No abstract

The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer.

IntroductionAlzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-β (Aβ) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein (“NPT088”) consisting of the active fragment of g3p and human-IgG1-Fc.MethodsAged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically.ResultsNPT088-lowered Aβ plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice.DiscussionThese observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aβ and tau, the hallmark pathologies of AD.

Signaling by stress-activated mitogen-activated protein kinase (MAPK) pathways influences translation efficiency in mammalian cells and budding yeast. We have investigated the stress-activated MAPK from fission yeast, Sty1, and its downstream protein kinase, Mkp1/Srk1, for physically associated proteins using tandem affinity purification tagging. We find Sty1, but not Mkp1, to bind to the translation elongation factor eukaryotic elongation factor 2 (eEF2) and the translation initiation factor eukaryotic initiation factor 3a (eIF3a). The Sty1-eIF3a interaction is weakened under oxidative or hyperosmotic stress, whereas the Sty1-eEF2 interaction is stable. Nitrogen deprivation causes a transient strengthening of both the Sty1-eEF2 and the Sty1-Mkp1 interactions, overlapping with the time of maximal Sty1 activation. Analysis of polysome profiles from cells under oxidative stress, or after hyperosmotic shock or nitrogen deprivation, shows that translation in sty1 mutant cells recovers considerably less efficiently than that in the wild type. Cells lacking the Sty1-regulated transcription factor Atf1 are deficient in maintaining and recovering translational activity after hyperosmotic shock but not during oxidative stress or nitrogen starvation. In cells lacking Sty1, eIF3a levels are decreased, and phosphorylation of eIF3a is reduced. Taken together, our data point to a central role in translational adaptation for the stress-activated MAPK pathway in fission yeast similar to that in other investigated eukaryotes, with the exception that fission yeast MAPK-activated protein kinases seem not to be directly involved in this process.

Mkp1 (MAPKAP kinase Schizosaccharomyces pombe 1) and Mkp2 are two members from fission yeast of the sub-class of putative MAPK-activated protein kinases in yeasts, the other known members being Rck1 and Rck2 from Saccharomyces cerevisiae. The Mkp1 protein is readily co-immunoprecipitated with Sty1 from S. pombe extracts; Mkp2 shows a weaker interaction with Sty1. In mkp1 mutants, conjugation and meiosis proceed more readily and rapidly than in wild-type cells, in analogy to what was previously found for S. cerevisiae rck1 mutants. Conversely, overexpression of mkp1 + delays meiosis. Mkp1 is phosphorylated in vivo in a sty1 + -dependent manner; this modification is removed when cells are starved for nitrogen, a condition that is conducive to entry into stationary phase and meiosis. Overexpression of mkp1 + , like a sty1 mutation, also causes vegetative cells to elongate. The level of Mkp1 phosphorylation drops as cells enter mitosis. We have localised Mkp1 to the cytoplasm, excluded from the nucleus, in vegetative cells. The Mkp1 protein accumulates in zygotic asci and is concentrated within spores. The mkp2 + gene has no noticeable impact on meiosis. Mkp2 is excluded from the nucleus in vegetative cells, and is concentrated at the septa of dividing cells. Mkp2 does not accumulate in meiotic cells.