What type of molecules help mediate protein folding?
What are two protein folding enzymes?
Protein disulfide isomerase (PDI) catalyzes disulfide bond formation in the ER, where an oxidizing environment allows (s-s) linkages; also catalyzes the reverse reaction – helps proteins to rapidly reach their most energetically stable conformations. Peptidyl prolyl isomerase catalyzes the transition of prolines between cis and trans conformations.
what is proteolysis?
cleavage of the polypeptide chain.
what is a biological example of proteolytic processing?
----Insulin, digestive enzymes and proteins involved in blood clotting cleaved in similar manner.
Insulin forms by two cleavages, first an N-terminal signal sequence that targets the polypeptide chain to the endoplasmic reticulum. Then an internal peptide is removed to yield mature insulin. Other proteins, like digestive enzymes and proteins involved in blood clotting are cleaved in a similar manner.
Where do many proteins enter for processing?
The rough ER
What is Glycosylation?
Glycosylation is the addition of carbohydrates to proteins to form glycoproteins. Glycoproteins are usually secreted or localized to the cell surface, however many nuclear and cytosolic proteins are glycosylated.
N-linked – carb is attached to N atom in side chain of asparagine; occurs in the ER. O-linked – carb is attached to the O atom in the side chain of serine or threonine, occurs in the Golgi.
What is N-linked glycosylation?
During cotranslational translocation, oligosaccharyl transferase glycosylates specific asparagine residues (in the sequence Asn‐X‐Ser/Thr). The oligosaccharide is assembled on a lipid carrier (dolichol phosphate) in the ER membrane and is transferred as an intact unit to the asparagine.
The three glucoses are used to mark the protein during its processing. All three glucoses removed in the ER indicates a properly folded protein.
Note: because topology is maintained and the glycosylation of proteins occurs inside the ER lumen, glycosylated proteins are rarely seen on the inner surface of the plasma membrane, rather they are almost always seen on the outer surface.
What are two chaperones that assist in glycoprotiens folding correctly?
----Calnexin and Calreticulin
Protein folding in the ER is slow and inefficient, and many are misfolded.
The chaperones calnexin and calreticulin associate with protein disulfide isomerase and peptidyl prolyl isomerase and assist glycoproteins to fold correctly.
A lost cause (irreversibly misfolded, folded incorrectly after repeated cycles) is targeted for degradation
Where do GPI anchors attach proteins?
----luminal side of the ER membrane
Lipid addition usually anchors proteins to the plasma membrane.
Some single pass transmembrane proteins exchange their transmembrane domain (C-terminus) for a glycosylphospatidylinositol that attaches the protein to the lumenal side of the ER membrane.
N-myristoylation – myristic acid (14 carbon fatty acid) is attached to an N-terminal glycine residue. The glycine is usually the second amino acid incorporated into the polypeptide chain, the initiator methionine is removed by proteolysis during translation. Many N-myristoylated proteins are associated with the cytosolic plasma membrane. Mutation of the glycine residue prevents myristoylation and association with the plasma membrane.
What is an example of preynaltaion addition of lipid?
Prenylation – prenyl groups are attached to the sulfur atoms in the side chains of cysteine residues. Many plasma membrane-associated proteins involved in the control of cell growth and differentiation are modified in this way, including the Ras oncogenes.
Three steps of prenylation:
1. Prenyl group (either farnesyl or geranylgeranyl) is added to a cysteine located 3 amino acids from the C terminus of the polypeptide chain
2. Amino acids following cysteine residue are removed
3. Methyl group added to carboxyl group of cysteine residue
Where is palmitic acid added in palmitoylation?
----Internal cysteine residue.
Palmitoylation – palmitic acid (a 16-C fatty acid) is added to sulfur atoms of the side chains of internal cysteine residues. Association of proteins with the cytosolic face of the plasma membrane.
What amino acids does phosphorylation and dephosphorylation affect?
Phosphorylation is reversible; it can activate or inhibit proteins in response to environmental signals. Protein kinases are often components of signal transduction pathways.
Protein kinases – catalyze protein phosphorylation; transfer phosphate groups from ATP to the hydroxyl groups of the side chains of serine, threonine, or tyrosine residues; usually specific for either serine and threonine or tyrosine residues.
Protein phosphatases – catalyze hydrolysis of phosphorylated amino acids; usuallyspecific for either serine and threonine or tyrosine residues
What is nitrosylation?
Nitrosylation – addition of NO groups to side chains of cysteine; bears some similarity to phosphorylation; can be reversed and may alter the activity of the protein that is modified.
What is the unfolded protein response? (UPR)
The UPR is a signal cascade that generates a strong increase in chaperone production (BiP and other chaperones) and increases enzymes that degrade misfolded proteins.
How does the ubiqudation process work for protein degradation?
Degradation is used as a form of regulation, but is also important for getting rid of faulty or damaged proteins.
Ubiquitin, a highly conserved, 76-aa protein, targets cytosolic and nuclear proteins for rapid proteolysis.
1. Ubiquitin is activated by being attached to ubiquitin-activating enzyme, E1.
2. Ubiquitin is transferred to a second enzyme, the ubiquitin-conjucating enzyme, E2.
3. Ubiquitin is transferred to the target protein by a ubiquitin ligase, E3. Cells have lots of E3s that recognize different substrate proteins and so determine the specificity of degradation.