Download - Ubiquitin proteolytic system
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
Small protein with big function
• small, heat-stable, compact globular protein (76 AA)
• Found only in eukaryotic organisms
• Highly conserved
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
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
Oncogenesis
• ↑ ubiquitin mediated destruction of Tumor suppressor gene products. (HPV – p53)
• ↓ ubiquitin mediated destruction of oncoproteins. (beta catenin)
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
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.
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
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