Ubiquitin

Moderator: Dr. Sarita Agarwal

Presentor: Dr. Karthikeyan

Overview

• Various ways of protein degradation

• Ubiquitin in contrast to lysosomes

• Discovery of Ubiquitin

• Nature of Ubiquitin gene and protein

• Signal sequences for ubiquitination

• 3 steps of Ubiquitination

• Proteasome assembly

contd.,

• Ubiquitin and Malignancies

• Use of Proteasome inhibitors in Malignancies

• Ubiquitin in cell cycle (cyclin) and gene expression (Histone)

• Ubiquitin like proteins in intracellular trafficking (SUMOylation)

• ERAD and Aggresomes

Topological diversity of proteolyticsystems

• Extracellular

– Digestive enzymes

– Blood Coagulation System

• Intracellular

– membrane secluded: Lysosomes

– Free Floating: Ubiquitin System (Cytosolic, nuclearand ER)

Proteins have variable life-spans

Enzyme Half-life Hours

Ornithine decarboxylase 0.2

RNA polymerase I 1.3

Tyrosine aminotransferase 2.0

Serine dehydratase 4.0

PEPcarboxylase 5.0

Aldolase 118

GAPDH 130

cytochrome c 150

• Lysosomes – Receptor mediated endocytosis & phagocytosis in

acidic pH

• Proteasomes: for intracellular proteins – transcription factors

– cyclins

– virus coded proteins

– improperly folded proteins

– damaged proteins

• Ubiquitin mediates degradation

of many but not all proteins.

Discovery

The Nobel Prize in Chemistry 2004

Aaron Ciechanover Avram Hershko Irwin Rose

Small protein with big function

• small, heat-stable, compact globular protein (76 AA)

• Found only in eukaryotic organisms

• Highly conserved

Ubiquitin Genes

• UBA52

• RPS27A

• UBB

• UBC

Ub genes typically exist in two states

• The ubiquitin gene (red) can be fused to a ribosomal protein gene (blue) giving rise to a translation product that is a Ub-ribosomal fusion protein.

Ub-C-term hydrolase

Ub genes can exist as a linear repeat

• Translation product iscomprised of a linearchain of Ub-moleculesfused together (apolyubiquitin molecule).

• Ub-C-term hydrolase cleaves the fusion proteins to yield Ubmonomers

Ub-C-term hydrolase

• Met1-Gln2-Ile3-Phe4-Val5-Lys6-Thr7-Leu8-Thr9-

Gly10-Lys11-Thr12-Ile13-Thr14-Leu15-Glu16-Val17-Glu18-Pro19-Ser20-Asp21-Thr22-Ile23-Glu24-Asn25-Val26-Lys27-Ala28-Lys29-Ile30-Gln31-Asp32-Lys33-Glu34-Gly35-Ile36-Pro37-Pro38-Asp39-Gln40-Gln41-Arg42-Leu43-Ile44-Phe45-Ala46-Gly47-Lys48-Gln49-Leu50-Glu51-Asp52-Gly53-Arg54-Thr55-Leu56-Ser57-Asp58-Tyr59-Asn60-Ile61-Gln62-Lys63-Glu64-Ser65-Thr66-Leu67-His68-Leu69-Val70-Leu71-Arg72-Leu73-Arg74-Gly75-Gly76

Primary structure

Signals for Degradation

• N – Degron

• PEST Sequence

• Signals in Hydrophobic core

• Masking and unmasking

Signals in Hydrophobic core

3 steps of ubiquitination

• Activation

• conjugation

• ligase action

Why couldn’t be done in a single step ?

• E1 (1)

• E2 (12-30)

• E3 (>200)

– HECT-type

– RING-type

– PHD-type

– U-box containing

Proteasome

26S proteasome

α subunit

β subunits

α subunit

20S core protein

Top view

Ubiquitin-Proteasome complex & Malignancies

Oncogenesis

• ↑ ubiquitin mediated destruction of Tumor suppressor gene products. (HPV – p53)

• ↓ ubiquitin mediated destruction of oncoproteins. (beta catenin)

↓ ubiquitin mediated destruction of β-catenin

P P

AUXIN – an eye opener

Death of Proteinsis Required for Life

P P

Ubiquitin in cell cycle and gene expression

Cyclin ubiquitination

• key regulators of the cell cycle.

• Cyclins themselves have no enzymatic activity but bind and activates Cdk.

• G1-S-G2-M cycle follows successive oscillations in the levels of cyclins, D, E, A & B.

• Precise control is achieved by ubiquitination

• Ubiquitination of protein does not always

imply degradation of proteins only.

Histone Ubiquitination and Gene Expression

• Histones are rich in lysine – potential site for ubiquitination.

• H2A mono-ubiquitination is a repressivemark.

• H2B mono-ubiquitination is an activationmark.

H2A mono-ubiquitination is a repressive mark.

Ubiquitin like modifiers

SUMOylation

• Post translational modification involved in nuclear-cytosolic transport

• Small Ubiquitin-like Modifier (or SUMO) proteins

• similar to ubiquitin but SUMO is not used to tag proteins for degradation

ERAD & Aggresomes

Endoplasmic-reticulum-associated protein degradation (ERAD)

• cellular pathway which targets misfolded proteins of ER for ubiquitination and subsequent degradation by proteasome.

• Molecular chaperones like calnexin/calreticulin try in correct folding of misfolded proteins. Terminally misfolded proteins are processed by mannosidase.

• translocated into cytosol for destruction

Aggresomes: cellular response to misfolded proteins

• occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins

Neurofibrillary tangles - Alzheimer's diseaseLewy bodies - Parkinson's diseasPick bodies - Pick's disease

Role of ubiquitin antibodies

Proteasome inhibitors

• Bortezomib

• Disulfiram

• Epigallocatechin-3-gallate

• Salinosporamide

• carfilzomib

Bortezomib

• Bortezomib - first therapeutic proteasome inhibitor

• Approved by FDA for treating relapsed multiple myeloma and mantle cell lymphoma.

• The boron atom in bortezomib binds the catalytic site of the 26S proteasome[4] with high affinity and specificity.

• may prevent degradation of pro-apoptotic factors, permitting apoptosis