BIOC 212 Final

What are Cdks?

Cyclin-dependent kinases (Cdks) are enzymes that phosphorylate target proteins, activating them to drive cell cycle forward

Phosphorylated = Active

Non-phosphorylated = Inactive

Cdk Activation & Role of Cyclins

Cdks are inactive alone; they must bind to cyclins to become partially active.

Cyclin binding induces a conformational change, partially activating the Cdk.

Cyclins are produced via protein synthesis and are phase-specific:

- Present only during the cell cycle phase they regulate.

- Destroyed when no longer needed

S-Cdk

what cyclin? what function? when is it active? how does it work?

Cyclin: S-cyclin

Function: Triggers DNA replication

Timing: S-phase

How it works:

Initiates origin firing by:

- Activating Mcm (DNA helicase)

- Recruiting DNA polymerase and replication machinery

Prevents re-replication by:

- Degrading Cdc6

- Exporting extra Mcm from the nucleus

M-Cdk

Cyclin: M-cyclin

Function: Triggers mitosis machinery

Timing: G2 & M-phase

Cdk Activity Through the Cell Cycle

G1 Phase:

- No Cdk activity at the beginning.

- Activity slowly builds toward the end.

S-phase onward:

- High Cdk activity (due to S-Cdk and M-Cdk complexes).

Cdk activity must be precisely controlled to ensure accurate cell cycle progression.

Regulation of Cdk Activity

1. Cdk-Activating Kinase (CAK)

Adds an activating phosphate to M-Cdk

Makes the active site more accessible → Fully activates the Cdk

2. Wee1 Kinase

Adds inhibitory phosphates to Cdk-cyclin complexes

Keeps complex inactive until the proper time (ex: delays mitosis)

3. Cdc25 Phosphatase

Removes inhibitory phosphates added by Wee1

Reactivates the Cdk-cyclin complex

Activated by Polo kinase

In mammals:

- Cdc25A, B, C exist.

- Cdc25C specifically activates Cdk1 at the start of mitosis

4. CKI Proteins (Cdk Inhibitors)

Bind directly to Cdk-cyclin complexes.

Hold them in an inactive state, even if the cyclin is bound

APC (Anaphase-Promoting Complex)

A regulated E3 ubiquitin ligase that triggers the metaphase-to-anaphase transition

Destroys:

• Securin → Activates separase, which cleaves cohesins to separate sister chromatids.

• M-cyclin → Lowers Cdk activity, enabling mitotic exit

Activated by Cdc20 during metaphase (via M-Cdk activity)

Later binds Hct1 after mitosis for continued cyclin degradation

If APC is absent: Cells can't exit mitosis; chromatids remain stuck at metaphase

Unlike SCF, APC itself is regulated, not just its substrates

Cycle Reset: After mitosis, APC is inactivated (due to loss of M-cyclin), allowing M-cyclins to build up for the next cycle

SCF Complex

An E3 ubiquitin ligase that targets phosphorylated proteins for degradation

Works with the proteasome, which actually degrades the proteins

Always active; regulated by phosphorylation of the substrates

- ex of targets: CKIs (ex: Siel in yeast, p27 in mammals)

Difference from APC: The substrate is regulated, not the E3 ligase

E3 Ubiquitin Ligases (General)

Attach ubiquitin to proteins, marking them for degradation by the proteasome.

Always active, but can only target phosphorylated proteins.

Cdk Complexes

G1-Cdk

- Activator: G1-cyclin

- Prepares cell for S-phase

- Phase: G1

G1/S-Cdk

- Activator: Cyclin E

- Initiates transition into S-phase

- Phase: End of G1

S-Cdk

- Activator: Cyclin A

- Triggers DNA replication, blocks re-replication

- Phase: S-phase

M-Cdk

- Activator: Cyclin B

- Triggers mitosis

- Phase: G2 → M-phase

Sic1 (Budding Yeast)

Inhibits S-Cdk in G1

Phosphorylated by Cdk1, which triggers Sic1 destruction and allows S-Cdk activation

Hct1-APC

Maintains APC activity after anaphase and throughout G1.

Keeps M-cyclin levels low to suppress Cdk activity.

Inhibited by Cdk activity.

Works with Sic1 to keep Cdk inactivated in G1.

