Hexokinase 1 attenuates type II death receptor-induced apoptosis

• Author / Creator

  Schindler, Anja

• Deregulated TNF signaling with elevated or decreased levels of TNF-induced apoptosis causes numerous inflammatory and cancerous diseases. Thus, there is a clear need to identify cellular proteins that regulate cell fate in the presence of TNF. RNA interference technology provides an excellent tool to address that problem. It allows the rapid generation of transient protein depletion “mutants” in cell culture, whose behaviour in the context of TNF can be examined. I developed a quantitative high-throughput siRNA assay to identify modifiers of TNF-induced cell death in HeLa cells and screened a set of nine hundred eighty six proteins, which includes the entire set of human kinases and phosphatases and several of their binding partners or related proteins. Of all gene products tested, loss of hexokinase 1 (HK1) resulted in the greatest elevation in TNF-induced death. In secondary assays, I demonstrated that the presence of HK1 attenuates TNF-induced apoptosis. Specifically, HK1 attenuates the processing of key caspases and caspase substrates, and decrease of the mitochondrial membrane potential. The predominantly mitochondrial localization of HK1 prompted me to examine whether HK1 impacted TNF-induced apoptosis at the mitochondria. I found that HK1 constitutively stabilized the mitochondrial membrane potential at least in part through the inhibition of the pro-apoptotic Bcl-2 effector proteins Bax and Bak. In line with these findings, HK1 attenuated Bax translocalization and oligomerization to and at the mitochondria in the absence and presence of an apoptotic stimulus. Finally, I found that attachment of hexokinases to the mitochondria is a prerequisite for mitochondrial integrity and essential for pro-survival functions of hexokinases in TNF-induced apoptosis. These data are the first loss-of-function reports to examine the involvement of HK1 in the transduction of extrinsic apoptotic cues and identify HK1 as a potential target in deregulated TNF signaling.

• Subjects / Keywords

  • SiRNA
  • TNF
  • Apoptosis
  • Cell viability
  • Hexokinase
  • RNAi
  • Mitochondria

• Graduation date

  Fall 2013

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3QH5F

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.