Post-translational
Modification
Ubiquitination
Introduction
• Goldknopf and his friends realized that histones can be
modified by the protein ubiquitin through Lys-linked
isopeptide bonds.
• The prevalence and importance of protein- based
modifications emerged in the 1980s, when landmark
studies connected the ATP-dependent ubiquitination of
substrates to their degradation by the 26S proteasome
• protein function can be regulated through chemical
modifications of amino acids
Ubiquitin….?
 First identified in 1975
 A 8.6 kDa protein
 Expressed in all eukaryotic cells and highly conserved
 A 76-amino acid protein
 Functions of ubiquitin and ubiquitination pathway were
elucidated in the early 1980s by Aaron Ciechanover, Avram
Hershko, and Irwin Rose
o Awarded Nobel Prize in Chemistry in 2004
 In mammals encoded by 4 different genes
 UBA52 and RPS27A genes ---monoubiquitin
 UBB and UBC genes--polyubiquitin precursor proteins
Ubiquitin….?
Key features-seven Lys residues and C-terminal tail
Ubiquitin….?
 Recognized by a hydrophobic surface that consists of Ile44,
Leu8, Val70, and His68 figure
• The Ile44 ---bound by the proteasome and most UBDs
---essential for cell division
 Another hydrophobic surface is centered on Ile36 and involves
Leu71 and Leu73 figure
• The Ile36 ---mediate ubiquitin-ubiquitin interactions
---recognized by HECTE3s, DUBs and UBDs
 Recognized through C-terminal tail figure
• β1/β2 loop containing Leu8 shows flexibility
o important for recognition by ubiquitin-binding proteins
Ubiquitin….?
 Insert 3 figures for
each structure
Ubiquitination
• Ubiquitination (or ubiquitylation) is an enzymatic posttranslational modification in which an ubiquitin protein is
attached to a substrate protein
• This process most commonly binds the last amino acid of
ubiquitin (glycine 76) to a lysine residue on the substrate.
• An isopeptide bond is formed between the carboxyl group
(COO−) of the ubiquitin's glycine and the epsilon-amino
group (ε-NH+3) of the substrate's lysine
• Trypsin cleavage of a ubiquitin-conjugated substrate leaves a
di-glycine "remnant" that is used to identify the site of
ubiquitination.
Ubiquitination
• Adding ubiquitin to electron-rich nucleophiles, sites
in a protein -----"non-canonical ubiquitination".
o The amine group N-terminus of the protein MyoD, 22
other proteins in multiple species and also in ubiquitin itself
o the sulfhydryl group on cysteine, and
o the hydroxyl group on threonine and serine.
• Ubiquitination requires three types of enzyme:
o E1s ubiquitin-activating enzymes
o E2s ubiquitin-conjugating enzymes
o E3s ubiquitin ligases– many(hundreds)
Ubiquitination
• Ubiquitin is attached to substrates by a complex 3-step cascades
1) Activation
 E1 (ubiquitin-activating enzyme)
 ATP dependent
 two-step reaction
 1st --- production of a ubiquitin-adenylate intermediate. The E1
binds both ATP and ubiquitin and catalyses the acyl-adenylation
of the C-terminus of the ubiquitin molecule.
 2nd transfers ubiquitin to an active site cysteine residue, with
release of AMP. This step results in a thioester linkage between
the C-terminal carboxyl group of ubiquitin and the E1
cysteine sulfhydryl group.
 The human genome contains two genes: UBA1 and UBA6
Ubiquitination
2) Conjugation
 E2 (ubiquitin-conjugating enzymes)
 catalyze the transfer of ubiquitin from E1 to the active
site cysteine of the E2 via a trans(thio)esterification
reaction.
 In order to perform this reaction, the E2 binds to both
activated ubiquitin and the E1 enzyme.
 Humans possess 35 different E2 enzymes, whereas
other eukaryotic organisms have between 16 and 35
 E2 have UBC (ubiquitin-conjugating catalytic) fold which is
highly conserved structure
Ubiquitination
3) Ligation
 E3 (ubiquitin ligases)
 Create an isopeptide bond between a lysine of the target
protein and the C-terminal glycine of ubiquitin.
