15 - Post-translational Modifications Flashcards
What is the importance of post translational modifications?
Still need modifications for the mature protein
Where do post translational modifications take place?
During or after assembly on the ribosome
What are the basic types of post translational modifications?
Add or remove residues, or change chemical nature of sidechain, or cleave peptide backbone
What led to the discovery of more types of post translational modifications?
More sophisticated methods
True or false: almost all amino acids can be modified
True: although some are more than others
What are the size of most post translational modifications?
50 - 100 Da
True or false: all PTMs occur at the same frequency
False: some are common, but some are rare
What are some examples of common PTMs?
Half of all proteins have initial Met removed, 60-90% of proteins in eukaryotic cytoplasm have N-terminal acetylation
What are some examples of rare PTMs?
Hydroxylation of certain Pro and Lys residues occur only in procollagen, only 0.1% of cellular phosphoproteins have pTyr
What determines nonenzymatic modifications?
The chemical environment
How selective are nonenzymatic modifications?
Nonselective (occur at any susceptible residue)
What are some examples of nonenzymatic PTMs?
Oxidation of Met or Cys, deamination of Asn/Gln to Asp/Glu, nitration of Tyr
How do cells use irreversible nonenzymatic PTMs?
Metabolic intermediates, environmental stress (oxidative stress)
True or false: all PTMs are reversible
False: some are irreverisble
What are some examples of irreversible PTMs?
Acetylation of N-terminus, proteolysis
What is a consensus sequence?
Short regions of protein sequence recognized by a modifying enzyme
What is an example of a nonspecific enzymatic PTM?
N-acetylation (only requires free N-terminus)
What is the consensus sequence for N-glycosylation?
Asn-X-Ser
What is the consensus sequence for phosphorylation (by PKA)?
Arg-Arg-X-Ser
What is the consensus sequence for farnesyl transferase?
Cys-A-A-X-COO-, A = alipathic
True or false: the presence of a consensus sequence means it must be modified
False: this needs to be shown experimentally
How can a modification be determined in vitro (3 ways)?
- Reaction with pure proteins (kinase + ATP, measure phosphorylation by SDS-PAGE)
- Synthetic peptide experiments (enzyme + peptide –> modified peptide)
- Reversal by chemical or enzymatic means
How can a modification by determined in vivo (3 ways)?
- Incorporate radioactive precursor (isolate, digest, map sites by mass spec or Edman sequencing)
- Inhibitor study
- Mutational analysis
What are the most convincing studies for determining if a PTM is present?
Using multiple approaches (radioactivity –> mutant, etc.)
What is the purpose of proteomic experiments?
Can identify multiple modified sites
How do proteomic experiments work?
Fractionation or affinity capture to enrich modified protein, then use mass spec to analyze masses and exact site of modification
What type of PTMs occur only in the ER?
Protein folding modifications
What PTMs (6) occur in the cytoplasm?
- Removal of initial Met
- N-terminal acetylation
- N-terminal myristoylation
- O-glycosylation with GlcNAc
- Addition of palmitoyl groups
- Virus polyprotein processing
What PTMs (1) occur in mitochondria/chloroplasts?
- Cleavage of signal peptide
What PTMs (6) occur in the ER?
- Cleavage of signal peptide
- Core glycosylation of Asn residues
- Addition of palmitoyl and glycosyl-phosphatidylinositol groups
- Carboxylation of Glu
- Hydroxylation of Pro and Lys in procollagen
- Disulfide bond formation
What PTMs (3) occur in the Golgi?
- Modification of N-glycosyl groups
- O-glycosylation with GalNAc
- Sulfation of Tyr residues
What PTMs (2) occur in secretory vesicles and granules?
- Amidation of C-terminus
2. Proteolytic processing of some precursors
True or false: all PTMs have physiological importance
False: not all PTMs have physiological importance
What is an example of a PTM that does not have physiological importance?
Artefact of isolation process (proteolytic digestion, disulfide scrambling) (shouldn’t have occurred)
How can a PTM change the activity of a protein?
Change in conformation, localization, or chemical property of side chain
What is an example of a PTM changing protein conformation?
