ubiquitin presentation- jacob patterson
TRANSCRIPT
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