M-Cdk activity in G1 Phase Entry

As cells exit mitosis, M-Cdk is inactivated

This allows Hct1 and Sic1 to accumulate → Cdk inactivation during G1

Positive Feedback Loops

1. M-Cdk Activation Loop

M-Cdk activates Cdc25 (a phosphatase) and inhibits Wee1 (a kinase), accelerating its own activation

2. Rb-E2F Loop

- G1-Cdk phosphorylates Rb, releasing E2F

- E2F:

• Activates transcription of G1/S- and S-cyclins, boosting Cdk activity

• Stimulates its own transcription, reinforcing the cycle

Origin Recognition Complex (ORC)

Binds to replication origins and stays bound throughout the cell cycle

Assembles pre-replicative complex when not phosphorylated

If phosphorylated, complex cannot assemble

Cdc6 is required for Mcm proteins to be loaded onto the ORC

- Mcm is a DNA helicase (unwinds DNA)

Mcm Proteins

DNA helicase components that load onto DNA at replication origins

Form helicase rings around DNA and are inactive until S-Cdk activation

Cdc6

Helps assemble the pre-replicative complex with ORC and Mcm

Must be expressed and imported into nucleus

After S-Cdk activation, Cdc6 is phosphorylated and degraded, preventing re-replication

E2F Transcription Factor

Activates transcription of:

- G1/S-cyclin (cyclin E)

- S-cyclin (cyclin A)

- Other genes required for S-phase

Positive feedback: E2F stimulates its own gene and further Rb phosphorylation

when Rb is phosphorylated, E2F is released

P21 Gene (CKI)

Activated by p53 in response to DNA damage

Binds and inhibits G1/S-Cdk and S-Cdk complexes

Function:

• Causes G1 arrest

• Prevents entry into S-phase

• Gives time for DNA repair

Yeast Cell Size Control During Starvation

During starvation, yeast cells slow the cell cycle to allow time for growth.

This prevents daughter cells from becoming too small, keeping size relatively stable.

Cell Size Coordination in Budding Yeast (Hypothetical Model)

Small yeast cells have a fixed number of Cln3-binding proteins (bound to DNA).

Cln3-BPs inhibit Cln3 activity

As the cell grows:

Total protein increases → Cln3 levels increase proportionally

Once Cln3 levels exceed the number of inhibitors, free Cln3 activates Cdks

This triggers entry into the next cell cycle

p53 Pathway

DNA damage → Activation of protein kinases → Phosphorylation of p53.

Normally, Mdm2 binds to p53, promoting its ubiquitylation and degradation.

Phosphorylation of p53 blocks Mdm2 binding, allowing p53 to accumulate.

Functions of p53

Transcription factor activated in response to DNA damage.

Activates genes like p21 (CKI), which:

• Arrests the cell cycle in G1 by inhibiting G1/S-Cdk and S-Cdk.

p53 can lead to:

• Cell cycle arrest or apoptosis, depending on the situation.

Regulation of p53:

• Mdm2 phosphorylation: Reduces p53 degradation, allowing it to stay active.

• p19ARF: Inhibits Mdm2, leading to higher levels of p53.

Growth Factors

Function: Stimulate cell growth (increase in size/mass), not cell division.

Examples:

• Insulin

• NGF (Nerve Growth Factor): Supports neuron survival and differentiation in post-mitotic (non-dividing) cells.

Mitogens

function? size? where? two major pathways?

Stimulate DNA replication and cell division.

Small proteins/peptides delivered through the bloodstream.

Bind to cell-surface receptors, activating two major pathways:

1. Ras → MAP kinase cascade → Myc (a transcription factor).

2. Myc → Cyclin D expression → Rb phosphorylation → E2F activation (promotes entry into S phase).

Myc Transcription Factor

Promotes growth and proliferation

Overexpression activates p19ARF, which inhibits Mdm2, leading to increased p53.

Can result in cell-cycle arrest or apoptosis, depending on context

When he grows, Mycat is overexpresses and says p19ARF, which inhibits Mydog’smeow, increasing p53 (ps3) usage

Necrosis

Accidental cell death (e.g., trauma, injury).

Cell explodes, releasing contents into the environment.

Can cause inflammation and tissue damage.

Apoptosis (Programmed Cell Death)

Controlled self-destruction in response to intrinsic or extrinsic signals.

The cell breaks down and is engulfed by a phagocyte.

Key function: match number of developing nerve cells to available survival signals.

- Target cells release limited survival factors → some nerve cells die.

Survival Factors

Molecules released by target cells to suppress apoptosis in neighboring cells.

Bind to receptors on the surface of other cells → activate internal survival pathways.

PKB (Protein Kinase B) Pathway

Activated by survival factor signaling

Promotes cell survival by phosphorylating:

1. Bad (pro-apoptotic Bcl-2 family protein)

• Normally Bad inhibits Bcl-2

• Phosphorylation causes Bad to release Bcl-2, allowing Bcl-2 to inhibit apoptosis and promote cell survival

2. Forkhead transcription factors

• Normally promotes apoptosis (unless phosphorylated)

• Phosphorylation inactivates them, promoting cell survival

Telomerase Function

Adds cap structures (telomeres) to chromosome ends.

In normal cells, telomeres shorten with each division → eventual senescence (~50 divisions).

In cancer cells, telomerase is permanently expressed, making them "immortal"

Cyclin D

G1 cyclin.

