization domain residing between the amino acid residues 423 and 457 of the P protein. Deletion of 12 amino acid residues within this coiled-coil motif (P Delta 439-450) completely abrogated oligomerization, whereas deletion in other regions outside the motif had no significant effect. The mutant P Delta 439-450 was both defective in mRNA synthesis in vitro and minigenome transcription in vivo. Interestingly, the mutant interacted with L to form L-P complex, albeit less efficiently, while its interaction with N protein to form N-P complex and with N-RNA template was similar to the wt P protein. Our results indicate that oligomerization provides a key function to the P protein in the transcription of HPIV 3 genome RNA.

A synapse is a stable adhesive junction between two cells across which information is relayed by directed secretion. The nervous system and immune system utilize these specialized cell surface contacts to directly convey and transduce highly controlled secretory signals between their constituent cell populations. Each of these synaptic types is built around a microdomain structure comprising central active zones of exocytosis and endocytosis encircled by adhesion domains. Surface molecules that may be incorporated into and around the active zones contribute to modulation of the functional state of the synapse

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways

Our studies of radiogenic carcinogenesis in mouse and human models of breast cancer are based on the view that cell phenotype, microenvironment composition, communication between cells and within the microenvironment are important factors in the development of breast cancer. This is complicated in the mammary gland by its postnatal development, cyclic evolution via pregnancy and involution, and dynamic remodeling of epithelial-stromal interactions, all of which contribute to breast cancer susceptibility. Microscopy is the tool of choice to examine cells in context. Specific features can be defined using probes, antibodies, immunofluorescence, and image analysis to measure protein distribution, cell composition, and genomic instability in human and mouse models of breast cancer. We discuss the integration of image acquisition, analysis, and annotation to efficiently analyze large amounts of image data. In the future, cell and tissue image-based studies will be facilitated by a bioinformatics strategy that generates multidimensional databases of quantitative information derived from molecular, immunological, and morphological probes at multiple resolutions. This approach will facilitate the construction of an in vivo phenotype database necessary for understanding when, where, and how normal cells become cancer

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.

The metabolite retinoic acid has been implicated as a key player during anterior-posterior patterning in vertebrate embryos. Recent studies in zebrafish extend this model by demonstrating the influence of retinoic acid on endoderm regionalization

Scn8a encodes an abundant, widely distributed voltage-gated sodium channel found throughout the central and peripheral nervous systems. Mice with different mutant alleles of Scn8a provide models of the movement disorders ataxia, dystonia, tremor and progressive paralysis. We previously reported that the phenotype of the hypomorphic allele of Scn8a, medJ, is dependent upon an unlinked modifier locus, Scnm1. Strain C57BL/6J carries a sensitive allele of the modifier locus that results in juvenile lethality. We now provide evidence that the modifier acts on the splicing efficiency of the mutant splice donor site. Mutant mice display either 90% or 95% reduction in the proportion of correctly spliced mRNA, depending on modifier genotype. The abundance of the channel protein, Na(v)1.6, is also reduced by an order of magnitude in medJ mice, resulting in delayed maturation of nodes of Ranvier, slowed nerve conduction velocity, reduced muscle mass and reduction of brain metabolic activity. medJ mice provide a model for the physiological effects of sodium channel deficiency and the molecular mechanism of bigenic disease.