Cellular Metabolism and Ethical Decision Making Notes

Week 13 – Cellular Metabolism, Part 1 Day 1

• Learning Objectives:
  • Describe how cells obtain energy sources.
  • Identify different types of membrane transport proteins based on energetics (passive vs. active transport), direction of transport, and concentration of transported molecules.
  • Analyze how different types of membrane transporters work together to transport glucose transport across the membrane of intestinal epithelial cells.
• Keywords:
  • Phagocytosis
  • Lysosome
  • Catabolism
  • Passive (diffusion) vs. active transport (primary vs. secondary)
  • Membrane transporters (channel vs. carrier vs. pump)

Cellular Metabolism Overview

• Cellular metabolism begins with nutrient uptake and concludes with nutrient oxidation.
• Figure 1 illustrates the major catabolic pathways associated with cellular metabolism.

Step 1: Acquiring Energy-Rich Molecules

• Cells and multicellular organisms obtain energy sources through various means.

Focus on Glucose Metabolism

• The discussion primarily focuses on glucose metabolism.
• For final oxidation, cells need to transport glucose into the cytoplasm.

Nutrient Uptake by Single-Cellular Heterotrophs

• Single-cellular heterotrophs mainly use phagocytosis (cell eating) to uptake nutrients.
• Engulfed material is digested within a lysosome, and macromolecules are transported to the cytoplasm.
• Lysosomal proteins are translated into the ER, then transported to the Golgi body, and finally fuse with endosomes.
• Lysosomal enzymes target the catabolism of macromolecules.
• Figure 2 depicts cellular ingestion, including phagocytosis and autophagy (self-eating to remove damaged organelles or proteins).

TopHat Question: Pseudopod Formation

• Actin drives pseudopod formation during phagocytosis, similar to protrusions during cell crawling.
• The correct answer is C. Actin.

Nutrient Availability for Heterotrophs

• Heterotrophic single-cell and multicellular organisms transport available nutrients, such as glucose, into the cell.
• Figure 3 shows cells of the digestive tract absorbing nutrients from food in the small intestine.
• Cells of the small intestine absorb molecules into the cytoplasm.

Can Glucose Cross the Cell Membrane?

• Glucose interacts with water molecules via hydrogen bonds.
• Membrane transporters are essential for transporting molecules across the plasma membrane.

Membrane Transporters Overview

• Membrane transporters are integral proteins that facilitate molecule transport across the plasma membrane.
• Passive Transport: Utilizes kinetic energy and diffusion to transport molecules down a concentration gradient.
• Active Transport: Uses energy (e.g., ATP) to transport molecules up a concentration gradient.
• Figure 9 illustrates passive vs. active transport.

Passive Transport: Simple and Facilitated Diffusion

• Passive transport includes simple diffusion and facilitated diffusion.
• Facilitated diffusion uses an integral membrane protein (channel or carrier) to transport solutes.

Channel Proteins

• Channel proteins create an opening or pore for molecule diffusion across the membrane.

Carrier Proteins

• Carrier proteins bind to the solute and change conformation to transport the solute across the membrane.
• Glucose transporters (GLUT) exemplify carrier-mediated facilitated diffusion.

Active Transport: Primary and Secondary

• Active transport uses specialized carrier proteins: pumps (primary) and other carrier proteins (secondary).
• Primary transporters use a direct energy source to transport a solute, indicated by option C.

Summary: Types of Membrane Transporters

• Passive Transport
  • Simple diffusion
  • Channel-mediated: Creates a pore for molecules.
  • Carrier-mediated: Molecules bind to the transporter protein.
• Active Transport
  • Primary (Pump-mediated): Uses ATP or energy.
  • Secondary (Carrier-mediated): Uses an existing electrochemical gradient.
• Figure 10 provides examples of membrane transporters.

TopHat Question: Channel vs. Carrier-Mediated Diffusion

• The key difference is that molecules must bind to a carrier-mediated protein, which then changes shape to move the molecule across the membrane.
• The correct answer is D.

Transporters Working Together

• Many cellular functions rely on multiple membrane transport proteins.
• Glucose absorption in human intestines involves three different transporters.

