Class 18 transcript

Multivalent interactions in signaling

Adapter proteins with multiple interaction domains engage in multivalent interactions

nucleating to form gel-like meshworks or biomolecular condensates near receptors on the plasma membrane.

provides spatial control and creates efficient "biochemical factories" by concentrating signaling molecules.

Scaffolding proteins

Proteins that sequester and organize multiple signaling molecules (e.g., kinases) into a complex.

Scaffolding proteins

• increase signaling precision (by preventing crosstalk)

• increase speed (by keeping molecules close together)

• limit signal amplification because molecules cannot freely diffuse.

Scaffolding proteins what are they

Proteins that sequester and organize multiple signaling molecules (e.g., kinases) into a complex.

Scaffolding proteins advantages

increase signaling precision (by preventing crosstalk)

increase speed (by keeping molecules close together)

Scaffolding proteins disadvantages

limit signal amplification because molecules cannot freely diffuse.

Scaffolding protein trade-offs

Benefits: precision (no crosstalk between pathways) and speed (molecules are pre-positioned). Downside: limited signal amplification (molecules are sequestered, not freely diffusing to activate many partners). Evolution favors precision/speed or amplification depending on cellular needs.

Tissue homeostasis

The balance between cell death, cell survival, cell proliferation, and differentiation that maintains functional tissues. Disruption (e.g., too much proliferation, too little apoptosis) can lead to cancer.

Apoptosis (morphological features)

Cells detach from ECM and round up, membrane becomes blebby/bubbly, DNA fragments and condenses, organelles (mitochondria, ER) fragment, but membranes remain intact (clean, tidy process).

Apoptotic cells are engulfed by neighbors without spilling contents.

Caspases

A family of proteases that execute apoptosis by cleaving proteins inside the cell.

Caspases are synthesized as inactive pro-caspases and must be cleaved to become active. Activation leads to fragmentation of DNA, cytoskeleton, organelles, and membrane blebbing.

Extrinsic apoptotic pathway

Apoptosis triggered by an external signal (e.g., from immune cells).

Example: cytotoxic T cell presents death ligand (Fas ligand) → binds death receptor (Fas) on target cell → receptor trimerizes → recruits adapter proteins → recruits pro-caspases → caspases cleave each other and activate → caspase cascade.

Common in immune system.

Death receptor (Fas)

A receptor on the plasma membrane that binds death ligand (Fas ligand) presented by cytotoxic T cells.

Upon binding, receptor trimerizes and initiates the extrinsic apoptotic pathway.

Death ligand (Fas ligand)

A ligand presented on the surface of cytotoxic T cells that binds to death receptor (Fas) on target cells, triggering the extrinsic apoptotic pathway to kill infected or abnormal cells.

Extrinsic pathway activation mechanism

1. Ligand binds receptor → receptor trimerizes. 2. Adapter proteins are recruited. 3. Pro-caspases are recruited and brought into close proximity. 4. Pro-caspases cleave each other, becoming active. 5. Active caspases diffuse and activate more caspases (massive amplification).

Intrinsic apoptotic pathway

Apoptosis triggered by internal signals (development, DNA damage, ER stress, hypoxia).

Key feature: involves mitochondria and release of cytochrome c.

Cytochrome c then activates a caspase cascade. Also called programmed cell death, critical for development (e.g., finger formation).

Cytochrome c (apoptosis function)

A mitochondrial protein normally involved in electron transport chain and ATP production. During intrinsic apoptosis, cytochrome c is released from mitochondria into the cytosol, where it binds Apaf-1 to form the apoptosome and activate caspases.

Apaf-1 (Apoptotic protease activating factor 1)

A protein that binds cytochrome c released from mitochondria.

Upon binding, Apaf-1 oligomerizes into a wheel-like structure (the apoptosome) with seven subunits, which recruits and activates pro-caspases.

Apoptosome

A wheel-like heptameric complex (7 subunits) formed by Apaf-1 upon binding cytochrome c.

The apoptosome recruits pro-caspases, bringing them into close proximity so they cleave and activate each other, initiating the caspase cascade.

Bax and Bak

Proteins embedded in the outer mitochondrial membrane that oligomerize to form channels (pores).

When activated, these channels open and release cytochrome c from the mitochondria into the cytosol, triggering intrinsic apoptosis.

They are pro-apoptotic.

BCL-2

A BCL-2 family protein embedded in the outer mitochondrial membrane that binds to Bax/Bak and blocks their channel activity, preventing cytochrome c release.

BCL-2 is anti-apoptotic.

The name comes from B-cell lymphoma, where it is often mutated (overexpressed).

BH3-only proteins

Pro-apoptotic

BCL-2 family proteins that contain only a single BH3 domain.