Promotes Rb phosphorylation → releases E2F, initiating S-phase gene transcription.

SCF E3 Ubiquitin Ligase

Tags CKIs (ex: p27, Cdk Inhibitors) for degradation

- promotes S-phase entry by targeting and degrading proteins that inhibit DNA replication, like CKIs

- Active when substrate is phosphorylated

Fibroblasts

Type of cells that grow well in cell culture

Require:

- Buffer: Maintains pH ~7

- Medium: Contains salts, glucose, and minerals

- Serum: Provides essential growth components

Serum

Derived from blood (supernatant after centrifugation).

Rich in:

- Growth factors

- Survival factors

- Mitogens (stimulate cell division)

Cell Growth Behavior

Normal cells:

- With buffer, medium, and serum, they proliferate until forming a monolayer (one layer covering the plate).

- Stop dividing upon contact (contact inhibition).

- Need continuous replenishment of medium and serum—depletion halts growth.

- Do not grow on top of each other.

Cancer cells:

- Grow in three dimensions.

- Continue dividing past the monolayer, forming bumps.

- Do not stop growing, ignoring contact inhibition

Tumor Types and Characteristics (Adenoma, Malignant)

Adenoma (Benign Tumor)

- Encased in extracellular matrix, keeping it localized

- Cells are immobile

- Usually small and hard to detect, but curable if found early

Malignant Tumor

- Extracellular matrix is degraded, so the tumor is not contained

- Invades surrounding tissue

- Cells can travel via lymphatic or blood system (metastasis).

Cancer Classifications

Carcinomas

- Originate from epithelial cells.

- Account for ~90% of all cancers.

- Common locations: Oral cavity & pharynx, Digestive organs, Respiratory system, Breast, Reproductive tract, Urinary organs

Melanoma

- Deadly skin cancer that spreads quickly if not caught early.

- Caused by sun exposure.

Leukemias & Lymphomas

- Cancers of blood or lymphatic cells.

- Characterized by liquid, over-proliferating cells.

- Can invade tissues and metastasize.

Sarcomas

- Cancers of: Central nervous system, Brain & spinal cord, Connective tissue, Muscle, Vasculature

- Low incidence but poor prognosis.

Chronic Myelogenous Leukemia (CML)

First solid link between cancer and DNA mutations

Discovery:

CML patients show abnormal chromosomes in blood:

- Chromosome 9 becomes longer

- Chromosome 22 becomes shorter

Philadelphia Chromosome:

Caused by a translocation:

- A piece of chromosome 22 fuses with chromosome 9

Creates a Bcr-Abl fusion gene:

- Abl (from chromosome 9) + Bcr (from chromosome 22)

- Produces a Bcr-Abl protein that is permanently active

Result: Uncontrolled cell proliferation → Leukemia

Clonal Evolution & Tumor Progression

Clonal Evolution

- Cells begin in a bump or benign tumor (e.g., wart) instead of a monolayer.

- If one cell acquires additional mutations, it may:

• Produce a protease that breaks down the basal lamina.

• Begin to invade deeper tissues, progressing toward malignancy.

Basal Lamina

- An extracellular matrix barrier.

- Separates:

• Outer epithelial cells from

• Inner connective tissue, blood vessels, and lymphatic vessels.

Selective Barriers

To become cancerous, cells must overcome:

- Low oxygen levels (hypoxia)

- Scarcity of proliferation signals

In normal cells:

- These barriers cause cells to differentiate or stop dividing.

In tumor precursors:

- Genetic instability increases the chance of acquiring mutations to bypass these barriers.

Balance of Instability

- If genetic instability is too high:

• Cells accumulate harmful mutations.

• Resulting in cell death or slower proliferation.

Stem Cells & Cancer

Normal Stem Cells

- Self-renewing tissue cells.

- Can differentiate into various cell types.

- Normally self-renew only once

- Produce daughter cells with limited proliferative capacity due to telomerase limits

Stem Cells & Tumors

If mutated:

- Stem cells may proliferate indefinitely

- Can contribute to tumor formation

Metastasis: Cancer's Dangerous Trait

Refers to dissemination of tumor cells to other locations in the body

Primary cause of death in cancer patients.

Secondary tumors form far from the primary site.

Steps in Metastasis

1. Tumor cells spread via blood or lymph.

2. Usually eliminated by the immune system.

3. If they survive:

- Adhere to blood vessels in another organ.

- Escape (extravasation) and proliferate in the new organ.

Detection

- After removing the primary tumor:

• Lymph nodes are biopsied to check for spread.

Hormonal Cancers

Breast Cancer: Driven by estrogen

Prostate Cancer: Driven by androgens

Higher incidence in patients with Western diets and lifestyles

Liver Cancer

Liver Cancer

Causes:

- Unhealthy lifestyle (diet/exercise)

- Lack of early screening (e.g., colonoscopy)

Lung Cancer

Rare before industrialization.

