Lec 15 - Protein folding and degradation

Where are cytosolic proteins synthesized and folded

On free ribosomes in the cytosol; folding occurs in the cytosol.

Where are membrane and secreted proteins synthesized and folded

Synthesized co-translationally into the ER via the translocon; folded in the ER lumen.

What directs ribosome–mRNA complexes encoding secretory/membrane proteins to the ER

N-terminal signal sequences (signal peptides).

Approximately what fraction of proteins are membrane/secreted

About 30%.

What is co-translational targeting

Delivery of the nascent polypeptide to the ER while translation is ongoing.

What is post-translational targeting

Delivery of fully synthesized proteins to destinations like nucleus, mitochondria, or peroxisomes.

What encodes the information needed for correct protein folding

The primary amino acid sequence.

What are chaotropic agents and examples

Mild denaturants that disrupt noncovalent interactions; examples include heat, urea, and beta-mercaptoethanol.

What happens to protein function upon denaturation

Loss of function due to structural disruption.

Under what conditions can denatured RNase refold and regain activity

Upon return to native conditions (e.g., temperature reduction and dialyzing away chaotropes) at low protein concentration.

Why does low protein concentration promote refolding in vitro

It minimizes aggregation that otherwise impairs refolding.

What is the hydrophobic effect in folding

Burying nonpolar side chains in the core while polar side chains face the aqueous environment.

Why is cellular folding challenging compared to dilute solutions

Molecular crowding increases aggregation risk, necessitating chaperone assistance.

What experimental approach revealed heat shock protein induction

Pulse-chase assay with radiolabeled amino acids (e.g., methionine).

Name environmental stressors that induce HSPs.

Heat, amino acid analogs, heavy metals, and energy metabolism inhibitors.

Name pathophysiological states that induce HSPs.

Fever/inflammation, hypertrophy, oxidative injury, ischemia, infection, and xenobiotics.

When else are HSPs elevated in unstressed cells

During specific cell cycle stages, with growth factors, development, and differentiation.

Define molecular chaperones.

Proteins that assist folding, refolding, and translocation without being part of the final structure.

What do chaperones preferentially bind on clients

Hydrophobic residues transiently exposed in nascent or misfolded proteins.

What outcomes can misfolding lead to

Aggregation, ubiquitination and proteasomal degradation, or association with neurodegenerative disease.

Where do cytosolic chaperones act

In the cytosol, during and after translation, shielding hydrophobics as they emerge from ribosomes.

What ER processes do chaperones participate in

Folding, the unfolded protein response (UPR), and ER-associated degradation (ERAD).

How do chaperones assist mitochondrial import

Cytosolic Hsp70 maintains a translocation-competent state; mitochondrial Hsp70 assists refolding inside.

Are chaperones ATPases

Yes; ATP binding/hydrolysis regulates client affinity and conformational changes.

What is Hsp40’s role with Hsp70

Delivers clients and activates Hsp70 ATPase to promote lid closure and high-affinity binding.

What structural "lid" closes over the client in Hsp70

An alpha-helical domain that clamps the client upon ATP hydrolysis.

What do NEFs do in Hsp70 cycles

Exchange ADP for ATP, converting to the low-affinity state for client release.

Which Hsp70 state has high client affinity

The ADP-bound (post-hydrolysis) state with the lid closed.

Which Hsp70 state has low client affinity

The ATP-bound state with rapid client exchange.

How do chaperonins promote folding

By sequestering clients in a hydrophilic chamber that shields hydrophobics and relieves crowding barriers.

What is GroEL/GroES architecture

GroEL is a dual-ring tetradecamer (two heptamer rings) capped by a heptameric GroES.

Where does an unfolded client first bind GroEL

The hydrophobic rim of GroEL before ATP-driven injection into the cavity.

What triggers GroES capping and client injection

Client binding plus ATP induces conformational changes that hide hydrophobics and admit the client.

What ejects the folded client from GroEL

ATP hydrolysis and allosteric changes coupled to binding at the opposite ring.

How many ATPs are needed per GroEL chamber cycle

Seven ATP per chamber per cycle.

What is the mammalian analog’s functional logic (Hsp90)

ATP binding closes a single chamber to fold the client; hydrolysis reopens to release it.

Are all chaperones chaperonins

No; all chaperonins are chaperones, but not all chaperones are chaperonins.

What reaction do PPIs catalyze

Cis–trans isomerization of peptide bonds involving proline residues.

Why is proline isomerization rate-limiting

The cyclic structure restrains the peptide bond, making isomerization energetically demanding.

Name three PPI classes.

Cyclophilins, FKBP binding proteins, and parvulins.

Which immunosuppressant binds cyclophilins

Cyclosporin A.

Which immunosuppressants bind FKBPs

FK506 (tacrolimus) and rapamycin.

Are cyclophilins/FKBPs essential for yeast viability

No; yeast lacking all cyclophilins and FKBPs can survive.

With which chaperone do PPIs often form complexes

Hsp90, potentially to coordinate isomerization and conformational maturation.

What is BiP and its role

An ER Hsp70 chaperone that binds nascent chains to prevent premature folding during translocation.

Is the ER lumen reducing or oxidizing

Oxidizing, favoring disulfide bond formation.

What does protein disulfide isomerase (PDI) do

Catalyzes formation and breakage/rearrangement of disulfide bonds between cysteines during folding and correction of misfolding.

Which amino acid forms disulfide bonds

Cysteine.

How is PDI re-oxidized after accepting electrons

By ER oxidoreductin-1 (Ero1), which transfers electrons to O2, generating H2O2.

What does PDI do when disulfides are mis-paired

Reduces or isomerizes incorrect disulfides to achieve the correct pairing.