Week 13 – Cellular Metabolism, Part 1 Day 2

• Learning Objectives:
  • Define a redox reaction and its importance in glucose metabolism.
  • Outline and analyze the regulation of biochemical pathways.
  • Define allosteric sites and analyze their impact on enzyme function.
  • Compare and contrast the role of equilibrium and irreversible reactions in glycolysis regulation.
  • Describe the cellular location of different steps in glucose metabolism.
• Keywords:
  • Enzymes
  • Pyruvate
  • Acetyl CoA
  • Catabolism vs. anabolism
  • Reduction vs. oxidation
  • Metabolic flux
  • Le Chatelier’s principle
  • Allosteric regulation
  • $\triangle G$ when zero vs. negative (equilibrium vs. irreversible)

Intermediate Products of Catabolism

• Once glucose is in the cytoplasm, pyruvate and acetyl-CoA are major intermediate products of catabolism (Stage 2).
• Figure 1 illustrates the major catabolic pathways.

Redox Reactions and Carbohydrate Catabolism

• Redox reactions are central to metabolic reactions, with carbohydrate catabolism being a major source of energy.
• Redox Reactions: Involve the transfer of electrons.
  • Loss of electrons: Oxidation
  • Gain of electrons: Reduction
• Energy released from electron transfer is used to synthesize ATP via oxidative phosphorylation.

TopHat Questions: Glucose Oxidation

• Carbon dioxide is produced after glucose is fully oxidized.
• The correct answer is D. Carbon dioxide.
• When coenzymes NAD+ and FAD+ accept electrons, they become more reduced.
• The correct answer is B. more reduced.

Biochemical Pathways

• Focus on carbohydrate metabolism, noting that some reactions are reversible.

Metabolic Flux

• Metabolic flux describes the rate at which products move through a biochemical pathway, often through different organelles.
• Cells control flux through various mechanisms.

Thermodynamics and Chemical Reactions

• $\Delta G$ represents the change in free energy of a reaction.
• Using $\Delta G$, we can predict if a reaction is:
  • Reversible ($\Delta G = 0$, at equilibrium)
  • Irreversible ($\Delta G$ is negative)
• These characteristics are important for regulating biochemical pathways.
• Enzymes, which are proteins, catalyze these reactions.

Regulation of Metabolic Flux

• Metabolic flux can be regulated in several ways:
  • For steps at equilibrium ($\Delta G = 0$), the concentration of products and reactants determines the reaction direction (Le Chatelier’s principle), and enzymatic activity maintains equilibrium.
  • For irreversible steps (\Delta G < 0), the rate of the reaction can be regulated through allosteric control of the enzyme.
  • Enzymes can also be regulated by post-translational changes (e.g., phosphorylation) or transcriptional changes (typically mediated by hormones).

Allosteric Regulation

• Allosteric activation or inhibition involves a molecule binding to an allosteric site (non-active site) of an enzyme, inducing a conformational change that alters its function.
• Figure 7 illustrates the active and allosteric sites of T. gondii bacterial pyruvate kinase.

Pyruvate Kinase Regulation

• Pyruvate kinase produces pyruvate as the final step in glycolysis and is allosterically regulated and modified by phosphorylation and acetylation.

TopHat Question: Mechanisms of Biochemical Control

• Allosteric regulation is the fastest form of regulation.
• The correct answer is A. Allosteric regulation.

Irreversible Steps in Glycolysis

• Steps 1, 3, and 10 have large -$\Delta G$ values, indicating they are highly regulated.
• Figure 4 shows $\Delta G$ values of enzyme reactions regulated during glycolysis/gluconeogenesis.

Glycolysis Overview

• Glycolysis is the first step in glucose catabolism.
  • Involves ten steps.
  • Seven steps are at equilibrium, with flux controlled by Le Chatelier’s principle.
  • Three steps have large -$\Delta G$ values, with flux controlled by enzyme regulation.

TopHat Questions: Glycolysis

• If hexokinase is inhibited, glucose levels would stay the same or increase.
• The correct answer is a. glucose levels would stay the same or increase.
• The biochemical reactions of glycolysis occur in the cytoplasm.
• The correct answer is B. Cytoplasm.