Increased due to:

- Smoking culture

- Pollution from industrial processes

Stomach Cancer

Previously common:

- Preserved foods (carcinogenic additives) before widespread refrigeration.

Decline due to:

- Fridge usage

- Monitoring of Helicobacter pylori (bacteria linked to ulcers & cancer)

- Better management of stomach pain

Ames Test

Used to assess if a compound is carcinogenic

Procedure:

1. Mix test compound with:

- Salmonella bacteria (defective in histidine production, won't grow without it)

- Homogenized mouse/rat liver extract (simulates liver metabolism)

2. Observe if bacteria begin to grow (indicating mutation & carcinogenic potential)

Cytochrome P-450: oxidase enzymes in liver convert non-carcinogens into carcinogens

Aflatoxin: A mold toxin that’s harmless until transformed by cytochrome P-450 into a DNA-binding carcinogen

Hepatitis B Virus (HBV)

DNA virus

Causes chronic liver inflammation → DNA damage → liver cancer

Epstein-Barr Virus (EBV)

A herpesvirus

Incorporates C-Myc oncogene into host cells:

- Stimulates host proliferation to aid viral replication

Can cause:

- Burkitt's lymphoma

- Nasopharyngeal cancer

HIV & HTLV

RNA retroviruses that permanently integrate into the host genome

Do not cause cancer directly

Cause immunodeficiency, allowing other viruses to proliferate and cause cancer

ex: HIV indirectly causes Kaposi's sarcoma

Carriers must stay on antiviral drugs for life

Prostate Cancer, Breast Cancer

Prostate Cancer

- Monitored via PSA (Prostate-Specific Antigen) blood test.

- High PSA levels = possible cancer.

Breast Cancer

- Monitored using mammography (X-ray imaging of breast tissue).

Cancer Risk Factors in Women

Reproductive Timing

- Later childbirth increases risk of: Breast cancer and Ovarian cancer

Hormonal Influence

- Before first childbirth, breast tissue is exposed to estrogen and reproductive hormones.

- Pregnancy causes the tissue to differentiate for milk production, reducing lifetime exposure.

p16

A cyclin-dependent kinase inhibitor (CDKI)

Blocks Cyclin D1-Cdk4 complex → prevents Rb phosphorylation

Activated under cellular stress to inhibit cell proliferation

Loss/inactivation of p16 allows uncontrolled proliferation

Considered a tumor suppressor

Rb (Retinoblastoma protein)

what does it do? what happens if its lost?

Inhibits entry into cell cycle when unphosphorylated

Cyclin D1-Cdk4 complex phosphorylates Rb, which:

- Releases its inhibition

- Allows cell cycle progression

Loss or mutation of Rb:

- Prevents cell cycle inhibition

- Encourages proliferation

- Rb is a tumor suppressor

Cyclin D1-Cdk4 Pathway Summary

p16 inhibits Cyclin D1–Cdk4 complex

Cyclin D1–Cdk4 phosphorylates Rb, inactivating it

Phosphorylated Rb releases E2F → activates cell cycle progression

Mutations in Rb or p16 remove this control → leads to uncontrolled proliferation

Oncogene Collaboration: Myc & Ras

Mouse models demonstrate that:

- Myc oncogene alone → increased tumor incidence

- Ras oncogene alone → increased tumor incidence

- Myc + Ras together → much higher tumor incidence than either alone.

• Tumor locations: mammary and salivary glands

• Synergistic effect: their combination promotes cancer more effectively than the sum of each alone

TGFβ (Transforming Growth Factor Beta)

A growth factor involved in colon cancer and many others

Normal Function:

- Binds to TGFβ receptor

- Activates transcription factor pair Smad3/Smad4 via phosphorylation

- Forms complex that moves to the nucleus and induces anti-proliferative genes (ex: p15)

Loss-of-function mutations in TGFβ receptor or Smad3/Smad4…

- results in unchecked proliferation, bypassing tumor suppression ➝ can lead to metastasis

HPV (Human Papillomavirus) and Cervical Cancer

Cervical Cancer

Preventable with Pap smears (Detects abnormally shaped cervical cells)

HPV (Human Papillomavirus)

Type: DNA virus (~8000 nucleotides, circular genome)

Transmission: Sexual contact

Infection Progression

Early Stage:

• Viral DNA remains extrachromosomal

• Causes benign growths (warts/lesions)

Malignancy Risk:

• If untreated, viral DNA integrates into host genome

• Triggers E6 and E7 oncoprotein expression

Viral Oncoproteins

E6 → Binds and degrades p53 (via ubiquitination)

E7 → Binds and inhibits Rb (tumor suppressor)

