2020 L8 Protein Degradation

Degradation Pathways

• Ubiquitin Proteasome System (UPS)

• Autophagosomal-Lysosomal Pathway (ALP)

• Both are very enzymatically selective

Pathway Determination

• Critical determinant = substrate size

  • UPS degrades single polypeptides, can fit into narrow channel of the proteasome

  • APL degrades larger structures e.g. protein aggregates, organelles or pathogens

UPS

• Major pathway for protein degradation, 80% of protein turnover

• Driven by ubiquitin as a degradation marker - ubiquitin is conjugated to a protein, signal that protein needs to be degraded.

  • Posttranslational modification - protein’s function altered after being synthesised

• Proteins tagged with ubiquitin have short half-lives (minutes to hours)

• Central to the UPS is the cytosolic 26s proteasome

26S Proteasome

• Large multi-subunit protease complex

• Composed of two subunits

  • 20S core protease houses peptidase activity

  • 19S regulatory particle - binds to core protease in ATP presence, contains ATPase domains

Ubiquitin-meidated degradation

• Ubiquitin-tagged substrates bind to regulatory particles, activating ATPase domains

• Conformational change following ATP hydrolysis allows access to the core protease

• Ubiquitin residues are removed and recycled, protease cleaves protein e.g. trypsin-like, chymotrypsin-like and caspase-like

Ubiquitin

• Small protein, contains C-terminal glycine - the site of ubiquitin attachment

• Conjugated to lysine residues in proteins e.g. Lysine 6, 11, 27 etc.

• Ubiquitin tags are diverse and dictate the outcome

  • Polyubiquitination (particularly of lysine 11 and 48) is most potent signal for degradation because it recruits shuttle factors

  • Monoubiqutination alters protein localisation and conformation

Ubiquitin cascade

• Conjugation occurs via enzyme cascade

  1. Ubiquitin activated by ATP, becomes bound to E1

  2. Ubiquitin transferred to E2 and finally to E3

  3. E3 enzymes are ubiquitin ligases, conjugate ubiquitin to a target protein

Additional Roles of UPS

• Small fraction of proteins are immediately targeted for degradation during translation

  • DRIP (defective ribosomal product hypothesis) -hypothesised to provide source of proteins for MHC-I presented antigenic peptides

• Ubiquitination occurs at stalled ribosomes, degrading nonsense mRNA and no-go decay proteins

UPS Molecular Chaperones

Molecular chaperones: stabilise misfolded proteins in a non-aggregated state - proteasome cannot degrade large proteins and aggregates

• E3 ligase CHIP interacts directly with Hsp70 and 90 via its tetratricopeptide domain

• Hsf1 upregulates several E3 ligases

• Hsf1 is ubiquitinated following proteotoxic stress

Endoplasmic Reticulum Associated Degradation (ERAD)

• Transports misfolded proteins from ER to cytosol

• E3 ligase Hrd1 ubiquitinates ER proteins, has a transcolon function

• Valosin (ubiquitin binding factor) contains protein VCP/p97 - transports proteins from ER to cytosol to be degraded

Cystic Fibrosis (CF)

• Lethal autosomal recessive disease caused by mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CTFR) gene

• Common in Europeans, 1 in 25 are carriers

• Associated w respiratory infection due to thickened mucus - CTFR transports chloride ions

CF Mutations

• Characterised by allelic heterogeneity - different mutations at the same gene locus can cause the same disease or phenotype. In cystic fibrosis, multiple mutations in the CFTR gene can lead to similar clinical symptoms.

  • Compound Heterozygosity - genetic condition in which an individual inherits two different mutations in the same gene, one from each parent. In cystic fibrosis, this means a patient may have two different mutations in the CFTR gene that both contribute to the disease.

