15 - Post-translational Modifications Flashcards
1
Q
What is the importance of post translational modifications?
A
Still need modifications for the mature protein
2
Q
Where do post translational modifications take place?
A
During or after assembly on the ribosome
3
Q
What are the basic types of post translational modifications?
A
Add or remove residues, or change chemical nature of sidechain, or cleave peptide backbone
4
Q
What led to the discovery of more types of post translational modifications?
A
More sophisticated methods
5
Q
True or false: almost all amino acids can be modified
A
True: although some are more than others
6
Q
What are the size of most post translational modifications?
A
50 - 100 Da
7
Q
True or false: all PTMs occur at the same frequency
A
False: some are common, but some are rare
8
Q
What are some examples of common PTMs?
A
Half of all proteins have initial Met removed, 60-90% of proteins in eukaryotic cytoplasm have N-terminal acetylation
9
Q
What are some examples of rare PTMs?
A
Hydroxylation of certain Pro and Lys residues occur only in procollagen, only 0.1% of cellular phosphoproteins have pTyr
10
Q
What determines nonenzymatic modifications?
A
The chemical environment
11
Q
How selective are nonenzymatic modifications?
A
Nonselective (occur at any susceptible residue)
12
Q
What are some examples of nonenzymatic PTMs?
A
Oxidation of Met or Cys, deamination of Asn/Gln to Asp/Glu, nitration of Tyr
13
Q
How do cells use irreversible nonenzymatic PTMs?
A
Metabolic intermediates, environmental stress (oxidative stress)
14
Q
True or false: all PTMs are reversible
A
False: some are irreverisble
15
Q
What are some examples of irreversible PTMs?
A
Acetylation of N-terminus, proteolysis
16
Q
What is a consensus sequence?
A
Short regions of protein sequence recognized by a modifying enzyme
17
Q
What is an example of a nonspecific enzymatic PTM?
A
N-acetylation (only requires free N-terminus)
18
Q
What is the consensus sequence for N-glycosylation?
A
Asn-X-Ser
19
Q
What is the consensus sequence for phosphorylation (by PKA)?
A
Arg-Arg-X-Ser
20
Q
What is the consensus sequence for farnesyl transferase?
A
Cys-A-A-X-COO-, A = alipathic
21
Q
True or false: the presence of a consensus sequence means it must be modified
A
False: this needs to be shown experimentally
22
Q
How can a modification be determined in vitro (3 ways)?
A
- Reaction with pure proteins (kinase + ATP, measure phosphorylation by SDS-PAGE)
- Synthetic peptide experiments (enzyme + peptide –> modified peptide)
- Reversal by chemical or enzymatic means
23
Q
How can a modification by determined in vivo (3 ways)?
A
- Incorporate radioactive precursor (isolate, digest, map sites by mass spec or Edman sequencing)
- Inhibitor study
- Mutational analysis
24
Q
What are the most convincing studies for determining if a PTM is present?
A
Using multiple approaches (radioactivity –> mutant, etc.)
25
What is the purpose of proteomic experiments?
Can identify multiple modified sites
26
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
27
What type of PTMs occur only in the ER?
Protein folding modifications
28
What PTMs (6) occur in the cytoplasm?
1. Removal of initial Met
2. N-terminal acetylation
3. N-terminal myristoylation
4. O-glycosylation with GlcNAc
5. Addition of palmitoyl groups
6. Virus polyprotein processing
29
What PTMs (1) occur in mitochondria/chloroplasts?
1. Cleavage of signal peptide
30
What PTMs (6) occur in the ER?
1. Cleavage of signal peptide
2. Core glycosylation of Asn residues
3. Addition of palmitoyl and glycosyl-phosphatidylinositol groups
4. Carboxylation of Glu
5. Hydroxylation of Pro and Lys in procollagen
6. Disulfide bond formation
31
What PTMs (3) occur in the Golgi?
1. Modification of N-glycosyl groups
2. O-glycosylation with GalNAc
3. Sulfation of Tyr residues
32
What PTMs (2) occur in secretory vesicles and granules?
1. Amidation of C-terminus
| 2. Proteolytic processing of some precursors
33
True or false: all PTMs have physiological importance
False: not all PTMs have physiological importance
34
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)
35
How can a PTM change the activity of a protein?
