HUANG - LECTURE 3 Flashcards
protein degradation
purposes of protein degradation
Regulated Responses: cytokines released in immune responses must be degraded after infection clearance.
Quality checkpoint: degrade misfolded, damaged, or foreign proteins.
Recycle: break down into amino acids to be reused in protein synthesis.
Metabolism: amino acids can feed into the Citric acid cycle, ketone body, and fatty acid metabolism
what are the different types of proteases
Different types of proteases
Serine proteases (Chymotrypsin): use serine at active site
Threonine, Cysteine, Aspartate, Glutamate.
Metalloproteases: require a metal ion for catalysis.
what are some protein degradation signals/half life determinants
- N-end rule:
N-terminal residue influences/associated with half-life:
Valine → long half-life
Arginine/glutamine → short half-life - PEST:
Sequences like PEST motifs mark proteins for degradation.
Can be exposed upon misfolding or conformational change. - Regulation can occur by: Ligands, folding, modification
- Cellular state
what are the two main pathways of degradation
Two main pathways of degradation: Lysosome & Proteasome
Lysosomal degradation:
Degrades endocytosed macromolecules and damaged organelles (autophagy).
Ubiquitin-Proteasome System (UPS):
Major cytosolic/nuclear degradation system.
what is the ubiquitination cascade
E1: Activating enzyme. It forms a thioester bond with ubiquitin (C-terminal to E1 cysteine group, ATP-dependent).
E2: Conjugating enzyme. Receives ubiquitin from E1. From the same thioester bond (also cysteine group of E2)
E3: Ligase. Recognizes substrate and transfers ubiquitin from E2 to lysine side chain of substrate.
Two different types of E3s
Adapter (RING-like): act as a scaffold, no thioester intermediate
Enzymatic (HECT-E3): forms intermediate thioester bond with ubiquitin before transferring it to the target protein.
what are the relative numbers of E1/E2 and E3 enzymes?
E1: 2/E2: ~30. /E3: >1000 (substrate-specificity)
what are some other proteins similar to ubiquitin
SUMO, NEDD8, ISG15
Also require multiple enzymes to target proteins
They usually regulate protein function or interactions, usually not targeted for degradation
what are the types of ubiquitination
- Mono-ubiquitination: single ubiquitin on one lysine.
- Multi-mono: multiple lysines each with one ubiquitin.
- Polyubiquitination:
A chain of ubiquitin molecules is formed on a single lysine residue of the substrate
Each ubiquitin is linked to the previous ubiquitin lysine via its C-terminal glycine
There are multiple lysines in ubiquitin molecules 🡪 different kinds of poly-ubiquitination
E.g. K48-linked → proteasome degradation.
K63-linked → signaling, trafficking, endocytosis ….
what is the 26S proteasome composed of
Composed of:
20S core (protease activity): 14 subunits
Two 19S regulatory caps (substrate recognition, unfolding, translocation): 20 subunits
Has ATPase, which is required to unfold the proteins, allowing it to transport through the 20S core to be degraded
what is the function of the 26S proteasome and what happens after
Degrades proteins into 2–25 amino acid peptides.
Peptides then can:
Be recycled into single amino acids
Be used for antigen presentation or other important functions
DUBs (Deubiquitinating enzymes): remove ubiquitin from substrates
mechanism of proteasome processing
- K48-linked polyubiquitinated proteins are recognized by the 19S regulatory subunit of the 26S proteasome.
- Requires ATP for binding, unfolding, and translocation into the 20S core.
- Protein is degraded into 2–25 amino acid peptides.
- Ubiquitin is removed and recycled by proteasome-associated DUBs.
- Proteasome-associated DUBs
USP14/UCH37:
Counteract E3 ligase
Remove ubiquitin chains before degradation, preventing recognition by the proteasome.
RPN11: Recycling of the ubiquitin. - Cytosolic DUBs:
Exist in the cytosol.
Balance E3 ligase activity: Difference between E3 ligase and DUBs determine the fate of proteins
what are mechanisms of regulation of degradation
Regulate E1, E2, E3’s
Regulate DUBs
Regulate ubiquitin
Ubiquitin Degradation Mechanisms
“Piggyback” Mechanism:
If DUBs do not remove ubiquitin chains efficiently, ubiquitin is degraded along with substrate.
Decreases overall cellular ubiquitin levels
Tail-up Ubiquitin:
A ~20 amino acid C-terminal extension is cleaved during proteasomal degradation.
Occurs independently of polyubiquitination.
Ubiquitin Self-Degradation (being ubiquitinated)
Ubiquitin can be ubiquitinated (K48-linked) and targeted for degradation itself.
what are some examples of targeted protein degradation technologies
- PROTAC (Proteolysis Targeting Chimeras) degraders:
Bivalent molecules:
One domain binds the protein of interest (POI).
Another domain binds an E3 ligase.
Brings POI into proximity of E3 ligase → promotes the ubiquitination and proteasomal degradation.
Enables degradation of previously undruggable targets - Molecular Glues:
Small molecules that bind POI and induce a conformational change.