• CTFR undergoes co- and post-translational folding and core glycosylation in ER

• Fully glycosylated in the Golgi and inserts in the apical membrane

F508Δ

• Deletion of phenylalanine at position 508 - common mutation of CTFR

• Nascent peptides misfolds and is immediately targeted to the proteasome for degradation

• No functional CTFR is localised to the membrane

• Chloride transport is inhibited + Na transport into the cell through ENaC (epithelial Na channels)

Juxtanuclear Quality Control Compartment (JUNQ)

• Centre of ubiquitination and degradation of UPS in response to cell - only one per cell

• Ubiquitinated proteins are sequestered into the JUNQ

• Protein used to be thought as a random process, but recent studies w fluorescent microscopy shows protein aggregation is tightly regulated - sequestered into inclusion bodies

• Many chaperone and proteasome complexes in JUNQ, concentrates proteins for degradation

• @ mitosis mother cell retains JUNQ through asymmetrical inheritance - therapies targeting this mechanism could mitigate neurodegenerative diseases by preventing the buildup of toxic aggregates that contribute to neuronal death.

ALS

• Eliminates large protein aggregates, protein complexes, organelles, pathogens

• Best characterised form: macroautophagy, where double membrane structure autophagosome, engulfs cell material

Autophagy Activation

• Macroautophagy initiated by ULK1 kinase, triggers a kinase cascade

• ATG8 protein complexes (LC3 and GABARAP in higher eukaryotes) begin to assemble

• ATG8 pcs bind to phophatidylethanolamine (phospholipid) on the phagophore

• Phagophore matures into autophagosome, fuses with a lysosome

Ubiquitin, UPS and ALP

• Many autophagy receptors contain a ubiquitin-binding domain, allowing ubiquitinates proteins/organellles to be processed by autophagy in addition to UPS

• Significant crosstalk between UPS and ALP

• Autophagy receptors e.g. p62 contain ubiquitin-binding domains, proteasome binding domains and LIR domains (phagophore)

• ALP can compensate if UPS is overwhelmed - decision of which to use governed by avidity of protein complex

• Heat shock can trigger high ubiquitin levels as UPS proceeds, which promotes autophagy

Lysosome

• Organelles composed of acidic lumen and lysosomal membrane

• Lumen is host to hydrolytic enzymes e.g. nucleases, proteases, phosphatases, lipases, sulfatases

• A vacuolar H+ ATPase transports H+ into the lumen

• Associated membrane proteins include SNARE proteins, LAMP1 and LAMP2

Glycocalyx

• Composed of glycoproteins and glycolipids, covalently attached to membrane of lysosome

• Protects the lysosome from attack by lytic enzymes in cytoplasm

Lysosomes in autophagy

• During macroautophagy autophagosomes fuse with lysosomes through LC3 proteins

• Molecular chaperones can also promote autophagy through CMA (chaperone-mediated autophagy)

• Proteins recognised through KFERQ motifs by certain heat shock proteins e.g. Hsc70

• LAMP2 assists in translocation of Hsc70-bound proteins into lysosomal lumen

Lysosomes in nutrient sensing

• Rag-GTPases and Ragulator localise mTORC1 to the lysosomal membrane

• mTORC1 central to protein, lipid and nucleotide synthesis, and regulates autophagy

• mTORC1 senses amino acid conc. through Sestrin 2 and Castor - bind leucine and arginine respectively

• high amino acids keep mTORC1 active

• Inactivation of mTORC1 through amino acid deprivation causes ULK1 to become active

Organelle degradation

• Driven by selective autophagy to remove defective organelles

• Mitophagy (autophagy turnover of mitochondria) can be ubiquitin-dependant or independant

  • Pink1/Parkin tag outer mitochondria proteins with ubiquitin

  • Mitophagy during erythrocyte maturation is ubiquitin independant

• Ribophagy is ubiquitin-dependant

• ER autophagy is ubiquitin-independant

Autophagy in Cancer

• Basal autophagy prevents DNA damage by degrading reactive oxidative species (ROS)

• Causes accumulation of LC3, increasing phagophore formation

• Cancer progresses > nutrient deprivation in tumour microenvironment promotes autophagy

• Bortezomib (proteasome inhibitor) successful for treating multiple myeloma

  • prolonged inhibition activates autophagy which cancer cells exploit

Insoluble Protein Deposits (IPOD)

• Centre of highly insoluble protein aggregates

• Spatially distinct from JUNQ, multiple can exist in a cell

• Proteins localised to IPODs are terminal

  • ATG8 proteins are highly associated

  • Hsp40 and 70 also localised

• Similar to JUNQ mother cell also retains the deposit