Phosphorylase
What is an example of a PTM changing localization?
Farnesylation of Ras
What is an example of a PTM changing chemical property of a side chain?
Carboxylation of Glu
What does farnesylation of Ras do?
Anchors Ras to the membrane (needs to signal from outside to inside)
How is a molecular barcode achieved?
By multisite PTMs (encode information)
What is the effect of multisite modifications?
Can have additive or antagonistic effects on other PTMs
True or false: one site can only have one possible PTM
False: there can be competing modifications at the same site
What are some examples of same site competition?
- Phosphorylation or GlcNAc on Ser/Thr
- Phosphorylation or sulfation on Tyr
- Lots of possible PTMs on Lys
- ADP ribosylation or methylation on Asp/Glu
What does SH2 interact with?
pTyr
What does HP1 chromodomain interact with?
Methylation of lysine
What does GCN5 bromodomain interact with?
Acetylation of lysine
What does Vps27 UIM interact with?
Ubitiquination of lysine
What does VHL-beta interact with?
Hydroxyproline
How do docking sites play a role in signal transduction?
RTKs can create pTyr, which act as docking sites for downstream signaling targets
What is proteolytic processing?
Cleavage of peptide bond
What proteins are commonly proteolytically processed?
Those destined for cellular organelles or secretion
What is proteolysis often used for?
Regulating the biological activity of a protein (only active when lysed)
What is a proprotein?
The inactive precursor that has yet to be lysed
What is a preproprotein?
The precursor of the proprotein, that has not had its signal peptide cleaved
When is the signal peptide cleaved for a preproprotein?
In the ER by a signal peptidase
What are some characteristics of proteolysis?
It is very specific (avoid indiscriminate proteolysis) and is irreversible
What are some examples of molecules that are proteolytically processed?
Peptide hormones, such as insulin
What is the structure of mature insulin?
A and B polypeptide chain, connected together by disulfide bonds
What is the structure of proinsulin?
Has an internal C peptide between the A and B polypeptide chain
How does proinsulin become insulin?
Cleavage of the C peptide releases the A and B peptide
What is the purpose of the C peptide in proinsulin?
Ensures proper folding and disulfide bond formation
Where does cleavage of the C peptide occur?
At pairs of basic residues (Lys-Arg and Arg-Arg)
What does POMC stand for?
Proopiomelanocortin
What is the structure of POMC?
It contains precursors of 8 different hormones
What is the significance of POMC?
It is a single precursor for many hormones, so it can coordinate actions, and produce different hormones
What determines which hormones are produced from POMC?
Based on the different processing enzymes produced by the (pituitary) cells
What are some examples of proteases?
Trypsin, chymotrypsin, and pepsin
What is a zymogen?
The inactive precursor of a protease
When is a zymogen converted into a protease?
When it is secreted and cleaved by a particular enzyme
What activates trypsinogen into trypsin?
Enterokinase
What activates chymotrypsinogen into chymotrypsin?
Trypsin
What are some examples of signaling pathways that use proteolysis?
Notch/Delta, Wnt/beta-catenin, NF-kB
How does Notch/Delta signaling work?
Cell adhesion/interaction leads to proteolysis to release intracellular domain to influence transcription
True or false: many PTMs occur at the N-terminus
True: there are many
What is deformylation?
The initial Met on prokaryotes is deformylated by a formylase
What is a formyl group?
OH - Fe - Cys / His / His
How many proteins in eukaryotes have N-terminal acetylation?
60-90%
What enzyme catalyzes N-terminal acetylation?
Ribosome-associated acetyltransferases
When can N-terminus acetylation occur?
Co-translationally or post-translationally, with or without preceding Met
What is the specificity of N-terminal acetylation?
Prefer small amino acids (Gly, Ala, Ser)
What is the biological role of N-terminal acetylation?
Regulate protein stability and interactions
How many proteins remove the initial Met?
About 50% in prokaryotes and eukaryotes
What enzyme removes the initial Met?
Ribosome-associated Met-aminopeptidase
What is the specificity of removal of initial Met?
Small residues at second position, large/charged residues unfavored
What is addition of a residue to the N-terminus?