Week 14 – Cellular Metabolism, Part 2 Day 1

• Learning Objectives:
  • Describe the cellular location of glucose metabolism steps.
  • List the inputs and outputs of glycolysis, the citric acid cycle, and the ETC.
  • Analyze the role of the inputs and outputs.
  • Describe energy transfer from glucose to create an H+ gradient used by ATP synthase.
  • Define ROS and how they are generated.
• Keywords:
  • Glycolysis
  • Citric acid cycle (CO2, NADH, FADH)
  • Pyruvate vs. Acetyl CoA
  • Electron transport chain (Complex I, II, III, IV, V)
  • Mitochondrial Inner Membrane Space
  • Mitochondrial Matrix
  • ROS (Reactive Oxygen Species)
  • Irreversible steps of glycolysis

Glycolysis and Pyruvate Conversion

• Glycolysis produces ATP, NADH, and pyruvate.
• Pyruvate is converted to Acetyl-CoA while being transported into the mitochondria.

Citric Acid Cycle and Oxidative Phosphorylation

• Acetyl-CoA (and other intermediates) are further oxidized by the citric acid cycle.
• Oxidative phosphorylation in the mitochondria drives the final step of ATP production.
• Figure 1 illustrates the major catabolic pathways.

The Citric Acid Cycle

• Before entering the Citric Acid Cycle:
  • Pyruvate is converted to Acetyl-CoA
  • Produces CO2 and NADH
• Most of the ATP in glucose catabolism is generated in the mitochondria via oxidation of NADH and FADH2.

Oxidative Phosphorylation and ATP Generation

• Oxidative phosphorylation in the mitochondria generates ATP via the transfer of electrons and protons.
• Protein complexes in the inner membrane of the mitochondria use electrons from reduced intermediates to create a proton gradient.
• ATP synthase uses this gradient to generate ATP via phosphorylation of ADP.

Complex I

• Complex I oxidizes NADH, transfers electrons to a coenzyme, and pumps protons into the intermembrane space.
• Electron transfer is driven by the electronegativity difference of complexes and coenzymes; each subsequent complex or coenzyme has a greater electronegativity than the previous one.
• NADH + H+ + CoQ => NAD+ + CoQH2 + 4H+ (Intermembrane space)
• 4 H+ ions are pumped from the matrix to intermembrane space

Complex II

• Complex II oxidizes FADH2, transfers the electrons to a coenzyme

Complex III

• Complexes III oxidizes CoQH2, transfers the electrons (e-) to another coenzyme (cytochrome c), and pumps protons (H+) into intermembrane space
• Protein complexes in the inner membrane of the mitochondria use the electrons from reduced intermediates to create a proton gradient. This gradient is then used by ATP synthase to generate ATP via phosphorylation of ADP.
• CoQH2 + 2 cytochrome c (Fe3+) + 2H+ (matrix) => CoQ + 2 cytochrome c (Fe2+) + 4 H+ (intermembrane space)
• 4 H+ ions are pumped from the matrix to intermembrane space

Complex IV

• Complexes IV oxidizes cytochrome c, transfers the electrons (e-) to oxygen, and pumps protons (H+) into intermembrane space
• The electrons are ultimately accepted by oxygen. The reduced oxygen binds to H+ to form water.
• 2 cytochrome c (Fe2+) + O2 + 4H+ (matrix) => 2 cytochrome c (Fe3+) + 2H2O + 2H+ (intermembrane space)
• 2 H+ ions are pumped from the matrix to intermembrane space

Complex V: ATP Synthase

• Complexes V (ATP synthase) uses the gradient created by the other complexes to produce ATP
• The electrons are ultimately accepted by oxygen. The reduced oxygen binds to H+ to form water.
  • ADP + Pi + H+ (intermembrane space) => ATP + H2O

Reactive Oxygen Species (ROS)

• During electron transport in the ETC, high-energy electrons can escape from the complexes.
  • Electrons can interact with oxygen to form reactive oxygen species (ROS).
  • Superoxide (O2-) is a common ROS.
  • ROS can damage proteins and DNA, leading to mutations and even apoptosis (cell death).

TopHat Questions: Electron Transport Chain

• The oxidation of FADH2 by the ETC results in fewer ATPs being produced compared to NADH because the electrons from NADH have more opportunities to move protons.
• The correct answer is A. The electrons from NADH have more opportunities to move protons.
• Inhibition of complex IV would result in an increase in ROS production and a decrease in NADH oxidation.
• The correct answer is D. A & C

Antioxidants and Free Radicals

• Antioxidants can help terminate reactions caused by free radicals.

