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CHE 541 > 15 - Post-translational Modifications > Flashcards

15 - Post-translational Modifications Flashcards

1
Q

What is the importance of post translational modifications?

A

Still need modifications for the mature protein

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2
Q

Where do post translational modifications take place?

A

During or after assembly on the ribosome

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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

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4
Q

What led to the discovery of more types of post translational modifications?

A

More sophisticated methods

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5
Q

True or false: almost all amino acids can be modified

A

True: although some are more than others

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6
Q

What are the size of most post translational modifications?

A

50 - 100 Da

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7
Q

True or false: all PTMs occur at the same frequency

A

False: some are common, but some are rare

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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

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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

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10
Q

What determines nonenzymatic modifications?

A

The chemical environment

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11
Q

How selective are nonenzymatic modifications?

A

Nonselective (occur at any susceptible residue)

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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

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13
Q

How do cells use irreversible nonenzymatic PTMs?

A

Metabolic intermediates, environmental stress (oxidative stress)

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14
Q

True or false: all PTMs are reversible

A

False: some are irreverisble

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15
Q

What are some examples of irreversible PTMs?

A

Acetylation of N-terminus, proteolysis

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16
Q

What is a consensus sequence?

A

Short regions of protein sequence recognized by a modifying enzyme

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17
Q

What is an example of a nonspecific enzymatic PTM?

A

N-acetylation (only requires free N-terminus)

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18
Q

What is the consensus sequence for N-glycosylation?

A

Asn-X-Ser

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19
Q

What is the consensus sequence for phosphorylation (by PKA)?

A

Arg-Arg-X-Ser

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20
Q

What is the consensus sequence for farnesyl transferase?

A

Cys-A-A-X-COO-, A = alipathic

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21
Q

True or false: the presence of a consensus sequence means it must be modified

A

False: this needs to be shown experimentally

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22
Q

How can a modification be determined in vitro (3 ways)?

A
  1. Reaction with pure proteins (kinase + ATP, measure phosphorylation by SDS-PAGE)
  2. Synthetic peptide experiments (enzyme + peptide –> modified peptide)
  3. Reversal by chemical or enzymatic means
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23
Q

How can a modification by determined in vivo (3 ways)?

A
  1. Incorporate radioactive precursor (isolate, digest, map sites by mass spec or Edman sequencing)
  2. Inhibitor study
  3. Mutational analysis
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24
Q

What are the most convincing studies for determining if a PTM is present?

A

Using multiple approaches (radioactivity –> mutant, etc.)

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25
Q

What is the purpose of proteomic experiments?

A

Can identify multiple modified sites

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26
Q

How do proteomic experiments work?

A

Fractionation or affinity capture to enrich modified protein, then use mass spec to analyze masses and exact site of modification

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27
Q

What type of PTMs occur only in the ER?

A

Protein folding modifications

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28
Q

What PTMs (6) occur in the cytoplasm?

A
  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
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29
Q

What PTMs (1) occur in mitochondria/chloroplasts?

A
  1. Cleavage of signal peptide
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30
Q

What PTMs (6) occur in the ER?

A
  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
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31
Q

What PTMs (3) occur in the Golgi?

A
  1. Modification of N-glycosyl groups
  2. O-glycosylation with GalNAc
  3. Sulfation of Tyr residues
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32
Q

What PTMs (2) occur in secretory vesicles and granules?

A
  1. Amidation of C-terminus

2. Proteolytic processing of some precursors

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33
Q

True or false: all PTMs have physiological importance

A

False: not all PTMs have physiological importance

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34
Q

What is an example of a PTM that does not have physiological importance?

A

Artefact of isolation process (proteolytic digestion, disulfide scrambling) (shouldn’t have occurred)

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35
Q

How can a PTM change the activity of a protein?

A

Change in conformation, localization, or chemical property of side chain

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36
Q

What is an example of a PTM changing protein conformation?

A

Phosphorylase

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37
Q

What is an example of a PTM changing localization?

A

Farnesylation of Ras

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38
Q

What is an example of a PTM changing chemical property of a side chain?

A

Carboxylation of Glu

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39
Q

What does farnesylation of Ras do?

A

Anchors Ras to the membrane (needs to signal from outside to inside)

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40
Q

How is a molecular barcode achieved?

A

By multisite PTMs (encode information)

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41
Q

What is the effect of multisite modifications?

A

Can have additive or antagonistic effects on other PTMs

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42
Q

True or false: one site can only have one possible PTM

A

False: there can be competing modifications at the same site

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43
Q

What are some examples of same site competition?

