Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College.
Addition:
There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
2. 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
3. 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
4. Topological diversity of proteolytic
systems
• Extracellular
– Digestive enzymes
– Blood Coagulation System
• Intracellular
– membrane secluded: Lysosomes
– Free Floating: Ubiquitin System (Cytosolic, nuclear
and ER)
5. 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
14. 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
15. Ub genes can exist as a linear repeat
• Translation product is
comprised of a linear
chain of Ub-molecules
fused together (a
polyubiquitin molecule).
• Ub-C-term hydrolase
cleaves the fusion
proteins to yield Ub
monomers
Ub-C-term hydrolase
40. 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
41. • Ubiquitination of protein does not always
imply degradation of proteins only.
42. Histone Ubiquitination and Gene
Expression
• Histones are rich in lysine – potential site for
ubiquitination.
• H2A mono-ubiquitination is a repressive
mark.
• H2B mono-ubiquitination is an activation
mark.
45. 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
47. 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
48. 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 disease
Lewy bodies - Parkinson's diseas
Pick bodies - Pick's disease
Role of ubiquitin antibodies
50. 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
Editor's Notes
Most of the cathepsins are found inside lysosomes except cathepsin K, which is secreted extracellularly after secretion by osteoclasts in bone resorption.
Extracellular long lived proteins
Ubiq – intracellular short lived regulatory proteins
There are only 3 differences in the sequence when Ub from yeast is compared to human Ub. This strong sequence conservation suggests that the vast majority of amino acids that make up Ub are essential as apparently any mutations that have occurred over evolutionary history have been removed by natural selection.
Titin , largest protein - connectin, molecular spring >30,000 AA
7 lysines – epsilon amino group participates in isopeptide bond
C-terminal glycine binds to epsilon amino group of lysine.
A degron is a specific sequence of amino acids in a protein that directs the starting place of degradation.
The N-degron. Alexander Varshavsky in1986carried out an elegant set of experiments (Science 234, 179-186) that showed a correlation between the half-life of a protein and its N-terminal residue. For example, proteins that have Ser as the N-terminal amino acid were long-lived with a half-life of more than 20 hours. In contrast, proteins with Asp as the N-terminal amino have a half-life of only 3 minutes. The mechanism that couples recognition of the N-terminal amino acid and the protein's half-life is still unknown. In is interesting to note, however, that the N-end rule applies to bacteria even though they do not contain ubiquitin.
2. Certain amino acid sequences appear to be signals for degradation. One such sequence is known as the PEST sequence because short stretch of about eight amino acids is enriched with proline, glutamic acid, serine, and threonine. An example is the transcription factor Gcn4p. This protein is 281 amino acids in length and the PEST sequence is found at positions 91-106. The normal half-life of this protein is about 5 minutes. But if the PEST sequence (and only the PEST sequence) is removed, the half-life increases to 50 minutes.
Some signals may also be subject to masking. A signal could be hidden if it is part of a protein-protein interaction. Or it may be masked by covalently attaching phosphate groups to the side chains of certain amino acids. Both of these mechanisms would thus allow for better control, as a protein degradation signal need only be unmasked to target it for degradation. Such reversible masking appears to be involved in the regulation of both transcription factor and cyclin concentrations.
3. Signals may also be buried in the hydrophobic core. This is why partially folded or abnormal, mutant proteins may be prone to degradation. When such proteins exist in their native state, the signals are hidden and the protein is thus long-lived. But in a partially unfolded state, the signals may be seen by the Ub machinery caused the protein to become tagged by Ub. This reaction appears to be hindered by chaperone activity.
Activation:
Ubiquitin-activating enzyme (E1) binds ATP-Mg2+ and ubiquitin. In the next step a catalytic cysteine on the E1 enzyme attacks the ubiquitin-AMP complex through acyl substitution, simultaneously creating a thioester bond and an AMP leaving group. Finally, the E1~ubiquitin complex transfers ubiquitin to an E2 enzyme through a transthioesterification reaction, in which an E2 catalytic cysteine attacks the backside of the E1~ubiquitin complex.
Conjugation: Ubiquitin-conjugating enzyme
Activated ubiquitin is then transferred to an E2 cysteine by transthioesterification. Once conjugated to ubiquitin, the E2 molecule binds one of several ubiquitin ligases
Ubiquitin ligase:
Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function.
Lid – substrate recognition
Base – substrate unfolding
Core - Proteolysis
β-catenin a subunit of cadherin complex is an oncoprotein. GSK-3 (a kinase) constitutively phosphorylates the β-catenin. Phosphorylated beta catenin is ubiquitinated and degraded.
similar to ubiquitin - formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein, around 100 amino acids, very little sequence identity with ubiquitin at the amino acid level, it has a nearly identical structural fold