Change in conformation, localization, or chemical property of side chain
36
What is an example of a PTM changing protein conformation?
Phosphorylase
37
What is an example of a PTM changing localization?
Farnesylation of Ras
38
What is an example of a PTM changing chemical property of a side chain?
Carboxylation of Glu
39
What does farnesylation of Ras do?
Anchors Ras to the membrane (needs to signal from outside to inside)
40
How is a molecular barcode achieved?
By multisite PTMs (encode information)
41
What is the effect of multisite modifications?
Can have additive or antagonistic effects on other PTMs
42
True or false: one site can only have one possible PTM
False: there can be competing modifications at the same site
43
What are some examples of same site competition?
1. Phosphorylation or GlcNAc on Ser/Thr
2. Phosphorylation or sulfation on Tyr
3. Lots of possible PTMs on Lys
4. ADP ribosylation or methylation on Asp/Glu
44
What does SH2 interact with?
pTyr
45
What does HP1 chromodomain interact with?
Methylation of lysine
46
What does GCN5 bromodomain interact with?
Acetylation of lysine
47
What does Vps27 UIM interact with?
Ubitiquination of lysine
48
What does VHL-beta interact with?
Hydroxyproline
49
How do docking sites play a role in signal transduction?
RTKs can create pTyr, which act as docking sites for downstream signaling targets
50
What is proteolytic processing?
Cleavage of peptide bond
51
What proteins are commonly proteolytically processed?
Those destined for cellular organelles or secretion
52
What is proteolysis often used for?
Regulating the biological activity of a protein (only active when lysed)
53
What is a proprotein?
The inactive precursor that has yet to be lysed
54
What is a preproprotein?
The precursor of the proprotein, that has not had its signal peptide cleaved
55
When is the signal peptide cleaved for a preproprotein?
In the ER by a signal peptidase
56
What are some characteristics of proteolysis?
It is very specific (avoid indiscriminate proteolysis) and is irreversible
57
What are some examples of molecules that are proteolytically processed?
Peptide hormones, such as insulin
58
What is the structure of mature insulin?
A and B polypeptide chain, connected together by disulfide bonds
59
What is the structure of proinsulin?
Has an internal C peptide between the A and B polypeptide chain
60
How does proinsulin become insulin?
Cleavage of the C peptide releases the A and B peptide
61
What is the purpose of the C peptide in proinsulin?
Ensures proper folding and disulfide bond formation
62
Where does cleavage of the C peptide occur?
At pairs of basic residues (Lys-Arg and Arg-Arg)
63
What does POMC stand for?
Proopiomelanocortin
64
What is the structure of POMC?
It contains precursors of 8 different hormones
65
What is the significance of POMC?
It is a single precursor for many hormones, so it can coordinate actions, and produce different hormones
66
What determines which hormones are produced from POMC?
Based on the different processing enzymes produced by the (pituitary) cells
67
What are some examples of proteases?
Trypsin, chymotrypsin, and pepsin
68
What is a zymogen?
The inactive precursor of a protease
69
When is a zymogen converted into a protease?
When it is secreted and cleaved by a particular enzyme
70
What activates trypsinogen into trypsin?
Enterokinase
71
What activates chymotrypsinogen into chymotrypsin?
Trypsin
72
What are some examples of signaling pathways that use proteolysis?
Notch/Delta, Wnt/beta-catenin, NF-kB
73
How does Notch/Delta signaling work?
Cell adhesion/interaction leads to proteolysis to release intracellular domain to influence transcription
74
True or false: many PTMs occur at the N-terminus
True: there are many
75
What is deformylation?
The initial Met on prokaryotes is deformylated by a formylase
76
What is a formyl group?
OH - Fe - Cys / His / His
77
How many proteins in eukaryotes have N-terminal acetylation?
60-90%
78
What enzyme catalyzes N-terminal acetylation?
Ribosome-associated acetyltransferases
79
When can N-terminus acetylation occur?
Co-translationally or post-translationally, with or without preceding Met
80
What is the specificity of N-terminal acetylation?
Prefer small amino acids (Gly, Ala, Ser)
81
What is the biological role of N-terminal acetylation?
Regulate protein stability and interactions
82
How many proteins remove the initial Met?
