The Ubiquitin The Ubiquitin Proteosome PathwayProteosome Pathway
Swati Pradhan
Mayura Dange
Vidyadhar Daithankar
OverviewOverview
BackgroundBackground Protein misfolding & degradationProtein misfolding & degradation Ubiquitin & proteosome structureUbiquitin & proteosome structure
Ubiquitin Proteosome PathwayUbiquitin Proteosome Pathway MechanismMechanism Structures of enzymes involved in pathwayStructures of enzymes involved in pathway
Pathogenic implication of defective pathwayPathogenic implication of defective pathway Biological functions of pathwayBiological functions of pathway Diseases & drug developmentDiseases & drug development
The Central DogmaThe Central Dogma
Translational Folding of a Translational Folding of a ProteinProtein
Chaperone Mediated Chaperone Mediated Protein Folding & Protein Folding &
MisfoldingMisfolding
Post-Translational Post-Translational ModificationModification
AcetylationAcetylation
GlycosylationGlycosylation
PhosphorylationPhosphorylation
UbiquitinationUbiquitination
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmdbooks&doptcmdl/Figure+6-79
Degradation of Misfolded Degradation of Misfolded ProteinsProteins
Lysosomal (extracellular) protein Lysosomal (extracellular) protein degradationdegradation– Protein degraded by lysosomal Protein degraded by lysosomal
enzymesenzymes
Cytosolic (intracellular) protein Cytosolic (intracellular) protein degradationdegradation– The Ubiquitin Proteosome pathwayThe Ubiquitin Proteosome pathway
Lysosomal degradationLysosomal degradation
Proteins delivered Proteins delivered via endocytosisvia endocytosis
Lysosomes Lysosomes – The cellular dust-The cellular dust-
binsbins– Contain many Contain many
hydrolytic enzymes hydrolytic enzymes ProteasesProteases LipasesLipases GlycosidasesGlycosidases
Cytosolic protein Cytosolic protein degradationdegradation
The Ubiquitin Proteosome PathwayThe Ubiquitin Proteosome Pathway
www.ihf.de/forschung/ popup/ubiquitin.html
2004 Nobel Prize in 2004 Nobel Prize in ChemistryChemistry
The discovery of ubiquitin-mediated The discovery of ubiquitin-mediated protein degradation protein degradation – Aaron CiechanoverAaron Ciechanover– Avram Hershko Avram Hershko – Irwin Rose Irwin Rose
Cells give a chemical "kiss of death" to Cells give a chemical "kiss of death" to proteins that need to be destroyed. proteins that need to be destroyed.
Targeting by UbiquitinTargeting by Ubiquitin
Despite help from chaperones, more Despite help from chaperones, more than 80% fold incorrectlythan 80% fold incorrectly
Proteins are dislocated back into the Proteins are dislocated back into the cytosolcytosol– Oligosaccharides are removedOligosaccharides are removed– Deglycosylation is catalyzed by N-glycanaseDeglycosylation is catalyzed by N-glycanase
One third of the newly made polypeptide One third of the newly made polypeptide chains are selected for degradationchains are selected for degradation
The Export of Misfolded The Export of Misfolded ProteinsProteins
UbiquitinUbiquitin
76 amino acids, 8.5 kDa 76 amino acids, 8.5 kDa proteinprotein
Heat stable Heat stable Folds into a compact Folds into a compact
globular structureglobular structure Found throughout the cellFound throughout the cell Found in all eukaryotic Found in all eukaryotic
cellscells Human and yeast Human and yeast
ubiquitin share 96% ubiquitin share 96% sequence identitysequence identity
Involved in many cellular Involved in many cellular processes processes
http://www.sanger.ac.uk/Users/sgj/thesis/html/node93.html
The ProteosomeThe Proteosome
Professional protein Professional protein degrading organellesdegrading organelles
An abundant ATP-An abundant ATP-dependent protease dependent protease
Constitutes nearly 1% of Constitutes nearly 1% of cellular proteincellular protein
Present in many copies Present in many copies throughout the cytosol throughout the cytosol and the nucleusand the nucleus
Consists of a central Consists of a central hollow cylinder (20S)hollow cylinder (20S)
Ends of the cylinder are Ends of the cylinder are associated with the 19S associated with the 19S capcap
http://walz.med.harvard.edu/Proteasome_Complexes/
The Structure of 20S The Structure of 20S ProteasomeProteasome
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3206
Types of UbiquitinationTypes of Ubiquitination
Mono-ubiquitinationMono-ubiquitination– Transcription, histone function, endocytosis and membrane traffickingTranscription, histone function, endocytosis and membrane trafficking
Lys48, Lys11 or Lys29 linked poly ubiquitinationLys48, Lys11 or Lys29 linked poly ubiquitination– Target proteins to the proteasomeTarget proteins to the proteasome
Lys63 linked poly ubiquitinationLys63 linked poly ubiquitination– Signaling, DNA repair, stress response, endocytosis and signal transductionSignaling, DNA repair, stress response, endocytosis and signal transduction
UBIQUITIN PATHWAYUBIQUITIN PATHWAY