A
  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
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44
Q

What does SH2 interact with?

A

pTyr

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45
Q

What does HP1 chromodomain interact with?

A

Methylation of lysine

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46
Q

What does GCN5 bromodomain interact with?

A

Acetylation of lysine

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47
Q

What does Vps27 UIM interact with?

A

Ubitiquination of lysine

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48
Q

What does VHL-beta interact with?

A

Hydroxyproline

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49
Q

How do docking sites play a role in signal transduction?

A

RTKs can create pTyr, which act as docking sites for downstream signaling targets

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50
Q

What is proteolytic processing?

A

Cleavage of peptide bond

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51
Q

What proteins are commonly proteolytically processed?

A

Those destined for cellular organelles or secretion

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52
Q

What is proteolysis often used for?

A

Regulating the biological activity of a protein (only active when lysed)

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53
Q

What is a proprotein?

A

The inactive precursor that has yet to be lysed

54
Q

What is a preproprotein?

A

The precursor of the proprotein, that has not had its signal peptide cleaved

55
Q

When is the signal peptide cleaved for a preproprotein?

A

In the ER by a signal peptidase

56
Q

What are some characteristics of proteolysis?

A

It is very specific (avoid indiscriminate proteolysis) and is irreversible

57
Q

What are some examples of molecules that are proteolytically processed?

A

Peptide hormones, such as insulin

58
Q

What is the structure of mature insulin?

A

A and B polypeptide chain, connected together by disulfide bonds

59
Q

What is the structure of proinsulin?

A

Has an internal C peptide between the A and B polypeptide chain

60
Q

How does proinsulin become insulin?

A

Cleavage of the C peptide releases the A and B peptide

61
Q

What is the purpose of the C peptide in proinsulin?

A

Ensures proper folding and disulfide bond formation

62
Q

Where does cleavage of the C peptide occur?

A

At pairs of basic residues (Lys-Arg and Arg-Arg)

63
Q

What does POMC stand for?

A

Proopiomelanocortin

64
Q

What is the structure of POMC?

A

It contains precursors of 8 different hormones

65
Q

What is the significance of POMC?

A

It is a single precursor for many hormones, so it can coordinate actions, and produce different hormones

66
Q

What determines which hormones are produced from POMC?

A

Based on the different processing enzymes produced by the (pituitary) cells

67
Q

What are some examples of proteases?

A

Trypsin, chymotrypsin, and pepsin

68
Q

What is a zymogen?

A

The inactive precursor of a protease

69
Q

When is a zymogen converted into a protease?

A

When it is secreted and cleaved by a particular enzyme

70
Q

What activates trypsinogen into trypsin?

A

Enterokinase

71
Q

What activates chymotrypsinogen into chymotrypsin?

A

Trypsin

72
Q

What are some examples of signaling pathways that use proteolysis?

A

Notch/Delta, Wnt/beta-catenin, NF-kB

73
Q

How does Notch/Delta signaling work?

A

Cell adhesion/interaction leads to proteolysis to release intracellular domain to influence transcription

74
Q

True or false: many PTMs occur at the N-terminus

A

True: there are many

75
Q

What is deformylation?

A

The initial Met on prokaryotes is deformylated by a formylase

76
Q

What is a formyl group?

A

OH - Fe - Cys / His / His

77
Q

How many proteins in eukaryotes have N-terminal acetylation?

A

60-90%

78
Q

What enzyme catalyzes N-terminal acetylation?

A

Ribosome-associated acetyltransferases

79
Q

When can N-terminus acetylation occur?

A

Co-translationally or post-translationally, with or without preceding Met

80
Q

What is the specificity of N-terminal acetylation?

A

Prefer small amino acids (Gly, Ala, Ser)

81
Q

What is the biological role of N-terminal acetylation?

A

Regulate protein stability and interactions

82
Q

How many proteins remove the initial Met?

A

About 50% in prokaryotes and eukaryotes

83
Q

What enzyme removes the initial Met?

A

Ribosome-associated Met-aminopeptidase

84
Q

What is the specificity of removal of initial Met?

A

Small residues at second position, large/charged residues unfavored

85
Q

What is addition of a residue to the N-terminus?

A

Transfer an amino acid from a charged tRNA to a peptide

86
Q

What is an example of addition to a terminal residue?

A

Arginyl tRNA protein transferase catalyzes transfer of Arg to proteins with N-terminal Glu or Asp to target them for degradation (ubiquitin)

87
Q

What does MAP stand for, and what does it do?

A

Methionine aminopeptidases, removal of initial Met

88
Q

What does NAT stand for, and what does it do?