 E3 enzymes function
 as the substrate recognition modules of the system
 can Interact with both E2 and substrate
 some activate the E2 enzymes
 E3 enzymes possess one of two domains:
 Homologous to the E6-AP carboxyl terminus (HECT) domain --transiently bind ubiquitin (thioester intermediate is formed with the
active-site cysteine of the E3)
 Really interesting new gene (RING) domain (or U-box domain) --catalyze the direct transfer from the E2 enzyme to the substrate
Ubiquitination
• In the ubiquitination cascade, E1 can bind with many E2s,
which bind with hundreds of E3s in a hierarchical way.
• E4 enzymes (ubiquitin-chain elongation factors)
o adding pre-formed polyubiquitin chains to substrate proteins
o For example, addition of a polyubiquition chain using p300
and CBP on monoubiquitylated tumor suppressor p53
• The end result of this process is
o the addition of one ubiquitin molecule (monoubiquitination)
or
o a chain of ubiquitin molecules (polyubiquitination) to the
substrate protein.
Monoubiquitination
• Multi-monoubiquitination is the addition of one ubiquitin
molecule to multiple substrate residues.
• is thought to be required prior to the formation of
polyubiquitin chains.
• Affects cellular processes such as
o Endocytosis
o Membrane trafficking
Polyubiquitination
• Formation of a ubiquitin chain on a single lysine residue on the
substrate protein
• These chains are made by linking the glycine residue of a
ubiquitin molecule to a lysine of ubiquitin bound to a
substrate.
• Ubiquitin is attached to the N-terminus of a 2nd ubiquitin fig
• Ubiquitin has 7-lysine residues and an N-terminus that serves
as points of ubiquitination; they are
• Lys48-linked chains---predominant (> 50% of all linkages)
o Role --- target proteins to the 26s proteasome for degradation(at
least four)
Polyubiquitination
• Lys63- linked chains---second abundant via performs
various non-degradative roles by binding with ESCRT-0
o Endocytic trafficking, inflammation, translation,
and DNA repair
• Remaining Lys6, Lys11, Lys27, Lys29 or Lys33 and Met1
o chains linked to this parts can induce proteasomal
degradation
• Researches to characterize this remaining residues led to the
discovery of highly linkage-specific enzymes and proteins
that assemble, recognize and hydrolyze each ubiquitin chain
type, i.e., ‘write’, ‘read’ and ‘erase’ this ubiquitin code
Polyubiquitination
• Structure of linked chains have specific effects on
the protein
• Lys29-, Lys33-, Lys63- & M1-linked chains form
open(linear) conformation chains fig
o do not interact with each other, except for the
covalent isopeptide bonds linking them together
• Lys6-, Lys11-, and Lys48-linked chains form closed
conformations fig
o have interfaces with interacting residues
• Proteins can specifically bind to ubiquitin via
ubiquitin-binding domains (UBDs)
Polyubiquitination
• The distances between individual ubiquitin units in
chains differ between lysine 63- and 48-linked chains.
• The UBDs exploit this by having
o small spacers between ubiquitin-interacting motifs that bind
lysine 48-linked chains
o larger spacers between ubiquitin-interacting motifs that
bind lysine 63-linked chains
• The machinery involved in recognizing polyubiquitin
chains can also differentiate between Lys63-linked
chains and M1- (induce proteasomal degradation)
linked chains,
Function of Ubiquitination
Transcriptional regulation
Histones---usually (mono-), rarely (poly)-ubiquitinated
o alters chromatin structure and allows the access of
enzymes involved in transcription.
o acts as a binding site for proteins that either activate or
inhibit transcription
o These effects can modulate the transcription of genes
Protein regulation
 Comprehensive proteomics studies identified tens-ofthousands of ubiquitination sites on thousands of proteins.
 Suggests most proteins will experience ubiquitination at some
point in their cellular lifetime
Function of Ubiquitination
Membrane proteins
 Multi-monoubiquitination
o Mark transmembrane proteins for removal
from membranes (eg. Receptors)-----How?
