Gene regulation Lecture No 5: Protein folding

and Ubiquitination

Dr. Mohamed KamalLecturer of Molecular Biology

E.mail: mk_saleh1980@yahoo.com

Protein foldingCorrect 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 .

*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

Protein folding

unfoldedprotein response (UPR)

ER Stress: It means overload of proteins in the ER.

Activate

i) protein translocation

ii) ERAD

iii) protein folding capacity

misfolded proteins

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 .

Protein folding

Failure of UPR Cell death )necrosis or apoptosis(

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

Protein folding

Regulate

Basic leucine-zipper (bZIP)-containingtranscription factors

Regulation of UPR:

Protein folding

Unstressed ER

BiP

IRE1, PERK or ATF6

Accumulation of unfoldedproteins

Free BiP

BiP-unfolded proteins binding

Activate UPR

Fig 1

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.

Science NewsPathway Identified in Human Lymphoma Points Way to New Blood Cancer TreatmentsScienceDaily (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 and disease

Protein foldingProtein 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 .

*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 folding

Protein UbiquitinationProtein Degradation

Normal protein turnover Elimination of misfolded proteins

Lysosomal10–20% of normal protein turnover

Protein Degradation

Proteasomalby

ubiquitination

Aberrations in this pathway provide a variety of pathological phenotypes

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

Protein Ubiquitination

E3 ligase is substrate specific

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

Protein Ubiquitination in Cancer

*20S proteasome subunit expression in Cancer

*E2 and some E3 ligases high expression in cancer

Protein Ubiquitination in Cancer

Protein Ubiquitination in Cancer