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)