Week 14 – Cellular Metabolism, Part 2 Day 2

• Learning Objectives:
  • Analyze energy status (high ATP vs. low ATP) of a cell to predict metabolic flux.
  • Identify major molecules and enzymes that regulate glycolysis.
    • How does flux mediate the regulation?
    • What steps are regulated?
    • Why are specific steps regulated (related to $\Delta G$) and explain which direction these steps function?
• Keywords:
  • Catabolic vs. anabolic
  • Glycolysis vs. glycogen
  • Irreversible steps of glycolysis
  • ATP, ADP, NADH, citrate, G-6-P
  • Energy status (high vs. low)

Glycogen Storage

• During times of high glucose, cells store glucose as glycogen via glycogenesis.
• Glycogen is a storage form of glucose in animal cells produced via anabolic reactions.
• The process requires the regulation of glycolysis to ensure glucose is stored when in excess.

Allosteric Regulators of Glycolysis

• Allosteric regulators of glycolysis direct the flux of glucose and glucose intermediates through glycolysis (low energy) or through glycogen synthesis (high energy).
  • G-6-P buildup indicates an excess of glucose.
  • Pi, ADP, and AMP indicate low energy in the cell.
  • ATP and citrate indicate high energy in the cell.

Regulation of Glycolysis

• Allosteric regulation of glycolytic enzymes. When interpreting this diagram, think about how the concentration of molecules relates to energy status. For example, if ATP is high, what does that say about the cell? Is more glycolysis needed?
• Lactate is produced in anaerobic respiration to regenerate NAD+ so glycolysis can continue.

TopHat Questions: Glycolysis Regulation

• Citrate allosterically inhibits phosphofructokinase because high levels of citrate indicate a surplus of substrate in the citric acid cycle, so inhibition will redirect glucose to storage.
• The correct answer is B.
• A mutation in phosphofructokinase that disrupts the allosteric binding site for ADP would mean that it would take longer to convert glucose to pyruvate during low-energy states compared to wildtype regulation.
• The correct answer is B.

Week 14 – Cellular Metabolism, Part 2 Day 3

• Learning Objectives:
  • Describe the energy and oxygen status of tumor cells.
  • Outline the two major factors that cause tumor cells to favor anaerobic vs. aerobic respiration.
  • Compare and contrast normal cellular metabolism and the Warburg Effect.
• Keywords:
  • Cell division and proliferation
  • Hypoxia
  • Warburg Effect (aerobic glycolysis)
  • Lactate
  • GLUT1
  • HIF1 and p53

Characteristics of Tumor Cells

• Tumor cells are characterized by rapid division (proliferation).

Aerobic vs. Anaerobic Respiration

• Energy-starved cells would favor anaerobic respiration (glycolysis to lactate production only).
• The correct answer is A.

The Warburg Effect

• Logic suggests that cancer cells would undergo aerobic respiration to maximize ATP output, however, cell division and proliferation increase the need for ATP and building blocks for new molecules (anabolism).
• Glycolytic intermediates provide many of those building blocks.

Cancer Metabolism

• Cancerous cells have altered gene expression that results in an increase in GLUT1 transporters, increasing glucose uptake.

Cancer Metabolism Summary

• Cancerous cells alter metabolism to favor anaerobic respiration EVEN WHEN OXYGEN IS PRESENT (The Warburg Effect).
  • Hypoxic conditions in tumors may help make this transition.
  • The benefit of producing macromolecule building blocks and reduced ROS favor glycolysis and lactate production.
  • High glucose transport into cancerous cells fuels rapid glycolysis.
  • This results in ATP levels similar to those produced by aerobic respiration.

Week 15 – Ethical Decision Making Day 1

• Learning Objectives:
  • Outline the framework for ethical decision making.
  • Apply the framework for ethical decision making to a range of ethical questions.
• Keywords:
  • Facts vs. opinions
  • Stakeholders
  • Bias
  • Moral feelings/judgments

Ethical Decision-Making Framework

• Gut Reactions: Initial thoughts related to the ethics of the scenario.
• Gather the Facts: Important questions to ask, facts vs. opinions, knowledgeable sources, and applicable laws or regulations.
• Stakeholders: Identify those directly impacted, consider diverse voices, and alternative viewpoints.
• Determine the Ethical Issue: Personal or group biases, multiple meanings of words, ethical values involved, and moral feelings or judgments.
• Explore Possible Solutions and Actions: Potential consequences, impacts on parties involved, prevention measures, and addressing moral judgments and feelings.

