1

Molecular Cell 53, 521–533, February 20, 2014

Hexokinase-II Positively Regulates Glucose Starvation-Induced Autophagy through TORC1 Inhibition

Waqas Ali

M.Phil., 1MA

Registration number: 2022-ag-20

​

​

Mammalian Molecular Genetics Lab (MMGL)

Presented to Dr. Muhammad Ali

2

Shigeki Miyamoto, PhD

​

Professor, Oncology

Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636, USA

• TPL2 kinase regulates the inflammatory milieu of the myeloma niche. Blood, 2014.
• Evaluation of glycomic profiling as a diagnostic biomarker for epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev, 2014. 23(4): p. 611-21.
• Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell, 2014. 53(4): p. 521-33.
• Pineal parenchymal tumor of intermediate differentiation: Treatment outcomes of five cases. Mol Clin Oncol, 2014. 2(2): p. 197-202.
• A novel pathway links oxidative stress to loss of insulin growth factor-2 (IGF2) imprinting through NF-kappaB activation. PLoS One, 2014. 9(2): p. e88052.
• Factors contributing to mortality and morbidity in pregnancy-associated intracerebral hemorrhage in Japan. J Obstet Gynaecol Res, 2014. 40(5): p. 1267-73.

Molecular and Cellular Pharmacology

Cellular & Molecular Biology

Molecular Biosciences

Cancer Biology

3

Autophagy signaling control

4

Two mechanism to explain of mTORC1 inhibition

5

Energy production through glycolysis and KREB cycle

• Increase in autophagy confers cardioprotection against energy depletion induced by starvation or ischemia ( Rabinowitz and White, 2010).

​

• HK-II plays a significant role in Akt-mediated mitochondrial protection ((Miyamoto et al., 2008).

​

• HK-II competes with apoptotic Bcl-2 family proteins, such as Bax and t-Bid, to prevent outer mitochondrial membrane rupture (Pastorino et al., 2002).

​

• Glucose deprivation activates autophagy through TORC1 inhibition (Moruno et al., ‎2012)

​

• Signal integration between glycolytic and TORC1/autophagy pathways has not been elucidated??

6

5-thio-glucose=5-TG

Kinase dead = KD

Hexokinase = HK

Rationale of the project

7

Fig.1

Conclusion:

• 16-hours of glucose starvation induces autophagy.
• 2-deoxy glucose (2DG) inhibits autophagy in even 16 h glucose starved cells.

Inhibition of HK-II by 2-Deoxy-D-glucose Decreases Glucose Deprivation-Induced Autophagy

8

Fig.1

Conclusion:

• Mitochondrial pathway is not involved in the glucose starvation induces autophagy.

Inhibition of HK-II by 2-Deoxy-D-glucose Decreases Glucose Deprivation-Induced Autophagy

9

Knockdown of HK-II Attenuates, but Overexpression of HK-II Potentiates, Autophagy Induced by Glucose Deprivation

Fig.2

Conclusion:

• HK-II knockdown inhibits glucose starvation induced autophagy
• HK-II overexpression induces glucose starvation induced autophagy

​

10

Knockdown of HK-II Attenuates, but Overexpression of HK-II Potentiates, Autophagy Induced by Glucose Deprivation

Fig.2

Conclusion:

• HK-II knockdown inhibits glucose starvation induced autophagy
• HK-II overexpression induces glucose starvation induced autophagy

​

11

Conclusion:

• Inhibition of glucose starvation induced autophagy increases cell death
• Overexpression of HK-II decreases cell death under glucose starvation

Knockdown of HK-II Increases, but Overexpression of HK-II Decreases, Cell Death Induced by Glucose Deprivation

Fig.3

12

Conclusion:

• Glucose starvation induces autophagy and inhibits mTORC1 activity
• 2-DG inhibits glucose starvation induced autophagy and enhances mTORC1 activity
• HK-2 overexpression inhibits mTORC1 kinase activity

Astrin inhibits mTOR-Raptor association and recruits Raptor to SGs

Fig.4

13

Conclusion:

• Glucose starvation induced autophagy does not depend on the kinase activity of HK-2

Role of HK-II Catalytic Activity in Regulation of Autophagy

Fig.5

14

Conclusion:

• 5-TG can’t induce autophagy through mTORC1 inhibition
• KD but not wild type HK-II has high potential of inducing autophagy

Role of HK-II Catalytic Activity in Regulation of Autophagy

Fig.5

5-thio-glucose=5-TG

Kinase dead = KD

Hexokinase = HK

15

Conclusion:

• HK-II and mTOR binds each other strongly under glucose starvation
• 2-DG decreases mTOR-HK-II binding even under glucose starvation

HK-II Directly Binds to TORC1, and Association Is Increased by Glucose Deprivation

Fig.6

16

Conclusion:

• Raptor is required for mTOR-HK-II binding
• 2-DG inhibits binding between mTOR and KH-II
• Glucose starvation enhances binding between mTOR and Raptor

HK-II Directly Binds to TORC1, and Association Is Increased by Glucose Deprivation

Fig.6

17

Summary and findings

Highlights

• In the absence of glucose, HK-II stimulates autophagy

• HK-II promotes autophagy by binding to, and inhibiting, TORC1

• The interaction with TORC1 is mediated by a TOS motif in HK-II

• Glucose-6 phosphate appears to suppress the autophagic role of HK-II

Impact:

Hexokinase-II regulates the switch from glycolysis to autophagy during nutrient starvation

Future Perspectives

• Gal9-AMPK interaction is important in medically relevent contexts e.g.
• In Mtb control
• In therapeutic uses such as metabolic disorders, cancer
• QC of cellular organelles
• Metabolic switching
• Cell physiology
• Autophagy
• Defense against intracellular pathogens

​

​

​

Limitations

• Check if any shortcoming

Thanks

​

​

Questions are welcomed