Enhances E3 ligase recognition and targeting for degradation.
role of proteasome in antigen processing
Degrades foreign proteins (e.g., from viruses or bacteria).
The resulting peptides (2–25 residues) are transported into the endoplasmic reticulum (ER), loaded onto MHC Class molecules, and finally presented on the cell surface for immune surveillance.
features of lysosomal degradation
Highly acidic organelle: Cells stomachs, pH 4.8.
Contains acid hydrolases
lysosome substrates vs Lysosome targeted proteins
Degrades Damaged organelles/Extracellular component via endocytosis
Generally Nonspecific, use enzyme protease called cathepsins
Antigen Presentation via Lysosome:
Degraded peptides are presented via MHC Class II pathway.
pulse chase experiments
Assess protein half-life and degradation kinetics.
Steps:
Incubate cells with radioactively labeled amino acids (pulse).
Proteins synthesized during “pulse” are labeled.
Replace media with non-radioactive amino acids (chase).
Track the decay of labeled protein over time.
IP the protein of interest, SDS-PAGE & autoradiography
Example: IRP2 protein has a half-life of ~6-8 hrs. under normal conditions, but <2 hrs. when FAC (a degradation trigger) is added.
cycloheximide (CHX) chase assay
Cycloheximide halts translation by blocking ribosomal elongation at the E site.
The degradation of existing proteins can be tracked over time by Western blot.
Example: RPPJK protein has a half-life of ~4–6 hrs; stabilization observed with P38 MAP kinase inhibitor (SB203580)
pulse chase or CHX?
Pulse-chase is more specific and less likely to cause secondary effects.
Cycloheximide chase is simpler but may alter cellular enzyme levels indirectly.
Short-term because it can cause adverse effects (inhibit general translation)
Pulse-chase is preferred because it does not inhibit general translation
how do some therapeutics inhibit the proteasome and the applications of that
MG132 /Bortezomib:
Peptide aldehydes that inhibit the 20S proteasomal catalytic core.
Covalently bind to active sites, inactivating proteolytic function.
Blocks protein degradation → accumulation of target proteins.
Example: Stabilization of p53 tumor suppressor protein and upregulation of its target gene PUMA.
Bortezomib is FDA-approved for multiple myeloma.
Despite p53 stabilization, proteasome inhibition may also:
Stabilize oncogenic proteins (e.g., MYC).
Not clear how proteasome inhibition benefits cancer.
CFTR protein characteristics
CFTR Protein:
A transmembrane chloride ion channel, pump Chloride ions from inside to the outside of the cell
Mutations lead to sticky mucus building up on the outside of the cells
Proper folding occurs in the ER (not efficient proper folding)
~70–80% of wild-type CFTR (not successfully folded) is degraded via ERAD (ER-associated degradation)
The remaining 20-30% of wild-type folded CFTR proteins are further modified in Golgi and secreted to the cell surface
∆F508 Mutation: Single amino acid deletion (phenylalanine at position 508).
Causes 100% misfolding, mutant CFTR is degraded via ERAD
western blot results of CFTR mutation
Wild type: Two bands
Band B: immature (ER)
Band C: Fully glycosylated, mature (Golgi-processed).
F508 Del Mutant: Predominantly Band B, little to no Band C.
Small Molecules to correct the folding problems:
e.g. Latondiune A corrects the folding problems
Restores 50% of chloride channel activity
features of parkinson’s disease
α-Synuclein: Neuronally expressed protein involved in synaptic vesicle regulation
Normally secreted or degraded
Mutations or misfolding overtime lead to protein aggregation
Aggregates are cleared via Proteasome/lysosome
Proteasomal degradation for cytoplasmic proteins (UPS Autophagy)
Lysosomal degradation by Neighboring neurons (via endocytosis) or Microglia (resident brain macrophages)
If have the mutation, enhanced protein aggregation leads to toxic buildup, neuroinflammation, and neurodegeneration.
alzheimer’s disease and the therapeutic approach
Alzheimer’s Disease
Tau Protein: Stabilizes microtubules in axons.
Mutations lead to tau tangles (intracellular aggregates).
Also leads to microtubule destabilization
Amyloid-β:
Normally cleared by microglia.
Mutations → increased aggregation and formation of amyloid plaques.
Plaques are highly stable and difficult to degrade.
Finally lead to neuroinflammation and neurodegeneration.
Therapeutic Approach:
Ultrasound stimulation activates microglia.
Enhances clearance of amyloid plaques in mouse models.
Visualized using Thioflavin staining or Aβ-specific antibodies.
prion disease characteristics
Prion Protein (Protease Resistant protein): Role in the nervous system, immune system, muscles, etc
Normal cellular form (PrPc): α-helix
Membrane proteins are anchored to the membrane by GPI. Engaged in cell communication
Pathogenic Form (PrPsc): beta-sheets – Prion form
β-sheet-rich, protease-resistant 🡪 Forms amyloid fibrils and plaques.
Amyloid fibrils are very stable
Can recruit additional proper folded cellular proteins, and spreads via seeding, leading to exponential plaque formation.
Source of the disease:
Scrapie (sheep) 🡪 Mad Cow disease 🡪 prion disease