Together: Inactivate key tumor suppressors → uncontrolled cell proliferation

HPV in Men can lead to penile cancer

Telomeres and Chromosomal Instability

Normal Telomere Function

- With each cell division, telomeres shorten

- Critically short telomeres signal DNA damage

- Triggers replicative senescence (cell cycle arrest)

Cancer and Telomeres

- If p53 or cell cycle checkpoints are mutated, cells ignore telomere damage

- Leads to breakage-fusion-bridge cycle, causing:

• Massive chromosomal damage

- Survival mechanism: telomerase reactivation

• Stabilizes chromosomes

• Allows further mutations ➝ contributes to cancer development

Familial Adenomatous Polyposis (FAP) and MLH Genes

FAP:

• Inherited monogenic cancer syndrome

• Caused by loss of one functional copy of a tumor suppressor gene (usually APC)

• Leads to formation of numerous colon polyps, with high risk of progression to cancer

MLH Genes:

• Part of the DNA mismatch repair (MMR) system

• Repair small replication errors (e.g., point mutations)

• Mutations in MLH (especially MLH1) are linked to Lynch syndrome, a different inherited colon cancer syndrome

Colon Cancer Development Sequence

1. Loss of APC/Wnt signaling pathway

2. ➝ Hyperproliferative epithelium

3. ➝ Loss of p53 ➝ Early adenoma

4. ➝ K-Ras activation ➝ Intermediate adenoma

5. ➝ Loss of Smad4 + other suppressors ➝ Late adenoma

6. ➝ Loss of p54 ➝ Carcinoma

7. ➝ Other unknown changes ➝ Metastasis

Capillary Structure and Angiogenesis

Capillary Structure

- Inner to outer: lumen → endothelial cells → basal lamina → connective tissue → smooth muscle

Angiogenesis

- Process of forming new blood vessels from existing ones

- Triggered when tissue needs more oxygen/nutrients

- Normal roles: wound healing, tissue repair

- In cancer: hijacked to feed tumors

VEGF (Vascular Endothelial Growth Factor)

Specific growth factor for blood vessel growth

Secreted under hypoxic conditions (low oxygen)

Tumors secrete VEGF to grow blood supply

HIF (Hypoxia-Inducible Factor)

Transcription factor that regulates angiogenesis

Low oxygen:

➝ HIF is stabilized

➝ Activates genes: VEGF, TGF-α, PDGF-β, EPO

High oxygen:

➝ HIF degraded via VHL (Von Hippel-Lindau) protein

Von Hippel-Lindau (VHL) Disease

Cause: Inherited mutation in VHL gene (1 defective allele at birth) (tumor supressor gene)

Two-hit hypothesis: Loss of second allele causes tumor formation

Tumor Features: Develop hundreds of vascular tumors in CNS and visceral organs

Mechanism:

- VHL loss → HIF not degraded → cells enter a "pseudo-hypoxic" state

- Triggers constant angiogenesis → supports tumor growth

Sporadic Cases:

- Somatic VHL mutations can cause tumors in non-inherited cases

- Sporadic kidney cancer: Highly aggressive, rising in incidence, may metastasize to bone

Drug Resistance:

- VEGF inhibitors work initially

- Tumors may switch to alternative angiogenic pathways

- Leads to resistance, requiring combination therapy

Oncogenes - Definition, Activation, Function, Behavior, and Viral Oncogenes

mutated forms of proto-oncogenes that gain new, active functions promoting cancer.

Activation Mechanisms:

- Point mutations or deletions, Gene amplification, Chromosomal rearrangements
(changes remove normal regulation like phosphorylation control)

Function:

- Stimulate cell proliferation

- Prevent cell death (anti-apoptotic)

- Promote continuous cell survival and division

- Often become targets for drugs or antibodies

Behavior:

- Dominant mutations → only one altered copy is enough

- Discovered early due to obvious effects in cell behavior

Viral Oncogenes:

- Some viruses hijack proto-oncogenes from host cells

- Viruses lack replication tools, so they force host cells to divide using these oncogenes

- This helps replicate viral DNA and spread infection

Studying Oncogenes - Assays

Contact inhibition assay:

- Normal cells stop growing in a monolayer.

- Oncogene-expressing cells grow in 3D/bumpy layers.

Growth in liquid culture:

- Only oncogene-expressing cells survive and proliferate.

