Download Protein Ubiquitination

Gene regulation
Lecture No 5: Protein folding
and Ubiquitination
Dr. Mohamed Kamal
Lecturer of Molecular Biology
E.mail:
[email protected]
Protein folding
Correct protein folding is essential to proper protein function.
Protein folding occurs mainly in the ER.
The status of protein folding act as a feedback to the protein
translation.
Signaling pathways emanate from the ER to regulate mRNA
translation.
These pathways prevent the accumulation of unfolded protein in the ER
by decreasing the load, increasing the ER folding capacity, and
increasing the degradation of misfolded proteins.
Protein folding
*High proportion of proteins are translocated into
the ER.
*Proteins are translocated into the ER lumen in an
unfolded state.
*Inhibition of translation initiation serves as an
effective means to limit the flow of proteins into the
ER .
Protein folding
*Translocation of nearly all proteins across the ER
membrane occurs co-translationally and is
usually directed by an N-terminal signal peptide.
*When the signal peptide is formed the signal
recognition particle (SRP) binds and stops
translational elongation until the ribosome docks
at the ER membrane.
Protein folding
Upon docking to the ER membrane, the polypeptide is simultaneously
synthesized and translocated across the lipid bilayer. This
mechanism prevents the polypeptide from improper localization in
the cytosol.
ER translocon: An aqueous pore formed by the Sec61
complex and the BiP/GRP78 (immunoglobulin-binding
protein/glucose-regulated protein).
Protein folding
Functions of BiP:
1- It has a peptide-dependent ATPase activity that is used to seal the
lumenal side of the aqueous pore to maintain the permeability
barrier between the ER and the cytosol when the ribosome is not
tightly attached.
2- BiP functions in post-translational translocation to ensure
unidirectional translocation of the elongating polypeptide across the
ER membrane.
Protein folding
ER Capacity: The protein concentration in the ER lumen is 100 mg/ml,
it is essential that protein chaperones facilitate protein folding by
preventing aggregation of protein folding intermediates and by
correcting misfolded proteins. BiP/GRP78 – uses the energy from
ATP hydrolysis to facilitate folding by preventing aggregation of
proteins within the ER.
Quality control: Only those polypeptides that are properly folded and
assembled in the ER can transit to the Golgi compartment. Proteins
that are misfolded in the ER are retained and eventually translocated
back through the ER translocon into the cytosol for degradation by
the 26S proteasome.
Protein folding
ER Stress: It means overload of proteins in the ER.
Activate
misfolded proteins
unfolded
protein response (UPR)
i)
protein translocation
iii)
ii)
ERAD
protein folding capacity
Protein folding
UPR is orchestrated by a general attenuation of
translation initiation, a selective translation of
a small subset of mRNAs encoding adaptive
functions, and transcriptional activation of a
large set of genes.
Failure of UPR
Cell death
(necrosis or apoptosis)
Protein folding
ER sensors: IRE1, PERK and ATF6
Protein folding
Combinatorial interactions of ER sensors generate diversity for
transcriptional induction of different subsets of UPR-responsive
genes.
Example: IRE1, PERK and ATF6
Regulate
Basic leucine-zipper (bZIP)-containing
transcription factors
Protein folding
Regulation of UPR:
Free BiP
BiP
Accumulation of unfolded
proteins
IRE1, PERK or
ATF6
Unstressed ER
Activate UPR
Fig 1
BiP-unfolded proteins
binding
Protein Folding and disease
Misfolded proteins accumulat in the cell
Accumulation of specific proteins cause
different diseases.
Example: Accumulation of αsynuclein increases
lewy bodies in the neurons and causes
Parkinson Disease.
Protein Folding and disease
Science News
Pathway Identified in Human Lymphoma Points Way to
New Blood Cancer Treatments
ScienceDaily (Nov. 21, 2012) — A pathway called the
"Unfolded Protein Response," or UPR, a cell's way of
responding to unfolded and misfolded proteins, helps tumor
cells escape programmed cell death during the
development of lymphoma.
Protein Folding and disease
Protein folding
Protein folding is a spontinous process.
Chaperons: Are a group of proteins playing
important role in protein folding.
Chaperons safeguard the folding of nascent
chains.
Most proteins bind chaperons, however it is
essential for the correct folding of a limited
number of proteins.
Protein folding
* Heat shock proteins (Hsp): They are
chaperons.
* Hsps stabelize the newly formed chains and
protect them from their hydrophobic characters.
* Hsp70-bound substrate must be transferred to •
a chaperonin complex for productive folding.
Protein folding
The chaperonins:
are large cylindrical protein
complexes consisting of two stacked rings of seven to
nine subunits each.
The chaperonin of the eukaryotic cytosol, termed TRiC or
CCT forms a cage-like structure.
Protein folding
Protein Ubiquitination
Protein Degradation
Normal protein turnover
Elimination of misfolded
proteins
Protein Degradation
Lysosomal
10–20% of normal protein turnover
Proteasomal
by
ubiquitination
Aberrations in this pathway provide a variety of pathological phenotypes
Protein Ubiquitination
The ubiquitination pathway:
The ubiquitin–protein ligase system includes three
enzymes, termed E1, E2, and E3, is absolutely
necessary, along with adenosine triphosphate (ATP), for
the conjugation of ubiquitin to proteins.
Protein Ubiquitination
E3 ligase is substrate specific
Protein Ubiquitination
Other functions for ubiquitination
- Internalization of proteins (binding to K63)
- nuclear export and translocation of proteins
into the cytoplasm (MonoUbiquitination)
Protein Ubiquitination
The Proteasome: is a protein complex
mediates protein degradation and is
localised in the nucleus and cytosol
Protein Ubiquitination
Proteasome confers trypsin-like activity.
Caspase-like effects.
It can cleave bonds on the carboxyl side of basic,
hydrophobic, or acidic amino acid residues.
Protein Ubiquitination in
Cancer
Protein Ubiquitination in
Cancer
* 20S proteasome subunit expression in
Cancer
* E2 and some E3 ligases high expression in
cancer
Protein Ubiquitination in
Cancer