Proteolysis & the Ubiquitin System

2 Major Proteolytic Pathways

• peptide bonds are cleaved by proteases for processing and degradation

• the proteosome and lysosome constitute 2 major proteolytic machineries in eukaryotic cells

  • proteasome: Degrades specific proteins tagged with ubiquitin (misfolded proteins) → produces short peptides

  • lysosome: breaks down proteins/organelles/aggregates and can go all to way to single AA’s for recycling

Ubiquitin Proteasome System

• degradation by the ubiquitin proteasome system is a 2-step process

• proteins to be degraded are first marked by ubiquitin (by ATP-dependent E1–E3 enzymes) and then degraded into short peptides by the proteasome

• proteasome is the degradation machinery for proteins will cleave off ubiquitin for recycling and then cleave the substrate into shorter peptides

Ubiquitin

• small, abundant, globular protein conserved across all eukaryotic cells

• 76 AA’s

• over 95% identical b/w yeast + human

• the di-glycine residue at the C-ter is critical

• ubiquitin belongs to a family of small proteins that can be conjugated (covalently linked) to substrate proteins via its carboxyl group at a glycine (G76)

Ubiquitylation

• the process that leads to the post-translational modification of conjugating ubiquitin to a substrate protein

• ubiquitylation occurs mostly on lysine residues

Ubiquitylation: Isopeptide bond

• when conjugated to a substrate, ubiquitin forms a stable isopeptide bond that has the same properties as a peptide bond

  • very stable and only cleaved by de-ubiquitinylating enzymes (deubiquitinase, DUBs, a type or protease)

  • b/w carboxyl group of G76 and amine of modified Lys on substrate

Ubiquitin Cascade

Ubiquitylation is mediated by a 3-step process (ubiquitin cascade)

• ubiquitin is first activated by the ubiquitin actiating enzyme (E1) in an ATP-dependent manner

  • E1 forms a thioester bond w/ ubiquitin and transfers it to E2 (b/w Cys of E1 active site and ubiquitin)

  • E1 Cys performs nucleophilic attack on ester bond => thioester bond

• E2 (ubiquitin conjugating enzyme): formation of thioester bond b/w Cys group of E2 and ubiquitin and carries ubiquitin

• Conjugation of ubiquitin onto substrate Lys by ubiquitin ligase E3 to form isopeptide bond

  • from the E2~Ub, ubiquitin is either directly transferred to the substrate w/ the help of a RING-containing E3 or via a 3rd thioester bond on a HECT or RBR E3 (classes of E3 enzymes)

E3 Transfer from E2: Ring

• most cases, the E3 ligase has a ring motif that recruits the E2 enzymes and then ubiquitin is transferred from Lys on E2 to Lys on substrate to form isopeptide bond

• E3 ligase acts as an intermediate to bring the substrate into vicinity of E2 and stimulate nucleophilic attack from deprotonated Lys on substrate to form isopeptide bond, and thus transfers ubiquitin from E2 to Lys of substrate

E3 Transfer from E2: HECT/RBR

HECT: homologous to E6AP C-ter

RBR: ring b/w ring domain

• both have Lys that forms an additional thioester bond b/w ubiquitin + Cys before transferring to Lys of substrate

Ubiquitin Cascade: Abundance of each enzyme

• the ubiquitin cascade has large arrays of proteins to be conjugate

• the specificity for a substrate is imparted by the E3 alone or in conjuction with the E2

  • each E3 ligase is regulated differently, so that not all proteins are ubiquitinylated at the same time

  • each E3 recognizes only a subset of substrates

• there are over 500 E3s encoded in the human genome each targeting a few to a large array of substrates

• there are also 300-400 proteins that have ubiquitin binding domains that can recognize ubiquitin molecules

Poly-ubiquitination

• multiple ubiquitin moieties can be added to form a poly-ubiquitin chain in a processive manner or via multiple substrate E3 association-dissociation cycles

• there are 7 Lys (+ the amino terminal) on ubiquitin that can be used to conjugate successive ubiquitin to form a poly-ubiquitin chain