Tumor Suppressor Genes: Function, Mutation, Mechanisms of Gene loss, and Challenges

Function: Prevent uncontrolled cell growth (e.g., p53, Rb, p16)

- Both copies must function properly to suppress tumors

Mutation & Loss:

- Loss-of-function mutations

- Two-hit hypothesis: Cancer forms when both alleles are lost/inactivated

- Some genes (like p53) show haploinsufficiency → partial function lost with just one mutation

Mechanisms of Gene Loss:

Non-disjunction (loss of a chromosome), Non-disjunction + duplication, Mitotic recombination, Gene conversion, Deletion, Point mutation, Epigenetic silencing

Drug Targeting Challenge:

- Unlike oncogenes, tumor suppressors are inactivated

- No active protein = "nothing to block" with drugs

Classic Example – Hereditary Retinoblastoma:

- Inherited 1 mutated Rb copy → only 1 copy left

- Loss of second copy → retinal tumor

- Proof of the two-hit hypothesis

Protein-Protein Interaction Assay

Aims to find proteins that interact with an unknown protein

Steps:

1. Clone the unknown protein into a plasmid for expression.

2. Add an epitope tag (antibody-binding sequence) to the N- or C-terminus.

3. Create an antibody specific to the tag.

4. Transfect cells with the plasmid to express the tagged protein.

5. Lyse cells to release proteins.

6. Add antibody + beads to bind the tagged protein.

7. Centrifuge to pellet the tagged protein + its binding partners.

8. Use mass spectrometry to identify interacting proteins.

Immunotherapy and Targeted Therapy

Goal: Train the immune system to recognize and attack cancer cells.

Method:

- Remove immune cells from the patient.

- Train them to recognize cancer.

- Reintroduce them to the body.

- Use checkpoint inhibitors to enhance immune response.

Challenge: Managing toxicity from overactive immune response.

Cellular Communication - Types of Signaling

Autocrine: Cell affects itself by secreting signals that it also receives.

Paracrine: Cells affect neighboring cells through signaling molecules.

Endocrine: Cells release hormones that travel through the bloodstream to distant cells.

Synaptic: Nervous system-specific, involves neurotransmitters across synapses.

Juxtacrine: Direct contact between adjacent cells (membrane-bound signaling).

Cell Surface Receptors

Receive signals from hydrophilic, protein-like molecules that can’t cross the membrane.

3 Main Classes:

1. Ion Channel-Linked Receptors

- Ligand binding opens/closes ion channels.

- Enable fast synaptic signaling.

2. G Protein-Coupled Receptors (GPCRs)

- Use a trimeric G protein as a signal relay.

- Ligand → GPCR conformational change → G protein activation → Regulates enzymes/ion channels → Intracellular signaling

- Do NOT have a kinase domain.

3. Enzyme-Linked Receptors

- Either have intrinsic enzymatic activity or are enzyme-associated

4 Classes of Enzyme-Coupled Receptors

1. Receptor Tyrosine Kinases (RTKs)

Structure:

- Extracellular domain: Large, glycosylated, binds ligand (ex: growth factor)

- Transmembrane domain: Short, hydrophobic

- Intracellular domain: Contains kinase activity

Autophosphorylation occurs on tyrosines when ligand binds

SH2 domains: Bind phosphorylated tyrosines

SH3 domains: Mediate protein-protein interactions (not phosphorylation-dependent)

2. Receptor Tyrosine Kinase-Associated Receptors

- Function through association with kinase proteins (not intrinsic kinase activity).

3. Receptor Serine/Threonine Kinases

- Similar to RTKs but phosphorylate serine/threonine residues.

4. Receptor Tyrosine Phosphatases

- Remove phosphate groups from tyrosines.

Intracellular Receptors

Bind small, hydrophobic signaling molecules (ex: hormones, vitamins) that cross the membrane

Often function as transcription factors due to a DNA-binding domain

Ras and GTPase Signaling

Ras is a monomeric GTPase:

- Active when bound to GTP

- Inactive when bound to GDP

Sos activates Ras by promoting GDP release.

GTPase-activating proteins (GAPs) enhance Ras's GTPase activity (promote GTP → GDP).

Comparing Kinase Receptors vs. GPCRs

Kinase Receptors

Signal Type: Ligand binding triggers phosphorylation

Mechanism: Phosphorylation creates docking sites for signaling proteins

Activation: Direct phosphorylation by receptor’s intrinsic kinase domain

Structure: Transmembrane receptor with built-in kinase activity

GPCRs (G-Protein Coupled Receptors)

Signal Type: Ligand activates a G protein

Mechanism: G protein stimulates enzymes or ion channels

Activation: Indirect via conformational change

Structure: No kinase domain, 7 transmembrane segments

Protein Phosphatases

Enzymes that remove phosphate groups from proteins.

Important because phosphorylation is unidirectional.

Overview of Metabolism

what is catabolism? what is anabolism?

The process by which living systems acquire and use free energy to carry out their functions

It integrates catabolism and anabolism:

- Catabolism: Breakdown of complex molecules (ex: proteins) into simpler ones (ex: amino acids) (Usually coupled with ATP production)

- Anabolism: Synthesis of complex molecules (ex: proteins) from simpler ones (ex: amino acids) (Requires an input of energy (ATP))

Integration: Intermediates from catabolism can serve as building blocks in anabolic pathways

Why is ATP favored as the energy currency?

