Transcript The Ubiquitin Proteosome pathway

The Ubiquitin Proteosome
Pathway
Swati Pradhan
Mayura Dange
Vidyadhar Daithankar
Overview
 Background
 Protein misfolding & degradation
 Ubiquitin & proteosome structure
 Ubiquitin Proteosome Pathway
 Mechanism
 Structures of enzymes involved in pathway
 Pathogenic implication of defective pathway
 Biological functions of pathway
 Diseases & drug development
The Central Dogma
Translational Folding of a Protein
Chaperone Mediated Protein
Folding & Misfolding
Post-Translational Modification

Acetylation

Glycosylation

Phosphorylation

Ubiquitination
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmdbooks&doptcmdl/Figure+6-79
Degradation of Misfolded Proteins

Lysosomal (extracellular) protein
degradation
– Protein degraded by lysosomal enzymes

Cytosolic (intracellular) protein
degradation
– The Ubiquitin Proteosome pathway
Lysosomal degradation


Proteins delivered
via endocytosis
Lysosomes
– The cellular dustbins
– Contain many
hydrolytic enzymes



Proteases
Lipases
Glycosidases
Cytosolic protein degradation

The Ubiquitin Proteosome Pathway
www.ihf.de/forschung/ popup/ubiquitin.html
2004 Nobel Prize in Chemistry

The discovery of ubiquitin-mediated protein
degradation
– Aaron Ciechanover
– Avram Hershko
– Irwin Rose

Cells give a chemical "kiss of death" to
proteins that need to be destroyed.
Targeting by Ubiquitin


Despite help from chaperones, more than
80% fold incorrectly
Proteins are dislocated back into the cytosol
– Oligosaccharides are removed
– Deglycosylation is catalyzed by N-glycanase

One third of the newly made polypeptide
chains are selected for degradation
The Export of Misfolded Proteins
Ubiquitin



76 amino acids, 8.5 kDa
protein
Heat stable
Folds into a compact
globular structure

Found throughout the cell

Found in all eukaryotic cells


Human and yeast ubiquitin
share 96% sequence
identity
Involved in many cellular
processes
http://www.sanger.ac.uk/Users/sgj/thesis/html/node93.html
The Proteosome

Professional protein
degrading organelles

An abundant ATPdependent protease

Constitutes nearly 1% of
cellular protein

Present in many copies
throughout the cytosol and
the nucleus

Consists of a central hollow
cylinder (20S)

Ends of the cylinder are
associated with the 19S cap
http://walz.med.harvard.edu/Proteasome_Complexes/
The Structure of 20S
Proteasome
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3206
Types of Ubiquitination

Mono-ubiquitination
– Transcription, histone function, endocytosis and membrane trafficking

Lys48, Lys11 or Lys29 linked poly ubiquitination
– Target proteins to the proteasome

Lys63 linked poly ubiquitination
– Signaling, DNA repair, stress response, endocytosis and signal transduction
UBIQUITIN PATHWAY
UBIQUITIN PATHWAY



Covalent Attachment
molecules
of
multiple
ubiquitin
Degradation of the tagged protein
3 Enzymes : Ub – Activating enzyme E1
Ub – Conjugating enzyme E2
Ub – Ligases E3
Hierarchical structure
Several E2 transfer Ub from E1 to E3 to which substrate protein is
bound

E3s catalyze covalent attachment to the substrate and recognize
the substrate

Ubiquitin Pathway
Ubiquitin Activating Enzyme E1

Adenylation

Thio-ester bond formation

E2 association
Mechanism

E1 activates C-terminus of
Ub by forming acyl -adenylate
intermediate

Catalytic Cys residue forms
thioester bond with Ub

Another Ub is adenylated

Transfer of Ub to E2 forming
a thioester bond
Ubiquitin Conjugating enzyme E2




Carries activated Ub from E1 to the
substrate
Cys residue positioned in a shallow groove
Relatively inflexible structure
Conserved Asn may be required for Hbond network OR plays a catalytic role in
isopeptide bond formation
Ub Ligases E3



Final target selection and specificity
Place activated Ub near Lys of substrate
Isopeptide formation of Gly of Ub with the є –NH2 Lys or
to the N-terminal residue of the substrate
Categories of E3 Ligases

HECT domain: Homologous to E6-AP C terminus

RING domain: Really Interesting New Gene
HECT Ub Ligases E3







Conserved 350 amino acids
Catalytic contribution
Forms thiol ester bond with Ub before transferring it to the
substrate
N lobe and C- lobe form ‘L’ or ‘inverted T’ shape
Flexibility of hinge loop is required for catalytic activity
C lobe accepts Ub form E2 and transfers it to the substrate
Sequential addition / Indexation
L – shaped E2/E3 complex
RING Ub Ligases E3







15th most common domain in Human genome
Conserved Cys and His Zn2+ co-ordinating residues
Interact directly with E2s
Allosterically activate E2 enzymes
Acts as molecular scaffold
Brings Ub-E2 and substrate closer
Increase # Lys in the vicinity of E2
Polyubiquitination


