The Role of Ubiquitin in Protein Degredation

and Signal Transduction

Consists of 76 amino acids, 8.5 kDa

Found in all eukaryotic cells (ubiquitously)

Highly ConservedUsed in post-translational

modification

Ubiquitin

26S ProteasomeAbundant in nucleus and

cytoplasmdestroys proteins marked

by Ubiquitin through Lysine 48-linked polyubiquitination

26S ProteasomeConsists of central hollow

cylinder (20S)4 stacked “rings” of 7

proteins eachCapped by regulatory

particles (19S) that recognize ubiquitin through ubiquitin binding domains (UBDs)

Core structure

Three types of Ubiquitination

MonoubiquitinationAdds one ubiquitin molecule to one substrate

protein residueRequired before a poly chain can begin to

formMembrane Trafficking, Transcription,

Endocytosis

PolyubiquitinationRequires one Ub linked to substrate before

chain begins to form.Chains made by linking Glysine residue of Ub

to a Lysine of a Ub bound to a substrate.Linking to different position on Ub leads to

different results.

Lysine 48-linked polyubiquitinationLinked by 48th amino

acid (Lysine)Marks proteins for

destructionRequires at least 4

Ub to be recognized by proteasome

Lysine 63-linked polyubiquitinationBinds to allow

coordination of endocytic trafficking

Bound to ESCRT-0 to prevent binding to proteasome

Ubiquitination

Ub activating enzyme (E1)E1 binds ATP and Ub.Transfers Ub to an active site cysteine

residue, releasing AMPThioester linkage between C-terminus of Ub

and E1 cysteine sulfhydryl groupOne E1 can transfer Ub to several different

E2 enzymes

Ub conjugating enzyme (E2)Ub is transferred from E1 to E2 through a

trans(thio)esterification reaction.Binding to both the activated Ub and the E1

enzyme before releasing E1.Each E2 can transfer Ub to a hundred

different E3 enzymes

Ub ligase enzyme (E3)Attaches Ub via

isopeptide bond to a lysine on target protein

E3 ligasesThe most varied of the three enzymes.Each E3 can attach to many different

substrate proteins. Different E2, E3 pairings will recognize

different proteins by distinct degradation signals.

Deubiquitinating enzyme (DUB)Use catalytic diads or

triads of cysteine, histidine, and asparagine to catalyze hydrolysis of the isopeptide bond

Deubiquitinating enzyme (DUB)Around 100 in the human

genomeSome cleave the whole

chain, some only cleave a set amount of Ubs

DUB USP5 selectively binds a 4-ubiquitin chain and severs it.

Ubiquitin in Protein Degradation

Ubiquitin in Protein DegradationAfter a protein is Ubiquitinated, it must be recognized by

the 19S regulatory particleUbiquitin Binding Domains exist to interpret signals from

Ubiquitinated substrates. ~20 different UBDs exist to bind to different specific shapes of Ubiquitin chains and different monoubiquitinated locations on a protein.

Ubiquitin in Protein DegradationNarrow gate formed by the N-terminus tails of

the alpha ring subunitsProtein must be partially unfolded, at least

their tertiary structureMust be deubiquitinated firstOrder not clearly known, depends of specific

substrate

ProteolysisThreonine-dependant

nucleophilic attackCentral chamber

releases typically 7-9 residue polypeptides.

Sometimes produce functioning molecules

Regulation of Protein Degradation

One means of controlling Ubiquitination is regulating the activation of E3 ligases.

Regulation of Protein Degradation

Another way for a protein to avoid degradation by the proteasome is to mask the residues that release the degradation signal.

Phosphorylating the area or creating an unstable N-terminus will let nearby E3’s know

NF-kB

A protein complex that controls transcription of DNA.

Synthesized as p105 and p100, C-termini inhibit entry into nucleus.

Ubiquitinated and processed by the Proteasome into their active forms, p50 and p52.

Circadian Rhythm and AgingUbiquitin is responsible for the degradation of

the “master circadian protein.”Also damaged proteins that arise due to

aging, stress, and oxidative damage.

Ubiquitin in Signal Transduction

RIP1Complexes with a polyubiquitin chainAs long as the signaling protein is

ubiquitinated, it acts to prevent cell death. Once deubuquitinated by the A20 enzyme,

RIP1 is free to drive forward the cell death process.

PCNAMonoubiquitination activates PCNA to restart DNA synthesis,

but very error prone. In yeast, lysine 63-linked polyubiquitination leads to an error

free pathway.

Epidermal Growth Factor ReceptorCell-surface receptor that auto-phosphorylates to activate

downstream activation cascade.Leads to DNA synthesis, cell proliferation, and cell

adhesion. Important for innate immune responseUbiquitination by Lysine 63-linkages required for

endocytosis and post endocytic sortingMutations to EGFR lead quickly to cancer, proper

ubiquitination prevents out of control mutations.

Defects in Ubiquitination PathwayTumor suppressor proteins like p53 and p27

are stabilized by UbiquitinDefects in Ubiquitin system accelerate

degradation of suppressors, increasing risk of cancer causing mutations

Defects in Ubiquitination PathwayAlzheimer'sHuntington’sParkinson’sKennedy’s Syndrome

(Spinobulbar Muscular Dystrophy)

Lewy BodiesParkinson’sDisplace other cell

components

Ubiquitin-like Proteins (UBLs)Little is known about most of themEnzyme cascade is almost the sameSUMO- Small Ubiquitin-like Modifier

Attaches in a manner similar to Ubiquitin, only used in signal transduction.

ISG15- Interferon Stimulated Gene 15Expressed in response to interferons or viral dsRNAUsed in JAK-STAT signalingpathway

ProkaryotesProkaryotic Ubiquitin-like Proteins (PUP)Attach to substrates in the same mannerOnly requires 2 enzymes

Prokaryotes