In cells, ATP:ADP ratio is ~10,000:1.

This creates a large disequilibrium, driving reactions forward and giving a large negative ΔG in practice.

True cellular concentrations determine the actual ΔG, not just standard conditions.

Regulation of Metabolic Pathways and Futile Cycle

Futile Cycle: Happens when opposing pathways run at the same time, wasting energy (ex: ATP used to make fatty acids from acetyl-CoA)

- Cells avoid this through multiple regulatory strategies:

Key Regulatory Mechanisms:

1. Exergonic Pathways:

- Only proceed when overall ΔG is negative (favorable).

2. Coupled Reactions:

- Unfavorable reactions (+ΔG) are driven forward by pairing with highly favorable ones (–ΔG).

3. Covalent Modifications:

- Enzyme activity is regulated by phosphorylation of amino acids.

4. Allosteric Regulation:

- Molecules bind to non-active sites, altering enzyme activity.

5. Compartmentalization:

- Opposing reactions are kept in separate locations within the cell (e.g., different organelles or membrane-bound spaces).

Overview of Glycolysis

Anaerobic breakdown of glucose (6C) into two pyruvates (3C).

Occurs in the cytosol of all cells (including liver and kidney).

Two phases:

Energy Investment Phase

Energy Generation Phase

Energy Investment Phase

1. Step 1: Hexokinase

Converts glucose → glucose-6-phosphate (G6P)

• 1st ATP used

• Traps glucose inside the cell

• Large ΔG drop

2. Step 3: Phosphofructokinase (PFK)

Converts F6P → FBP (fructose-1,6-bisphosphate)

• 2nd ATP used

• Rate-limiting step

• Inhibited by high ATP

• Large ΔG drop

3. Step 4: Aldolase

Splits FBP → GAP + DHAP (two 3-carbon sugars)

• DHAP is converted to GAP → 2 GAP molecules continue through glycolysis

Energy Generation Phase of Glycolysis

Step 6: GAP → 1,3-BPG

• Unfavorable (+ΔG)

• Coupled with Step 7 to proceed forward

Step 7: 1,3-BPG → 3PG + ATP

• Substrate-level phosphorylation

• Highly favorable (–ΔG), drives Step 6

• Occurs twice (once per GAP) → 2 ATP total

Steps 8 & 9: 3PG → 2PG → PEP

• Conversion via enolase

Step 10: PEP → Pyruvate (via pyruvate kinase)

• Generates 2 more ATP

• Large –ΔG makes this step highly favorable

Total ATP Yield per Glucose from Glycolysis

-2 ATP (investment)

+4 ATP (generation)

+2 NADH → ~5 ATP (2.5 ATP/NADH)

Total: 7 ATP

NAD+/NADH and Lactate dehydrogenase (LDH)

Glycolysis requires NAD+ regeneration to proceed

NADH is an electron carrier (NAD + 2 electrons).

In anaerobic conditions (in cytosol):

- Lactate dehydrogenase (LDH) converts pyruvate to lactate and regenerates NAD+

- Prevents NADH buildup and ensures the GAP → BPG reaction (step 6) can occur (Regenerates NAD⁺ so glycolysis can continue without oxygen)

Major ΔG drops in Glycolysis

1. Hexokinase (Step 1)

2. PFK (Step 3)

3. Pyruvate kinase (Step 10)

These steps drive the pathway and are difficult to reverse (though the liver and kidney can under specific conditions).

Three Phases of Metabolism

1. Glycolysis - Cytoplasm

2. Conversion of Pyruvate to Acetyl-CoA - Mitochondria

3. Oxidation of Acetyl-CoA - Mitochondria

Phosphoryl Potential

Phosphates bound to metabolites are unstable and energetically favorable for transfer.

This "willingness" to transfer phosphate is what drives phosphorylation reactions in metabolism.

Hexokinase Regulation

Catalyzes the first step of glycolysis:
Glucose → Glucose-6-phosphate (G6P)

Main regulation: G6P inhibits hexokinase (negative feedback)

Purpose: Prevents excess glucose use when G6P accumulates (ex: if glycolysis slows down)

Gluconeogenesis vs Glycolysis

Glycolysis breaks down glucose for energy.

Gluconeogenesis is the opposing pathway, generating glucose from non-carbohydrate sources.

Both pathways are regulated to avoid futile cycling (wasting energy)

Phosphofructokinase (PFK) and PFK2

PFK is the rate-limiting enzyme of glycolysis.

Catalyzes: F6P → F1,6 BP (FBP)

Highly regulated by various metabolites and energy status:

PFK2: Can both phosphorylate F6P → F2,6 BP and hydrolyze F2,6 BP back to F6P.