Poly Ub chain synthesized by adding Ub moieties to
Lys of the previous Ub
Another enzyme E4 may be catalyzing this step
Deubiquitination





Thiol proteases
Ubiquitin processing (UBP) enzymes
Removes Ub from polyubiquinated proteins
Ubiquitin carboxy terminal hydrolases (UBH)
Regenerates monomeric Ub
Pathological implication of defective
ubiquitin-proteosome pathway
 Ubiquitin proteasome pathway is ubiquitous & targets
many processes and substrates.
 Several complex processes are mediated via degradation
or processing of specific proteins.
 Aberrations in these systems associates with pathogenic
conditions either directly or indirectly.
Biological function of Ubiquitin
Proteosome pathway
Consequences of Defects in
Ubiquitination
Pathological Conditions Associated with
Ubiquitin Proteosome Pathway
– Malignancies
– Neurodegenerative disorders
– Genetic disease
Cystic fibrosis, Angelman’s syndrome & Liddle’s
syndrome
– Immune and inflammatory responses
Malignancies

Oncoproteins like NMyc, c-Myc, c-Fos, are substrates of U-P pathway.

Destabilization of tumor suppressor genes like p53 and p27.

Extremely low levels of p53 in uterine cervical carcinoma.

Prostate, Colorectal and breast cancer:
– Tumor suppressor protein p27 is CDK inhibitor of the cell cycle.
– Healthy individuals have high levels of p27. Mitogenic stimuli
rapidly degrades the protein.
– Cancer patients has low levels of p27 in quiescent cells.
–
Defects in ubiquitin system accelerates degradation of suppressor.
– Strong correlation of low levels of p27 and aggressiveness of cancer.
Skp2
Polyubiquitination
Degradation of P27
Cell Cycle Regulators and Cancer
Defect in ubiquitin
pathway ( Skp2)
Neurodegenerative disorders
 Alzheimer's disease
 Parkinson's disease
 Huntington’s disease
Formation of
inclusion bodies
 Spinocerebellar ataxias
 Spinobulbar muscular dystrophy (Kennedy’s syndrome)
(Ref: http://w3.dbb.su.se/~oliveberg/images/bildstrat1.jpg)
 Accumulation of ubiquitin may be secondary reflecting unsuccessful
attempts of ubiquitination.
 Abnormal protein associate with each other forming aggregates.
 Hypothesis: Aggregated proteins inhibit ubiquitin proteosome pathway.
Parkinson’s disease and Lewy Bodies
( Ref: http://www.neurodegeneration.uni-goettingen.de/index.html?/en/p311.html)
Liddle’s Syndrome
 Hereditary form of hypertension.
 Caused due to deletion of proline rich (PY) region in the β and γ subunits of
epithelial Na+ channel (hENaC).
 HECT domain of E3 binds to PY motif of hENaC.
 Mutation in PY motif leads to stabilization of channel complex and E3 ligase
cannot bind to PY motif.
 Increased expression of hENaC channel causing excessive reabsorption of
sodium and water.
Stabilization of channel
Angleman syndrome
 Ubiquitin system is considered to be involved in brain development.
 Defective synthesis of gene coding for E3 ligase E6-AP
 Characteristic symptoms involve mental retardation, seizures, out of
context frequent smiling and laughter.
 Brain proteins that could be stabilized by mutation have not been
identified.
Cystic fibrosis
 Gene codes for a protein, CFTR, which is chloride ion channel.
 Small fraction of protein matures to the cell surface.
 Mutation in protein ΔF508, CFTRΔF508 doesn't reach the cell surface.
 Ubiquitination degrades mutant CFTRΔF508, resulting in complete
lack of cell surface expression.
Immune and inflammatory responses
 Ubiqutin proteosome pathway is involved in processing of antigenic proteins.
 Epitopes are presented on class I MHC molecule generating T cell immune
response.
Ubiquitin proteosome
pathway
Native protein
Foreign protein
CLASS I MHC molecule
No immune response
Immune response
Drug Development for Ubiquitin
Dysfunction
 Inhibition of enzymes common to entire pathway would target the
process non- specifically.
 Narrow window between benefits and toxicity needs to be
identified.
 Develop completely specific E3 ligase inhibitors that would affect
the pathways of interests.
 Better approach would be development of small molecules that
would be specific for substrates.
Conclusions
 Ubiquitylation plays a fundamental role of protein degradation at cellular level.
(Levels of proteins in nucleus, cytoplasm, ER lumen and transmembrane protein are
kept in check by ubiquitin proteosome pathway.)
 Ubiquitylation is highly complex, temporally controlled and tightly regulated process.
 Enzymologically Ubiquitination is more complex pathway compared to other post
translational modification.
 Mechanism of catalysis by E3 ligase still remains unclear.
 Elucidation of complete catalytic mechanism of ubiquitylation will provide considerable
insight on cellular functions.
Questions
extra
Extra
www.mekentosj.com/ubiquitin/proteasome.html
Extra (The Central
Dogma)