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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2014)

The post-translational modification of serines and threonines of intracellular proteins by N-acetylglucosamine (O-GlcNAc) is becoming increasing implicated in processes which control embryonic stem cell self-renewal and transcriptional regulation of pluripotency. While O-GlcNAc and its requirement for embryonic development were described nearly 30 and 15 years ago, respectively, our molecular understanding of this dependence is severely lacking. Historically, this is due to the lack of strategies to directly detect O-GlcNAcylated proteins and unambiguously identify their sites of modification. The work reported here utilizes recent advancements in lectin-based O-GlcNAcylated peptide enrichment strategies, coupled with electron transfer dissociation mass spectrometry, to identify and characterize O-GlcNAc modified proteins in embryonic stem cells. This work has established that many transcriptional regulatory proteins responsible for stem cell maintenance are O-GlcNAc modified. One such example, SOX2, a transcription factor essential for establishing and maintaining pluripotency, possesses a complex modification occupancy pattern where the major form is O-GlcNAcylated. This thesis reveals that SOX2 O-GlcNAcylation is inhibitory for pluripotency transcriptional networks. Our results suggest a potential crosstalk between O-GlcNAc and phosphorylation in regulating SOX2 function, where the O-GlcNAc may prevent a nearby phosphorylation. Although O-GlcNAc signaling is essential for stem cell self- renewal we show that the connection between O-GlcNAc and transcriptional regulation, especially with respect to pluripotency, is more complex than previously appreciated.

    Cover page: Detection and Characterization of O-GlcNAc in Embryonic Stem Cells
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)
    Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
      Cover page: A Neo-Substrate that Amplifies Catalytic Activity of Parkinson’s-Disease-Related Kinase PINK1
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2013)
      Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
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