A

N-acetyltransferases, add acetyl group to N-terminus

89
Q

What is Gla?

A

Glu carboxylated at gamma position

90
Q

What is the structure of Gla?

A

Two carboxyl groups at terminal carbon

91
Q

In what proteins does carboxylation of Glu occur?

A

Proteins involved in blood clotting and bone structure

92
Q

What enzyme catalyzes Glu carboxylation, and where is it found?

A

Vitamin K-dependent carboxylase, found in the membrane of the ER

93
Q

What is the function of Gla?

A

Calcium binding (binds more tightly)

94
Q

What is proline hydroxylation?

A

Add a hydroxyl group to proline (or Lys)

95
Q

Where is the hydroxyl group added in Pro?

A

At the gamma position (2 carbons from the alpha carbon)

96
Q

What is the consensus sequence for Pro hydroxylation?

A

X-Pro-Gly

97
Q

What is the importance of gamma Pro hydroxylation?

A

Stabilizes alpha helix (other site for H-bonds, helps for helix/crosslinking)

98
Q

What is the importance of delta-hydroxyl-Lys?

A

Important for glycosylation and for forming cross links

99
Q

When is HIF1alpha expressed?

A

Under hypoxia (low oxygen conditions)

100
Q

What happens to HIF1alpha in well oxygenated cells?

A

It is hydroxylized, which signals it for destruction

101
Q

What is the significance of Lys acetylation?

A

Enhances gene expression through histone modifications

102
Q

How does acetylation open up a histone?

A

Histone is positive, and acetylation removes positive charge (less association between histone and DNA)

103
Q

What enzyme catalyzes histone acetylation?

A

HATs (histone acetyltransferases)

104
Q

What enzyme catalyzes histone deacetylation?

A

HDACs (histone deacetylases)

105
Q

What is a nucleosome?

A

DNA wrapped around 8 histone proteins (200 bp)

106
Q

What do chromatin remodeling complexes do?

A

Alter chromatin structure (multisite modifications)

107
Q

What competing reactions occur in histone remodeling?

A

Lys acetylation and Lys methylation (also Ser phosphorylation)

108
Q

What is the significance of Lys4 on Histone 3?

A

It must be trimethylated to act as a docking site for other cellular proteins

109
Q

Where does disulfide bond formation occur?

A

In the lumen of ER

110
Q

Why is disulfide bond formation in the ER?

A

More oxidizing than cytosol

111
Q

What is the significance of disulfide bonds?

A

Linked with 3D protein folding

112
Q

What does PDI stand for?

A

Protein disulfide isomerase

113
Q

What does PDI do?

A

Assists in disulfide bond formation and isomerization

114
Q

What is the structure of PDI?

A

2 active sites with pairs of Cys residues

115
Q

What is the mechanism for PDI?

A

It has pairs of Cys residues which can help reorganize disulfide bonds

116
Q

Where does ADP-ribosylation occur?

A

In the cytosol or nucleus

117
Q

What is ADP-ribosylation?

A

Add ADP-ribose (through NAD) to a protein

118
Q

What is the specificity of ADP-ribosylation?

A

Add to N atoms of Arg, His, Asn, Lys, the carboxyl of Glu, and the alpha carboxyl of Lys

119
Q

True or false: ADP ribosylation can only occur once

A

False: it can occur multiple times - poly(ADP-ribose)

120
Q

How does cholera toxin work?

A

It ADP-ribosylates a G-protein to lock it in the “on” position

121
Q

True or false: ADP-ribosylation is only pathological

A

False: it also has normal physiological roles

122
Q

What does phosphorylation control?

A

Metabolism, hormone action, cell growth/cancer, gene expression, and memory

123
Q

How does phosphorylation work?

A

Take a phosphate from ATP and add it to a protein

124
Q

What is a kinase?

A

An enzyme that adds a phosphate to a protein

125
Q

What is a phosphatase?

A

An enzyme that removes a phosphate from a protein

126
Q

What are the two broad categories of protein kinases?

A

Tyr, and Ser/Thr

127
Q

Which is the most common type of kinases?

A

Ser/Thr kinases

128
Q

What is the same with all kinases?

A

Conserved catalytic core of 270 amino acids

129
Q

What kinases are seen in prokaryotes?

A

pHis and pAsp

130
Q

What is an example of a pHis pathway?

A

Bacterial chemotaxis

131
Q

How does bacterial chemotaxis work?

A

CheA (His kinase) phosphorylates itself and CheY (response regulator) to alter motion (2 compartment system)

CHE 541 (20 decks)

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