They bind to and inhibit anti-apoptotic BCL-2 proteins, thereby "releasing the brake" and allowing Bax/Bak to open cytochrome c channels.

Bad is a BH3-only protein.

Double-negative regulation (apoptosis)

Bad (pro-apoptotic) inhibits BCL-2 (anti-apoptotic).

BCL-2 inhibits Bax/Bak (channel formers).

Therefore, Bad activation leads to cytochrome c release and apoptosis. This "double-negative" allows tight control: multiple layers of regulation prevent accidental apoptosis.

Mitochondrial outer membrane permeabilization (MOMP)

The process by which Bax/Bak channels open the outer mitochondrial membrane, releasing cytochrome c and other pro-apoptotic factors into the cytosol.

MOMP is a commitment point in intrinsic apoptosis.

PI3 kinase (PI3K)

A lipid kinase that phosphorylates the membrane phospholipid PIP2 to produce PIP3 (phosphatidylinositol (3,4,5)-trisphosphate).

PI3K is activated by RTKs and is a key player in the cell survival pathway.

Unlike typical kinases, it phosphorylates lipids, not proteins.

PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate)

A phospholipid second messenger produced by PI3 kinase from PIP2.

PIP3 is anchored to plasma membrane

serves as docking site for proteins with PH domains, including the kinases PDK1 and Akt.

PH domain (Pleckstrin Homology domain)

A protein domain that binds specifically to PIP3 on the plasma membrane.

PH domain-containing proteins (e.g., PDK1, Akt) are recruited to the membrane when PIP3 is produced, enabling their activation.

Akt (Protein Kinase B)

A kinase recruited to the plasma membrane via its PH domain binding to PIP3.

Akt is activated by PDK1 and mTORC2.

Active Akt phosphorylates many substrates, including the BH3-only protein Bad (inactivates it), to promote cell survival (by inhibiting apoptosis).

PDK1 (Phosphoinositide-dependent kinase 1)

A kinase recruited to the plasma membrane via its PH domain binding to PIP3.

PDK1 phosphorylates and activates Akt, contributing to the cell survival pathway.

Bad phosphorylation by Akt

Akt phosphorylates the BH3-only protein Bad.

Phosphorylated Bad is bound and sequestered by the protein 14-3-3.

This prevents Bad from inhibiting anti-apoptotic BCL-2, allowing BCL-2 to block Bax/Bak and inhibit cytochrome c release.

Thus, Akt activation promotes survival.

14-3-3 protein

A protein that binds and sequesters phosphorylated Bad, preventing Bad from interacting with and inhibiting anti-apoptotic BCL-2.

This keeps Bad inactive, promoting cell survival.

PTEN phosphatase

A phosphatase that removes the phosphate group from PIP3, converting it back to PIP2.

PTEN antagonizes PI3K signaling, turning off the survival pathway.

PTEN is a tumor suppressor; mutations in PTEN are common in cancer, leading to uncontrolled survival signaling.

Apoptosis detection methods

1. Cytochrome c localization (GFP-cytochrome c: diffuse cytosolic signal = released = apoptosis). 2. Caspase cleavage by Western blot (pro-caspase = large band; cleaved fragments = smaller bands). 3. TUNEL assay (labels DNA fragments). 4. FRET-based caspase sensors.

TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling)

A common assay that labels free DNA ends generated during DNA fragmentation in apoptosis. More labeling = more DNA fragments = apoptosis. Used to detect apoptotic cells in tissues (e.g., developing mouse paw between digits).

FRET-based caspase sensor

A FRET probe consisting of CFP and YFP linked by a peptide sequence containing a caspase cleavage site. Intact = FRET (YFP emission). Caspase cleavage separates CFP and YFP → loss of FRET. Allows live-cell imaging of caspase activation with single-cell and temporal resolution.

FRET advantages over Western blot

FRET provides: 1) Single-cell resolution (Western blot is population-based). 2) Live-cell imaging (temporal information). 3) Spatial localization. 4) Quantitative information (FRET ratio). Western blot only gives population average at a single time point.

PIP3 FRET reporter (PH-GFP)

A GFP-tagged PH domain (binds PIP3).

In basal state, GFP is diffuse in cytosol. Upon EGF stimulation (activating PI3K), PIP3 is produced on plasma membrane → PH-GFP translocates to membrane (visible as green ring).

Signal turns off when PIP3 is depleted (by PTEN or other phosphatases).

PIP3 signaling duration

Upon EGF stimulation, PIP3 is rapidly produced on the plasma membrane, recruiting PH-domain proteins.

The signal is transient; PIP3 is depleted over time by phosphatases (e.g., PTEN), causing PH-GFP to return to the cytosol. This transient signal ensures controlled survival signalin