Practice Ethical Decision Making

• Pick an ethical dilemma and practice using the framework.

Week 15 – Ethical Decision Making Day 2

• Learning Objectives:
  • Outline basic epigenetic mechanisms related to maternal diet and fetus genome.
  • Compare and contrast white vs. brown adipose tissue.
  • Apply the framework for ethical decision making to a range of ethical questions.
• Keywords:
  • Brown vs. white adipose
  • Adipogenesis
  • DNA methylation
  • Heterochromatin vs. euchromatin

Epigenetics, Maternal Diet, and Disease

• Ethical Dilemma: Epigenetic changes influence the development of white, beige, and brown adipocytes, resulting in changes in body adiposity and risk for metabolic diseases.

Ethical Scenario: Maternal Diet and Epigenetics

• Scenario: The American Association of Pediatricians recommends pregnant people eating three or more meals a week at fast food restaurants can predispose their children to obesity and other potential metabolic disorders. They also propose an exit form that requires pregnant people to sign off that they will consider the food they consume during pregnancy in light of its implications of fetal epigenetic reprogramming.

Week 15 – Ethical Decision Making Day 3

• Learning Objectives
  • Describe the function of the CRISPR system in its organism of origin.
  • Compare and contrast the different components of the CRISPR system.
  • Describe Identify potential problems that still exist with CRISPR.
  • Analyze the potential ethical concerns with CRISPR.
• Day 3 – Keywords
  • cluster of regularly interspaced repeats
  • crRNA, tracrRNA, guide RNA (gRNA)
  • Cas 9
  • Germ-line vs. somatic
  • CCR5

What is CRISPR?

• CRISPR
  • Clustered
  • Regularly
  • Interspaced
  • Short
  • Palindromic
  • Repeats
• CRISPR is a molecular process that acts as a viral defense in bacteria

CRISPR, a bacterial Immune System

• Phage (virus) inject their genome into host
• CAS proteins recognize it as viral DNA
• A small piece is taken from phage DNA (protospacer) and incorporated into the bacterial genome
• This creates A cluster of regularly interspaced repeats – a memory of past viral infections

How it Works?

• If the virus attacks again…CRISPR acts as an immune system
• The stored bits of viral DNA are transcribed into RNA called crRNA
• crRNA binds with another structural RNA (tracrRNA, nonspecific) to for a guide RNA or gRNA
• The guide RNA uses sequence complementarity to guide Cas9 to the viral DNA. (Remember SNRNPS, tRNAs and miRNAs?)
• Cas 9 cleaves (cuts) the target nucleotides (DNA or RNA) and inactivates it

What can we do with CRISPR?

• We can make our own gRNA specific to gene of interest

• Removing errors in DNA and fixing the double-strand DNA or…

• Adding new DNA and fixing the double-strand DNA

  TOPHAT QUESTION

• The correct answer to this question according to the PowerPoint is B Germline mutation

Potential Problems With CRISPR

• Remaining Hurdles: how many mismatches can the guide RNA tolerate
• Off-site targeting – similar to primer binding in PCR
• Could lead to unintended genomic editing or a mosaic of edited vs. unedited cells

What is it Good For?

• Potential Applications
• Basic Science – Understanding how genes function and interact – Genetic screens
• Therapeutic tool – A subset of known diseases caused by a single nucleotide polymorphism.
  • Duchenne muscular dystrophy
  • Hemophilia
  • Huntington’s Disease
  • Sickle-cell anemia
• Innovative crops – Withstanding drought, enhanced yield, pathogen resistance

Initial Thoughts

• Okay to cure…
  • Parkinson’s Disease?
  • Baldness?
  • Shortness?
  • Short lifespan?
  • Mandated gene editing?
  • Tailness?

What is CCR5?

• CCR5 normally binds to cytokines (ligands) that initiate cell signaling pathways related to inflammation in the body.

Are There Ethical Concerns

• Ethical Concerns
  • Is using CRISPR on germ line cells unethical?
    • Changes are passed on to future generations
    • Changes the human gene pool
    • Are you perfect? Would you be allowed to exist in this future?
  • Is not using CRISPR unethical?
    • If it is possible to end suffering, how can we not?