1. Measuring Cholesterol: From Man to Yeast and Back
Peter Espenshade
Department of Cell Biology

2. Cellular Control of Metabolism
Cells require tight regulation of metabolism to maintain
homeostasis and viability.
Tell story of temperature control in your house. Heating and cooling system is meant to maintain temperature stability or homeostasis and it does so by controlling two sets of reactions, heat and the air conditioning. My lab is interested in understanding how cells control different reactions to maintain this stability. In particular, in any one of these systems there must be some device that actually measures what the temperature is so that the system knows whether to turn on the heat or the AC.
Jürgen Berger/Max Planck Institute
Metabolism = the chemical reactions in a cell or organism
Homeostasis = control of a system keep it stable or constant

3. Cells Adapt to Their Environment to Maintain Homeostasis
Central Vein
Low oxygen
 O2
Hepatic Artery
High oxygen
Eukaryotes require environmental oxygen for essential metabolic processes. However, cells exist in microenvironments with varying oxygen supply and thus initiate biological responses to maintain homeostasis. Show here is Liver lobule. The cells near hepatic arteries are well oxygenated and cells near the central veins are poorly oxygenated. These cells adapt to different oxygen supply. Understanding the mechanism underlining oxygen sensing and regulation is important not only for normal physiology but also for some disease.
Adapted from

4. Tumor Cells Adapt to Their Environment to Maintain Homeostasis
Eukaryotes require environmental oxygen for essential metabolic processes. However, cells exist in microenvironments with varying oxygen supply and thus initiate biological responses to maintain homeostasis. Show here is Liver lobule. The cells near hepatic arteries are well oxygenated and cells near the central veins are poorly oxygenated. These cells adapt to different oxygen supply. Understanding the mechanism underlining oxygen sensing and regulation is important not only for normal physiology but also for some disease.
Semenza, 2011
Semenza, 2008

5. Control of Cholesterol and Fat Synthesis
Acetyl-CoA, NADPH, O2, ATP
Cholesterol
& Fat
Synthesis
SREBP
Multiple pathways are known to regulate cholesterol synthesis. One example of a negative feedback regulation is the control of the SREBP transcription factor by the end product cholesterol. SREBP activates transcription of cholesterol biosynthetic enzymes and cholesterol inhibits SREBP to reduce pathway flux when synthesis is sufficient. An example of a feed-forward mechanism is that of AMP activated kinase which senses energy supply and anticipates whether there will be enough substrate for synthesis. Our lab is interested in understanding mechanisms by which cells sense these molecules and how they adapt their metabolism in response to changes in their supply. Our studies in this area began with efforts to understand how cells sense levels of cholesterol to regulate the SREBP transcription factors. To introduce SREBP,
Cholesterol
& Fat

6. SREBP Controls Synthesis of Cholesterol and Fat
Normal Liver
High SREBP
Korn et al., J. Clin. Invest. 1998, 102:

7. Loss of SREBP Prevents Diabetic Fatty Liver
SCAP is a new therapeutic target for fatty liver. Corrects steatosis in rodent models of diabetic, high fat and high carbohydrate models of fatty liver and hypertriglyceridemia.
Moon et al Cell Metab. 15:

8. Talk Outline Yeast SREBP is controlled by oxygen
SREBP is required for virulence of
pathogenic fungi.
What can this tell us about human cancer?
Talk is broken into three parts. First I would like to give you an introduction to the SREBP pathway in fission yeast. Then tell you what learned about the regulation and physiological function of SREBP in yeast. And finally I will finish with a fun little story about the function of SREBP in fungal pathogenesis.

9. Conservation of SREBP in Fungi
This slide shows a brief summary of the conservation of SREBP. Found in fungi, but not the commonly studied budding yeast S. cerevisiae, so we began this work in the fission yeast S. pombe.

