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

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

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 http://www.siumed.edu/

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

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

SREBP Controls Synthesis of Cholesterol and Fat Normal Liver High SREBP http://www.ncbi.nlm.nih.gov/pmc/articles/PMC509159/pdf/1022050.pdf Korn et al., J. Clin. Invest. 1998, 102:2050-2060.

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. 2012 Cell Metab. 15: 240-246.

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.

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.

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

Control of Cholesterol and Fat Synthesis X 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

Conservation of SREBP in Fungi

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.

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

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

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.

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

SREBP is a Conserved Virulence Factor and Antifungal Drug Target

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

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

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

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

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.

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