Faculty Bibliography

Notch-mediated cell-cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor

The proapoptotic factors Reaper, Hid, Grim, and Sickle regulate apoptosis in Drosophila by inhibiting the antiapoptotic factor DIAP1 (Drosophila inhibitor of apoptosis 1). Heat, UV light, x-rays, and developmental signals can all increase the proapoptotic factors, but the control of transcription of the diap1 gene is unclear. We show that in imaginal discs the single Drosophila STAT protein (STAT92E) when activated can directly increase DIAP1 through binding to STAT DNA-binding sites in the diap1 promoter. The STAT92E contribution to DIAP1 production is required for cell survival after x-irradiation but not under unstressed conditions. Because DIAP1 prevents apoptosis after a variety of stresses, STAT92E may have a role in regulating stress responses in general

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the up-regulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response

Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity

Development of a functional organ requires the establishment of its proper size as well as the establishment of the relative proportions of its individual components. In the zebrafish heart, organ size and proportion depend heavily on the number of cells in each of its two major chambers, the ventricle and the atrium. Heart size and chamber proportionality are both affected in zebrafish fgf8 mutants. To determine when and how FGF signaling influences these characteristics, we examined the effect of temporally controlled pathway inhibition. During cardiac specification, reduction of FGF signaling inhibits formation of both ventricular and atrial cardiomyocytes, with a stronger impact on ventricular cells. After cardiomyocyte differentiation begins, reduction of FGF signaling can still result in a deficiency of ventricular cardiomyocytes. Consistent with two temporally distinct roles for FGF, we find that increased FGF signaling induces a cardiomyocyte surplus only before cardiac differentiation begins. Thus, FGF signaling first regulates heart size and chamber proportionality during cardiac specification and later refines ventricular proportion by regulating cell number after the onset of differentiation. Together, our data demonstrate that a single signaling pathway can act reiteratively to coordinate organ size and proportion

Skin cancer is one of the most preventable groups of malignancies; however, skin cancer incidence continues to rise in the United States. The relationship between skin cancer and ultraviolet (UV) radiation is well known. Many interventions to prevent skin cancer by reducing exposure to UV radiation have been employed throughout the United States. Studies show an increase in knowledge and awareness regarding sun exposure and skin cancer. Unfortunately, sun protection interventions are slow at effecting behavioral change. In this review, we examine current barriers facing youth today in regards to sun protection practices, appropriate age groups to target for intervention, proposed methods of sun protection, the influence of role models in changing sun protective behavior, the stages of behavioral change, and characteristics and techniques of sun protection programs that can not only increase knowledge but actually elicit changes in sun protection behavior

Although the iconic mammoth of the Late Pleistocene, the woolly mammoth (Mammuthus primigenius), has traditionally been regarded as the end point of a single anagenetically evolving lineage, recent paleontological and molecular studies have shown that successive allopatric speciation events must have occurred within Pleistocene Mammuthus in Asia, with subsequent expansion and hybridization between nominal taxa [1, 2]. However, the role of North American mammoth populations in these events has not been adequately explored from an ancient-DNA standpoint. To undertake this task, we analyzed mtDNA from a large data set consisting of mammoth samples from across Holarctica (n = 160) and representing most of radiocarbon time. Our evidence shows that, during the terminal Pleistocene, haplotypes originating in and characteristic of New World populations replaced or succeeded those endemic to Asia and western Beringia. Also, during the Last Glacial Maximum, mammoth populations do not appear to have suffered an overall decline in diversity, despite differing responses on either side of the Bering land bridge. In summary, the 'Out-of-America' hypothesis holds that the dispersal of North American woolly mammoths into other parts of Holarctica created major phylogeographic structuring within Mammuthus primigenius populations, shaping the last phase of their evolutionary history before their demise

The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn (34)). Using synthetic peptides, we have previously investigated the role of Asn (34) in the context of simple hydrophobic transmembrane peptides. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-Ala sequence (Leu 12) or Leu residues at the native positions found in PLB (Leu 9). Asn-containing Leu 9 and Leu 12 peptides were synthesized with a single Asn residue located either one amino acid (N+/-1) or one turn of the helix (N+/-4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle.

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida, that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse zona pellucida consists of three glycoproteins that are synthesized solely by growing oocytes and assemble into long fibrils that constitute a matrix. Zona pellucida glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing sperm to undergo acrosomal exocytosis, and preventing sperm from binding to fertilized eggs. Many features of mammalian and non-mammalian egg coat polypeptides have been conserved during several hundred million years of evolution.