What triggers the UPR

Excess unfolded proteins causing chaperone depletion at ER membrane sensors.

Name the three key UPR sensors. PERK, ATF6, and IRE1.

What does PERK do

Phosphorylates eIF2 to inhibit most translation while enabling preferential translation of ATF4.

What does ATF6 do upon activation

Translocates and is cleaved to a transcription factor that upregulates UPR target genes.

What does IRE1 do to XBP1 mRNA

Cleaves/splices it to produce active XBP1 transcription factor.

What are major cellular outcomes of UPR activation

Induction of chaperones, ER membrane expansion, translation inhibition, and ERAD gene induction.

What is "distended ER" a sign of

Marked ER expansion during stress/UPR.

What is ERAD

ER-associated degradation, which clears misfolded ER proteins via ubiquitin-proteasome and autophagy pathways.

What is retrotranslocation

Movement of misfolded proteins from ER lumen/membrane back to the cytosol for degradation.

When is ERAD engaged

After UPR fails to restore proper folding and misfolded proteins persist.

What is ubiquitin

A 76-amino-acid protein tag that targets substrates to the proteasome.

What does E1 do

Activates ubiquitin in an ATP-dependent step and carries it for transfer to E2.

What does E2 do

Accepts ubiquitin from E1 and collaborates with E3 to transfer ubiquitin to the substrate.

What does E3 do

Binds the substrate and positions it for ubiquitin transfer from E2; determines substrate specificity.

What is polyubiquitylation

Attachment of multiple ubiquitin units (often in chains) that signal proteasomal degradation.

What do 19S caps do

Recognize ubiquitinated substrates, remove ubiquitin via deubiquitinating enzymes (DUBs), unfold, and translocate substrates into 20S.

Name the three proteolytic activities in the 20S core. .

Trypsin-like, chymotrypsin-like, and caspase-like activities

What happens to ubiquitin after substrate entry

It is removed and recycled by DUBs before substrate degradation.

Is the proteasome membrane-bound

No; it is a large cytosolic multi-subunit complex.

Why must Cyclin B be degraded near mitosis completion

To prevent re-entry into mitosis and allow cell cycle reset.

Therapeutic rationale for proteasome inhibitors in cancer

Induce proteotoxic stress and apoptosis by preventing degradation of damaged/misfolded proteins.

Example proteasome inhibitor used clinically

Bortezomib (Velcade).

What is PrPC versus PrPSc

PrPC is the normal isoform; PrPSc is the misfolded, infectious isoform with beta-rich structure.

How can prions impair protein homeostasis

PrPSc aggregates can occlude proteasome entry, inhibiting degradation.

Name human and animal prion diseases.

Human: CJD and Kuru; Animal: BSE (mad cow) and scrapie.

What neuropathology is typical of prion diseases

Spongiform changes due to neuronal death.

Two hallmark lesions in Alzheimer’s disease

Amyloid-beta plaques and tau neurofibrillary tangles.

Key aggregating protein in Parkinson’s disease

Alpha-synuclein (Lewy bodies).

A protein aggregate implicated in Huntington’s disease

Mutant huntingtin.

A misfolded enzyme linked to some ALS cases

Superoxide dismutase (SOD1).

Aggregating peptide in type 2 diabetes islets

Amylin (islet amyloid polypeptide).

Structural protein aggregation in cataracts

Crystallins.

Example of localized iatrogenic amyloidosis

Injection-site insulin amyloid.

Major proteins in systemic amyloidoses

AL (immunoglobulin light chain), AA (serum amyloid A), and transthyretin (senile systemic).

Define "molecular crowding."

High macromolecule concentration in cells that enhances aggregation risk and impairs spontaneous refolding.

Define "quality control" in the ER.

Mechanisms detecting misfolded/unassembled proteins and directing UPR/ERAD responses.

What are "immature glycans" in ER QC

Incompletely processed N-linked glycans that can flag folding intermediates or misfolded species.

What is "chaperone depletion"

Sequestration of chaperones by unfolded proteins, freeing UPR sensors and triggering signaling.

Define "retrotranslocon."

A conduit that exports misfolded ER proteins back to the cytosol for degradation.

Define "DUBs."

Deubiquitinating enzymes that remove ubiquitin chains from substrates at the proteasome.

Why is ATP/ADP ratio important for chaperones

High cellular ATP favors client release when needed, enabling progression through folding pathways.

What is "preferential translation" in UPR

Despite eIF2-mediated global translation inhibition, select mRNAs like ATF4 are translated.

Hsp70 cycle: list the four key steps.

Hsp40 delivers client -> ATP hydrolysis on Hsp70 closes lid (high affinity) -> NEF exchanges ADP->ATP -> client release (low affinity).

GroEL/GroES cycle: list the three key steps.

Client binds GroEL rim + ATP -> GroES capping and client injection -> ATP hydrolysis and allosteric switch ejects folded client while loading the opposite ring.

PDI/Ero1 redox cycle: summarize.

PDI accepts electrons to form/reshuffle disulfides (becomes reduced) -> Ero1 re-oxidizes PDI using O2, producing H2O2.

UPR integrated outcomes: list three.

More chaperones, ER expansion, and global translation inhibition with ERAD induction.

ERAD route: outline.

Detect misfolded client -> retrotranslocate to cytosol -> ubiquitylation (E1/E2/E3) -> 26S proteasome degradation.

Ubiquitin conjugation: sequence the enzymes.

E1 activates Ub (ATP) -> E2 carries Ub -> E3 binds substrate and facilitates Ub transfer from E2.

Size of ubiquitin

76 amino acids.

26S proteasome core composition

20S core with two alpha and two beta heptameric rings; flanked by two 19S regulatory particles.