  • the first ubiquitin that is attached to the substrate is now conjugated by another ubiquitin and so on (Add another ubiquitin onto the previous ubiquitin)

Deubiquitinase

• i.e ubiquitin protease, DUB, that can break isopeptide bonds

• can reverse the ubiquitin cascade reaction in an ATP-independent manner

• generates free ubiquitin moiety that can be recycled into the system

Polyubiquitylation & the Ubiquitin Code

• substrates can be modified in different manners depending on the combination of E2 & E3, and sometimes a DUB

• different functions are associated to different type of ubiquitin linkages that form the ubiquitin code

• diff messages are associated to diff types of ubiquitin chains

• polyubiquitylation of histone H2As is utilized as a key message during DNA repair process, where it can recruit specific proteins to the damaged sites to continue DNA repair

Proteasome

• small 2.5 megDa multi subunit complex over 30 proteins

• the 26S proteasome is composed of one 20s core complex and 1- 2 19S regulatory particle(s)

• targets for degradation of poly-ubiquitylated proteins

Proteasome: 20S

• the 20S has a hollow barrel shape; the barrel shape is formed by 4 diff rings

• the 7 ⍺-subunits (outer rings) form the gate that controls entry to the 20S

  • the ⍺-subunits will be in ca closed conformation when 20S alone is in the cell, preventing entry of proteins

• in absence of 19S, proteins typically cannot access the inside chamber

• proteolytic activity resides inside the cavity

• 3/7 β-subunits are Thr proteases that have caspcase/trypsin/chemotrypsin-like activities

  • β1: capase-like, cleaves after acidic residues

  • β2: trypsin-like, cleaves after basic residues

  • β-5: chemotrypsin-like, cleaves after hydrophobic residues

• with 3 distinct proteases, the 26S proteosome can degrade any type of polypeptide

Proteasome: 19S Subunits

• 19S subunits regulate entry of the substrate into the 20S chamber

  • function: unfold the substrates to mediate the translocations and also mediate the opening the ⍺-subunits so that the polypeptide can enter the 20s

• 19s base region sites on top of the 20S

• 6 ATPases form the base

• the rest of the lid subunit will mediate recognition and processing before entry

Proteasome Mechanism

• the 19S recognizes polyubiquitinated proteins (substrate) via ubiquitin binding proteins (Rpn10, Rpn13) (1)

• the substrate is position at the 19S base; disordered regions begin to translocate into the ATPase ring (2)

  • when this occurs, there’s a rearrangement of 19s subunits so that there is an active site of deubiquinase that sites ontop of the ATPase ring

• Rpn11 deubiquitinase removes the ubiquitin chain (3) from the substrate and is recycled (not degraded)

  • coupled to translocation

• substrate unfolding (ATP-dependent) allows full translocation of substrate into the 20S and substrate protein is unfolded

• the β-subunits of the 20S cleave the protein into shorter peptides, which diffuse out and are further degraded into AA’s for recyling

Proteasome: Target Pathways

• there are 2 major classes of proteasome substrates

• proteins targeted by a protein quality control pathway and proteins degraded by a regulated pathway

Ubitquitin Attachments

• thioester and isopeptide

• both are covalent bonds and are resistant to SDS denaturation

• thioester is more labile and dissociates upon exposure to a reducing agent (e.g. DTT)

• isopeptide can only be cleaved by a specialized protease

• thioester is first catalyzed in an ATP-dependent manner by E1 or following the transfer from E1 to E2 (and E2 to E3 in some cases)

• the isopeptide is formed following the nucleophilic attack of the thioester by the deprotonated lysine

Inhibition of Proteasome Proteolytic Activity

• there are several potent inhibitors of the proteasome

• there are 3 proteasome inhibitors, as well as two small molecule docking substrates to E3 (revlimid and pomalidomide) that are used to treat some cancers

In Vitro Ubiquitination Reactions

• require E1, E2, E3, ATP, ubiquitin and a substrate and can be inhibited by specific elements/ mutations

In vitro Proteasomal Degradation Assays

• require the 26S proteasome, a poly-ubiquitinated substrate and ATP

• they can also be inhibited by specific elements