• When tagged with ubiquitin,
• the protein subcellular localization is altered
• targeting for destruction in lysosomes
• a negative feedback mechanism because often the
stimulation of receptors by ligands increases their rate
of ubiquitination and internalisation
 lysine 63-linked polyubiquitin chains
o in the trafficking some membrane proteins
Function of Ubiquitination
Genomic maintenance
 Proliferating cell nuclear antigen (PCNA) ---protein involved
in DNA synthesis
 Under normal physiological conditions PCNA is sumoylated
 When DNA is damaged by ultra-violet radiation or chemicals,
the SUMO molecule that is attached to a lysine residue is
replaced by ubiquitin
 Monoubiquitinated PCNA recruits polymerases for repair but
prone to error resulting in the synthesis of mutated DNA
 Lysine 63-linked polyubiquitination of PCNA allows a less
error-prone
• bypass mutation by the template switching pathway.
Function of Ubiquitination
Infection and immunity
• Regulate immune signal transduction pathways at all stages
o
o
o
o
o
steady-state repression,
activation during infection and
attenuation upon clearance
Without this regulation resulting in chronic disease or death
Alternatively hyper-activate the immune system resulting autoimmune
damage in organs and tissues
• Viruses can easily overcome host cells by blocking the
ubiquitin, system which have high role in cells, to support
their own replication
• In humans the retinoic acid-inducible gene I (RIG-I) protein
o primary immune system sensor for viral and other invasive RNA
Malfunction of Ubiquitination
Genetic disorders
• Neurodegenerative disorders: Alzheimer's and Parkinson's
o lesions of different transcripts of ubiquilin-1.
o Higher levels of ubiquilin in the braindecrease malformation
of amyloid precursor protein (APP) trigger Alzheimer's disease
o lower levels of ubiquilin-1 in the brain have been associated with
increased malformation of APP
• A frameshift mutation in ubiquitin B abnormal peptide, UBB+1
accumulation in Alzheimer's disease
• Angelman syndrome is caused by a disruption of UBE3A, which
encodes a ubiquitin E3 ligase enzyme
• 3-M syndrome is an autosomal-recessive growth retardation disorder
associated with mutations of the Cullin7 E3 ubiquitin ligase
Increased ubiquitination activity
Cervical cancer
 Human papillomavirus (HPV) are known to hijack cellular
ubiquitin-proteasome pathway for viral infection and replication.
1. The E6 proteins of HPV will bind to the N-terminus of the
cellular E6-AP E3 ubiquitin ligase,
2. Redirecting the complex to bind p53, a well-known tumor
suppressor gene that inactivation is found in many types of
cancer
3. p53 undergoes ubiquitination and proteasome-mediated
degradation.
4. The E7 proteins of HPV will bind to Rb, also a tumor suppressor
gene, mediating its degradation.
 Loss of p53 and Rb in cells allows limitless cell proliferation
Increased ubiquitination activity
p53 regulation
• MDM2 a gene encodes for a RING E3 Ubiquitin ligase
• downregulation of p53 activity
• MDM2 targets p53 for ubiquitination and proteasomal
degradation thus keeping normal cell condition
• Overexpression of MDM2 causes loss of p53 activity and
therefore allowing cells to have a limitless replicative potential
Efp (estrogen-inducible RING-finger protein)
• E3 ubiquitin ligase
• Its overexpression has been shown to be the major cause
of estrogen-independent breast cancer
Deubiquitination
• Deubiquitinating enzymes (DUBs)
• Removing ubiquitin from substrate protein
• They are cysteine proteases that cleave the amide bond between
the two proteins
• They are highly specific
• They can cleave both isopeptide (between ubiquitin and lysine)
and peptide bonds (between ubiquitin and the N-terminus).
• Cleave polyubiquitin to produce active ubiquitin or form
monoubiquitin
• Recycle ubiquitin that has been bound to small nucleophilic
molecules during the ubiquitination process.
Ubiquitination
Further ubiquitin modifications
• Ubiquitin is also be modified by
other modifications. E.g.
o by SUMO (small ubiquitin-like
modifier family)
• SUMOylation of (poly)ubiquitin is
conceptually similar to
polyubiquitin chain formation
• Ubiquitin is also modified by
acetylation
• Mining of available datasets
indicates that 6 out of 7 ubiquitin
Lys residues can become acetylated
Further ubiquitin modifications
• Ubiquitin phosphorylation on---• provide additional regulation in
the ubiquitin system
• For these modifications
o Writers--- kinases and
acetyltransferases,
o Eraser--- phosphatases and
deacetylases and
o Readers--- phospho-ubiquitin
(phosphoUb) or acetylubiquitin (AcUb) binding
domains
Human and Yeast Ub ----highly conserved