Adjusts F2,6 BP levels to fine-tune glycolysis activity

F2,6 BP allosterically activates PFK1

PFK Inhibitors and Activators

Inhibitors (signal high energy or pathway backup):

• ATP: High levels = no need for more ATP

• Citrate: From CAC (Oxaloacetate + Acetyl-CoA); signals cycle backup

• NADH, Acetyl-CoA: Indicate high energy, inhibit glycolysis

- PFK inhibition causes upstream intermediates like G6P to accumulate → Excess G6P is diverted to glycogen synthesis

Activators (signal low energy):

• ADP & AMP: From ATP use; stimulate glycolysis

• Fructose-2,6-bisphosphate (F2,6-BP):

  • Made by PFK2 (bifunctional enzyme)

  • Strong allosteric activator of PFK1

  • Fine-tunes existing PFK1 activity

Feed-Forward Activation

A metabolic strategy to accelerate glycolysis when glucose is abundant:

1. Glucose → G6P

2. G6P → F6P

3. F6P → F1,6 BP (via PFK)

4. F1,6 BP activates pyruvate kinase (enzyme in final step of glycolysis)

ensures glycolysis proceeds efficiently when upstream intermediates accumulate

Pyruvate Dehydrogenase Complex (PDC)

where is it located? what does it do? reaction mechanism? ATP yield?

Location: Mitochondria

Function: Converts pyruvate → acetyl-CoA for entry into the Citric Acid Cycle (CAC)

Reaction: Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

ATP Yield

- Produces 1 NADH per pyruvate → 2.5 ATP

- 2 pyruvates per glucose, so 5 ATP total from PDC per glucose

- Massive multi-enzyme complex

- Composed of many proteins, notably: E1 (Pyruvate Dehydrogenase, ~30 heterotetramers), E2, E3

Pyruvate Translocase (MPC)

Brings pyruvate into the mitochondria

Uses proton support (H⁺ symport) to sustain the electrochemical gradient

Pyruvate is negatively charged, so H⁺ is co-transported to balance charge

PDC Reaction Steps & Cofactors

1. E1 + TPP (Thiamine Pyrophosphate)

• Decarboxylates pyruvate → releases CO₂

• Forms hydroxyethyl-TPP intermediate

• CO₂ release makes this step irreversible

2. E2 + Lipoamide

• Accepts hydroxyethyl group → forms acetyl-lipoamide

• Coenzyme A (CoA)

  • Replaces lipoamide to produce acetyl-CoA

3. E3 + FAD

• Transfers electrons from reduced lipoamide to FAD → FADH₂

• Then to NAD⁺ → NADH

• Lipoamide is regenerated to continue the cycle

Regulation of PDC

1. Allosteric Feedback Inhibition

- Inhibitors: Acetyl-CoA, NADH (products of the reaction)

- Signal high energy, preventing further pyruvate decarboxylation.

2. Covalent Regulation (Phosphorylation)

- PDH Kinase:

• Phosphorylates E1 → inactivates PDC

• Activated by: ATP, NADH, Acetyl-CoA

• Inhibited by: ADP, Pyruvate

- PDH Phosphatase:

• Dephosphorylates E1 → reactivates PDC

Why Use Multi-Enzyme Complexes?

Minimized substrate diffusion: Faster reactions

Channeled intermediates: Protect unstable intermediates

Limits side reactions

Coordinated control: Turning off one enzyme effectively stops the entire complex

Citric Acid Cycle (CAC)

Location: Mitochondria

Function: Oxidizes Acetyl-CoA → CO₂ and produces high-energy electron carriers (NADH, FADH₂)

Key Characteristics:

Amphibolic: Involves both catabolism and anabolism

• Anabolic: Produces intermediates for biosynthesis

• Catabolic: Oxidizes acetyl-CoA for energy

Reaction: Acetyl-CoA + 3 NAD⁺ + FAD + GDP + Pi → 2 CO₂ + 3 NADH + FADH₂ + GTP + CoA

Citric Acid Cycle (CAC) ATP Yield (Per Acetyl-CoA)

3 NADH → 7.5 ATP

1 FADH₂ → 1.5 ATP

1 GTP → 1 ATP

Total per cycle: 10 ATP

Per glucose (2 acetyl-CoA): 20 ATP

Steps of the CAC

1. Citrate Synthase: Acetyl-CoA + Oxaloacetate → Citrate

- First committed step 2-8. Series of enzyme-catalyzed steps regenerating oxaloacetate

2. Malate Dehydrogenase: Malate → Oxaloacetate

- Completes the cycle

Irreversible Steps with Large Negative ΔG

1. Citrate Synthase (Step 1)

2. Isocitrate Dehydrogenase (Step 3)

3. ⍺-Ketoglutarate Dehydrogenase Complex (Step 4)

- All other steps are reversible.