Transfer an amino acid from a charged tRNA to a peptide
What is an example of addition to a terminal residue?
Arginyl tRNA protein transferase catalyzes transfer of Arg to proteins with N-terminal Glu or Asp to target them for degradation (ubiquitin)
What does MAP stand for, and what does it do?
Methionine aminopeptidases, removal of initial Met
What does NAT stand for, and what does it do?
N-acetyltransferases, add acetyl group to N-terminus
What is Gla?
Glu carboxylated at gamma position
What is the structure of Gla?
Two carboxyl groups at terminal carbon
In what proteins does carboxylation of Glu occur?
Proteins involved in blood clotting and bone structure
What enzyme catalyzes Glu carboxylation, and where is it found?
Vitamin K-dependent carboxylase, found in the membrane of the ER
What is the function of Gla?
Calcium binding (binds more tightly)
What is proline hydroxylation?
Add a hydroxyl group to proline (or Lys)
Where is the hydroxyl group added in Pro?
At the gamma position (2 carbons from the alpha carbon)
What is the consensus sequence for Pro hydroxylation?
X-Pro-Gly
What is the importance of gamma Pro hydroxylation?
Stabilizes alpha helix (other site for H-bonds, helps for helix/crosslinking)
What is the importance of delta-hydroxyl-Lys?
Important for glycosylation and for forming cross links
When is HIF1alpha expressed?
Under hypoxia (low oxygen conditions)
What happens to HIF1alpha in well oxygenated cells?
It is hydroxylized, which signals it for destruction
What is the significance of Lys acetylation?
Enhances gene expression through histone modifications
How does acetylation open up a histone?
Histone is positive, and acetylation removes positive charge (less association between histone and DNA)
What enzyme catalyzes histone acetylation?
HATs (histone acetyltransferases)
What enzyme catalyzes histone deacetylation?
HDACs (histone deacetylases)
What is a nucleosome?
DNA wrapped around 8 histone proteins (200 bp)
What do chromatin remodeling complexes do?
Alter chromatin structure (multisite modifications)
What competing reactions occur in histone remodeling?
Lys acetylation and Lys methylation (also Ser phosphorylation)
What is the significance of Lys4 on Histone 3?
It must be trimethylated to act as a docking site for other cellular proteins
Where does disulfide bond formation occur?
In the lumen of ER
Why is disulfide bond formation in the ER?
More oxidizing than cytosol
What is the significance of disulfide bonds?
Linked with 3D protein folding
What does PDI stand for?
Protein disulfide isomerase
What does PDI do?
Assists in disulfide bond formation and isomerization
What is the structure of PDI?
2 active sites with pairs of Cys residues
What is the mechanism for PDI?
It has pairs of Cys residues which can help reorganize disulfide bonds
Where does ADP-ribosylation occur?
In the cytosol or nucleus
What is ADP-ribosylation?
Add ADP-ribose (through NAD) to a protein
What is the specificity of ADP-ribosylation?
Add to N atoms of Arg, His, Asn, Lys, the carboxyl of Glu, and the alpha carboxyl of Lys
True or false: ADP ribosylation can only occur once
False: it can occur multiple times - poly(ADP-ribose)
How does cholera toxin work?
It ADP-ribosylates a G-protein to lock it in the “on” position
True or false: ADP-ribosylation is only pathological
False: it also has normal physiological roles
What does phosphorylation control?
Metabolism, hormone action, cell growth/cancer, gene expression, and memory
How does phosphorylation work?
Take a phosphate from ATP and add it to a protein
What is a kinase?
An enzyme that adds a phosphate to a protein
What is a phosphatase?
An enzyme that removes a phosphate from a protein
What are the two broad categories of protein kinases?
Tyr, and Ser/Thr
Which is the most common type of kinases?
Ser/Thr kinases
What is the same with all kinases?
Conserved catalytic core of 270 amino acids
What kinases are seen in prokaryotes?
pHis and pAsp
What is an example of a pHis pathway?
Bacterial chemotaxis
How does bacterial chemotaxis work?
CheA (His kinase) phosphorylates itself and CheY (response regulator) to alter motion (2 compartment system)