Covalent Attachment of multiple ubiquitin moleculesCovalent Attachment of multiple ubiquitin molecules
Degradation of the tagged proteinDegradation of the tagged protein
3 Enzymes : Ub – Activating enzyme E13 Enzymes : Ub – Activating enzyme E1 Ub – Conjugating enzyme E2Ub – Conjugating enzyme E2 Ub – Ligases E3Ub – Ligases E3
UBIQUITIN PATHWAYUBIQUITIN PATHWAY
Hierarchical structureHierarchical structure
Several E2 transfer Ub from E1 to E3 to which substrate Several E2 transfer Ub from E1 to E3 to which substrate protein is boundprotein is bound
E3s catalyze covalent attachment to the substrate and E3s catalyze covalent attachment to the substrate and recognize the substraterecognize the substrate
Ubiquitin PathwayUbiquitin Pathway
Ubiquitin Activating Ubiquitin Activating Enzyme E1Enzyme E1
AdenylationAdenylation
Thio-ester bond Thio-ester bond
formationformation
E2 associationE2 association
MechanismMechanism
E1 activates C-terminus E1 activates C-terminus
of Ub by forming acyl -of Ub by forming acyl -
adenylate intermediateadenylate intermediate
Catalytic Cys residue Catalytic Cys residue
forms thioester bond with forms thioester bond with
UbUb
Another Ub is adenylatedAnother Ub is adenylated
Transfer of Ub to E2 Transfer of Ub to E2
forming a thioester bondforming a thioester bond
Ubiquitin Conjugating Ubiquitin Conjugating enzyme E2enzyme E2
Carries activated Ub from E1 to the Carries activated Ub from E1 to the substratesubstrate
Cys residue positioned in a shallow Cys residue positioned in a shallow groovegroove
Relatively inflexible structureRelatively inflexible structure Conserved Asn may be required for Conserved Asn may be required for
H- bond network OR plays a catalytic H- bond network OR plays a catalytic role in isopeptide bond formationrole in isopeptide bond formation
Ub Ligases E3Ub Ligases E3
Final target selection and specificityFinal target selection and specificity Place activated Ub near Lys of substratePlace activated Ub near Lys of substrate Isopeptide formation of Gly of Ub with the Isopeptide formation of Gly of Ub with the є –NHє –NH22
Lys or to the N-terminal residue of the substrateLys or to the N-terminal residue of the substrate
Categories of E3 LigasesCategories of E3 Ligases
HECT domainHECT domain: Homologous to E6-AP C : Homologous to E6-AP C
terminusterminus
RING domainRING domain: Really Interesting New Gene: Really Interesting New Gene
Conserved 350 amino acidsConserved 350 amino acids Catalytic contributionCatalytic contribution Forms thiol ester bond with Ub before transferring it to the Forms thiol ester bond with Ub before transferring it to the
substratesubstrate N lobe and C- lobe form ‘L’ or ‘inverted T’ shapeN lobe and C- lobe form ‘L’ or ‘inverted T’ shape Flexibility of hinge loop is required for catalytic activityFlexibility of hinge loop is required for catalytic activity C lobe accepts Ub form E2 and transfers it to the substrateC lobe accepts Ub form E2 and transfers it to the substrate Sequential addition / IndexationSequential addition / Indexation
HECT Ub Ligases E3HECT Ub Ligases E3
L – shaped E2/E3 complexL – shaped E2/E3 complex
1515thth most common domain in Human genome most common domain in Human genome Conserved Cys and His ZnConserved Cys and His Zn2+ 2+ co-ordinating residuesco-ordinating residues Interact directly with E2sInteract directly with E2s Allosterically activate E2 enzymes Acts as molecular scaffold Brings Ub-E2 and substrate closer Increase # Lys in the vicinity of E2
RING Ub Ligases E3RING Ub Ligases E3
PolyubiquitinationPolyubiquitination
Poly Ub chain synthesized by adding Ub Poly Ub chain synthesized by adding Ub moieties to Lys of the previous Ubmoieties to Lys of the previous Ub
Another enzyme E4 may be catalyzing this stepAnother enzyme E4 may be catalyzing this step
DeubiquitinationDeubiquitination
Thiol proteasesThiol proteases Ubiquitin processing (UBP) enzymesUbiquitin processing (UBP) enzymes Removes Ub from polyubiquinated proteinsRemoves Ub from polyubiquinated proteins Ubiquitin carboxy terminal hydrolases (UBH)Ubiquitin carboxy terminal hydrolases (UBH) Regenerates monomeric UbRegenerates 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.
Cell Cycle Regulators and Cell Cycle Regulators and CancerCancer
Defect in ubiquitin pathway ( Skp2)
Degradation of P27 Skp2 Polyubiquitination
Neurodegenerative disorders
Alzheimer's disease
Parkinson's disease
Huntington’s disease
Spinocerebellar ataxias
Spinobulbar muscular dystrophy (Kennedy’s syndrome)
Formation of inclusion bodies
(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.
( Ref: http://www.neurodegeneration.uni-goettingen.de/index.html?/en/p311.html)
Parkinson’s disease and Lewy Bodies
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
extraextra
Extra Extra
www.mekentosj.com/ubiquitin/proteasome.html
Extra (The Central Extra (The Central Dogma)Dogma)
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