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Isolation and characterization of neuronal substrates of the ubiquitin proteasome system

  • Author(s): Keil, Jeffrey McCartney
  • et al.
Abstract

The covalent attachment of ubiquitin to proteins is one of the most common regulatory mechanisms in mammalian cells. It regulates numerous cellular processes through a combination of proteolytic and non-proteolytic forms of ubiquitination. In a neuronal context, the UPS has long been implicated in a variety of neurological disorders, and more recently, in normal neuronal function. Given the potential importance of the UPS in a neuronal framework, a logical step towards better understanding the scope of its function in neurons would involve an examination of the neuronal targets of the UPS. This research began with a large-scale identification of ubiquitinated, neuronal proteins. Using transgenic, His-6-ubiquitin tagged mice, in combination with denaturing nickel-affinity chromatography, we were able to specifically enrich for large amounts of ubiquitinated proteins. We identified over 385 unique proteins, of which 278 passed stringent filters of potential false-positives. With an approximate proteome of 1200 proteins, we concluded that at least 25% of all synaptic proteins were ubiquitinated at a detectable level. Following the screen, I conducted a more in-depth analysis of the more interesting targets. One of the more promising candidates identified was stromal interaction molecule I (STIM1). Characterization of neuronal STIM1 revealed that it was expressed throughout development with stable levels of expression in mature neurons. Furthermore, subpopulations of STIM1 were identified on the surface of hippocampal neurons. Ectopically expressed STIM1 rapidly redistributed into punctate clusters in response to thapsigargin (TG)-induced store-depletion. Proteasome inhibitors significantly increase surface STIM1 levels and peak Ca2+ influx upon TG -induced SOCE. Additionally, overexpression of POSH, an E3 ligase, leads to a decrease in STIM1 surface populations. Together, these results provide compelling evidence for previously undescribed roles of the ubiquitin-proteasome system (UPS) in the regulation of STIM1 and SOCE function. Examination of neuronal Rac1-GEF, kalirin-5, reveals that its stability is affected by CamKII and PP1/2 activity, in a proteasome-dependent manner. The role of kalirin-5 in the brain is yet unknown, but UPS-mediated regulation may provide a platform from which to pursue future studies

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