10. SREBP Responds to Oxygen in Fungi[ O2 ]
SREBP
The main finding from these studies is that oxygen signals to SREBP in fungi to control the hypoxic response. Today I will tell you about two new oxygen sensing mechanisms that act to regulate the activity of this hypoxic transcription factor.
Gene expression required
for low oxygen growth

11. Control of Cholesterol and Fat SynthesisX
Acetyl-CoA, NADPH, O2, ATP
Cholesterol
& Fat
Synthesis
SREBP
Multiple pathways are known to regulate cholesterol synthesis. One example of a negative feedback regulation is the control of the SREBP transcription factor by the end product cholesterol. SREBP activates transcription of cholesterol biosynthetic enzymes and cholesterol inhibits SREBP to reduce pathway flux when synthesis is sufficient. An example of a feed-forward mechanism is that of AMP activated kinase which senses energy supply and anticipates whether there will be enough substrate for synthesis. Our lab is interested in understanding mechanisms by which cells sense these molecules and how they adapt their metabolism in response to changes in their supply. Our studies in this area began with efforts to understand how cells sense levels of cholesterol to regulate the SREBP transcription factors. To introduce SREBP,
Cholesterol
& Fat

12. Conservation of SREBP in Fungi

13. Cryptococcus neoformans
Human opportunistic fungal pathogen
Infects ~30% of patients with AIDS in Africa and Southeast Asia
Common diseases:
- meningoencephalitis
- pneumonia
It is conserved, hypoxic transcription factor. Hypothesized that cells may encounter a hypoxic environment in the host and therefore may require Sre1 for growth in host.

14. Cryptococcus neoformans
21% O2
14% 3%
As an extension of our studies in fission yeast, we have examined the function of Sre1 in a pathogenic fungus that is from a different phyla from S. pombe, Crytpococcus neoformans.
Hypothesis: SREBP is required for growth in animals.
June Kwon-Chung – NIH, NIAID

15. SREBP is Required for C. neoformans Infection
7 days post-infection via tail vein

16. Conservation of SREBP in Fungi
To conclude, I wanted to say that Sre1 is a conserved hypoxic regulator. While it is absent in yeast, we have performed similar studies in the pathogen C. neoformans and others have extended our studies to Aspergillus fumigatus. In both organisms, Sre1 is a hypoxic transcription factor. Interestingly, … and we are pursuing it as a potential antifungal target.

17. SREBP is Required for A. fumigatus
Low Oxygen Growth
Wild-type
sre1D
Hypoxia 1% O2, 5% CO2, 94% N2
Willger et al. PLoS Pathogens 2008

18. SREBP is a Conserved Virulence Factor and Antifungal Drug Target

19. Tumor Cells Adapt to the Environment to Maintain Homeostasis
Eukaryotes require environmental oxygen for essential metabolic processes. However, cells exist in microenvironments with varying oxygen supply and thus initiate biological responses to maintain homeostasis. Show here is Liver lobule. The cells near hepatic arteries are well oxygenated and cells near the central veins are poorly oxygenated. These cells adapt to different oxygen supply. Understanding the mechanism underlining oxygen sensing and regulation is important not only for normal physiology but also for some disease.
Semenza, 2011
Semenza, 2008

20. Is SREBP a Therapeutic Target
for Pancreatic Cancer?
Tumor Hypoxia (Low O2)
SREBP
Seed funding from Goldman Pancreatic Cancer Center led to grant from the Pancreatic Cancer Action Network
Cholesterol & Fat Synthesis
Tumor Growth

21. CRISPR Knockout of SREBP Pathway
Figure 1. Pa03c cells lacking SCAP.

22. Is the SREBP Pathway a Therapeutic Target
for Pancreatic Cancer?
Tumor Hypoxia (low O2)
SREBP
Drugs
Seed funding from Goldman Pancreatic Cancer Center led to grant from the Pancreatic Cancer Action Network
Cholesterol & Fat Synthesis
Tumor Growth

23. Summary
Yeast SREBP is controlled by oxygen and required for fungal pathogenesis.
SREBP may be required for pancreatic tumor growth and a new therapeutic target for this devastating disease.
- Studies of a simple yeast can provide important insights into human disease.

24. AHN-JHU Cancer Research Fund
CURRENT LAB MEMBERS
Risa Burr
Sara Clasen
He Gu
Jiwon Hwang
Meredith McGuire
Sumana Raychaudhuri, PhD
Wei Shao, PhD
Shan Zhao
COLLABORATORS
C. neoformans
June Kwon-Chung – NIH
Pancreatic cancer
Anirban Maitra – SKCCC
Zeshaan Rasheed – SKCCC
espenshadelab.com
FUNDING
AHN-JHU Cancer Research Fund