About 50% in prokaryotes and eukaryotes
83
What enzyme removes the initial Met?
Ribosome-associated Met-aminopeptidase
84
What is the specificity of removal of initial Met?
Small residues at second position, large/charged residues unfavored
85
What is addition of a residue to the N-terminus?
Transfer an amino acid from a charged tRNA to a peptide
86
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)
87
What does MAP stand for, and what does it do?
Methionine aminopeptidases, removal of initial Met
88
What does NAT stand for, and what does it do?
N-acetyltransferases, add acetyl group to N-terminus
89
What is Gla?
Glu carboxylated at gamma position
90
What is the structure of Gla?
Two carboxyl groups at terminal carbon
91
In what proteins does carboxylation of Glu occur?
Proteins involved in blood clotting and bone structure
92
What enzyme catalyzes Glu carboxylation, and where is it found?
Vitamin K-dependent carboxylase, found in the membrane of the ER
93
What is the function of Gla?
Calcium binding (binds more tightly)
94
What is proline hydroxylation?
Add a hydroxyl group to proline (or Lys)
95
Where is the hydroxyl group added in Pro?
At the gamma position (2 carbons from the alpha carbon)
96
What is the consensus sequence for Pro hydroxylation?
X-Pro-Gly
97
What is the importance of gamma Pro hydroxylation?
Stabilizes alpha helix (other site for H-bonds, helps for helix/crosslinking)
98
What is the importance of delta-hydroxyl-Lys?
Important for glycosylation and for forming cross links
99
When is HIF1alpha expressed?
Under hypoxia (low oxygen conditions)
100
What happens to HIF1alpha in well oxygenated cells?
It is hydroxylized, which signals it for destruction
101
What is the significance of Lys acetylation?
Enhances gene expression through histone modifications
102
How does acetylation open up a histone?
Histone is positive, and acetylation removes positive charge (less association between histone and DNA)
103
What enzyme catalyzes histone acetylation?
HATs (histone acetyltransferases)
104
What enzyme catalyzes histone deacetylation?
HDACs (histone deacetylases)
105
What is a nucleosome?
DNA wrapped around 8 histone proteins (200 bp)
106
What do chromatin remodeling complexes do?
Alter chromatin structure (multisite modifications)
107
What competing reactions occur in histone remodeling?
Lys acetylation and Lys methylation (also Ser phosphorylation)
108
What is the significance of Lys4 on Histone 3?
It must be trimethylated to act as a docking site for other cellular proteins
109
Where does disulfide bond formation occur?
In the lumen of ER
110
Why is disulfide bond formation in the ER?
More oxidizing than cytosol
111
What is the significance of disulfide bonds?
Linked with 3D protein folding
112
What does PDI stand for?
Protein disulfide isomerase
113
What does PDI do?
Assists in disulfide bond formation and isomerization
114
What is the structure of PDI?
2 active sites with pairs of Cys residues
115
What is the mechanism for PDI?
It has pairs of Cys residues which can help reorganize disulfide bonds
116
Where does ADP-ribosylation occur?
In the cytosol or nucleus
117
What is ADP-ribosylation?
Add ADP-ribose (through NAD) to a protein
118
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
119
True or false: ADP ribosylation can only occur once
False: it can occur multiple times - poly(ADP-ribose)
120
How does cholera toxin work?
It ADP-ribosylates a G-protein to lock it in the "on" position
121
True or false: ADP-ribosylation is only pathological
False: it also has normal physiological roles
122
What does phosphorylation control?
Metabolism, hormone action, cell growth/cancer, gene expression, and memory
123
How does phosphorylation work?
Take a phosphate from ATP and add it to a protein
124
What is a kinase?
An enzyme that adds a phosphate to a protein
125
What is a phosphatase?
An enzyme that removes a phosphate from a protein
126
What are the two broad categories of protein kinases?
Tyr, and Ser/Thr
127
Which is the most common type of kinases?
Ser/Thr kinases
128
What is the same with all kinases?
Conserved catalytic core of 270 amino acids
129
What kinases are seen in prokaryotes?
pHis and pAsp
130
What is an example of a pHis pathway?
Bacterial chemotaxis
131
How does bacterial chemotaxis work?
CheA (His kinase) phosphorylates itself and CheY (response regulator) to alter motion (2 compartment system)
