Posttranslational Modification of Proteins

1
Posttranslational Modification of Proteins

• This refers to reactions that occur
  co-translationally (during protein synthesis) or
  posttranslationally (after protein synthesis)
• There are more than 50 types of posttranslational
  modifications well cover a selected group of
  them
• The most common modification is phosphorylation
  and dephosphorylation, and well devote future
  lectures to this topic
• Posttranslational reactions are divided into two
  main categories
• Those that have a signal peptide are targeted to
  the ER
• Those that lack a signal peptide are targeted
  initially to the cytosol

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Protein Targeting
Nascent polypeptide/ribosome
- Signal Seq
Endoplasmic Reticulum
Cytosol
Plasma Membrane
Golgi
Secretory Vesicles
Mitochondria
Nucleus
Lysosomes
Plasma Membrane
Cytosolic Pathway Secretory
Pathway No signal peptide With
a signal peptide
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Cytosolic Pathway

• Proteins that lack a signal peptide at the amino
  terminus are not translocated into the Golgi and
  are not processed
• These proteins are synthesized on free ribosomes
  not associated with the rough endoplasmic
  reticulum
• Final cell location
• Cytosol, e.g., hexokinase
• Nucleus, e.g., DNA polymerase
• Mitochondrion, e.g., cytochrome c
• Other modifications in the cytosol
• Acetylation (2C) Prenylation (15 or 20C)
• Myristoylation (14 C) Palmitoylation (16C)

4
NLS (Nuclear Localization Sequence)

• The nucleus is surrounded by a nuclear envelope
• Inner nuclear membrane
• Outer nuclear membrane
• Macromolecules are translocated through a nuclear
  pore
• All proteins found in the nucleus are synthesized
  in the cytosol and are translocated through the
  nuclear pore into the nucleus
• Histones, DNA polymerases, RNA polymerases
• Transcription factors, splicing factors

5
NLS (Nuclear Localization Sequence)

• Nuclear proteins contain an NLS
• One or two sequences (patches) rich in lysine and
  arginine
• Can be found anywhere in the protein at the
  N-terminus, in the middle, or at the C-terminus
• PKKKRKV is an example PKNKRKV is inactive
• Attachment of this sequence to normally cytosolic
  proteins results in the import of such mutated
  proteins into the nucleus
• The nucleoplasminin story
• It is required for chromatin assembly
• It contains two patches that are required for
  nuclear import
• A Lys-Arg pair
• Four lysines located 10 amino acids further
  downstream
• KRPAATKKAGQAKKKK, where the key residues are
  underlined

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NLS (Nuclear Localization Sequence)

• Mechanism
• Proteins with an NLS bind to importins that take
  them to the nuclear pore
• The alpha subunit of importin binds the NLS
• The beta subunit binds to the nuclear pore
• A Ran GTPase interacts with the protein-importin
  complex and energizes nuclear translocation with
  GTP hydrolysis
• The NLS is not removed proteolytically. Why?
• Nuclear Export Sequence (NES)
• Newly synthesized ribosomes (RNA and protein)
  bear nuclear export sequences
• This may involve signals on both proteins and RNA
• Nuclear Retention Signal (NRS)
• Found in proteins that bind to immature RNAs in
  the nucleus
• mRNAs that contain introns
• Pre-tRNAs

7
Nuclear Import and Export

• Importin binds to cargo and interacts with
  nucleoporins
• It is a nuclear import receptor
• It binds to basic NLSs
• RanGTP occurs in the nucleus, RanGDP in the
  cytosol
• RanGTP dissociates import complexes
• RanGTP forms export complexes
• Ran guanine nucleotide exchange factor (RanGEF)
  occurs in the nucleus
• RanGTPase activating protein (RanGAP) occurs in
  the cytosol
• Important points
• Ran is a GTPase
• RanGTP is nuclear
• RanGDP is cytosolic

8
Mitochondria and Protein Import

• Powerhouse of the cell
• Krebs cycle, beta oxidation, pyruvate
  dehydrogenase
• Contain DNA, RNA, ribosomes
• Synthesize about 20 mitochondrial proteins
• Most mitochondrial proteins are synthesized in
  the cytosol and imported

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Anatomy of the Mitochondionand Protein Import
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Import of Proteins

• Amino-terminal sequence (10-70 aa) contains
  positively charged, ser/thr, and hydrophobic
  amino acids but no common sequence
• Cyt c has an internal targeting sequence
• Hsp70 keeps proteins in an unfolded state
• Translocation through TOM (transport outer
  membrane)
• The fit is snug during transport
• Ions and other small molecules do not leak across
  the membrane
• Voltage gradient is required for transport across
  the inner membrane by TIM
• Presequence is cleaved by a signal protease
• Mit Hsp 70 and 60 aid in translocation and
  facilitates folding

11
Insertion of mit membrane proteins

• Proteins targeted for mitochondrial membranes
  contain hydrophobic stop sequences that halt
  translocation through the TOM or TIM complexes

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Sorting proteins to the intermembrane space

• I Through Tom into inner mitochondrial space
• II From Tom to Tim with hydrophobic stop
  sequences that are cleaved
• III Into matrix
• Remove hydrophilic basic sequence
• Exposes hydrophobic sequence that directs protein
  to inner mitochondrial space

13
Protein Import and the Peroxisome

• Peroxisomes oxidize lipids (fatty acids gt 18
  carbon atoms)
• Peroxisomes contain a single lipid bilayer
  membrane
• Unlike mitochondria, peroxisomes lack DNA
• All proteins are encoded by nuclear genes
• Peroxisome Targeting Signal (PTS)
• Type I PTS1
• C-terminus
• Ser-Lys-Ala (Dont memorize)
• Type II PTS2
• Rare (4 in humans)
• At or near the N-terminus
• RLXXXXXH/QL (Dont memorize)
• Peroxins deliver peroxisomal proteins to the
  target and insert them into the matrix or the
  membrane (mechanism ?)
• Take home message there are peroxisome targeting
  signals

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Membrane Localization Signals I

• Posttranslational attachment of lipids to
  proteins creating non-membrane spanning integral
  membrane proteins that will reside on the
  cytoplasmic surface of the plasma membrane of
  subcellular membranous organelle
• Myristoylation (14C)
• N-terminal processing
• Met-aminopeptidase often removes N-terminal Met
• If residue after methionine is a glycine, a
  myristoyl group can be attached via an amide
  linkage, blocking the amino terminal group
• The lipophilic myristoyl group can be inserted
  into the membrane
• Several of the alpha subunits of heterotrimeric
  G-proteins possess this modification
• Not all proteins that are N-myristoylated are
  attached to membranes
• MyristoylCoA H2N-Gly-protein ?
  myristoyl-CO-N(H)-Gly-protein CoA the high
  energy thioester is used to drive the synthesis
  of the low energy amide linkage

15
Protein Prenylation II

• A 15 carbon farnesyl group or a 20 carbon
  geranylgeranyl group is added to proteins that
  contain a C-terminal CaaX box
• C is cysteine
• a represents aliphatic residues (not Alanine)
• X represents leucine for geranylgeranyl groups
  and Met, Ser, Ala for farnesylation
• Ras is farnesylated
• Part of the Raf-MEK-ERK pathway
• Mutated in 25 of all human cancers
• Inhibition of Ras farnesylation is a targeted
  anticancer target
• The gamma subunit of many G-gamma proteins is
  geranylgeranylated
• These modifications promote membrane binding
• Know what a CaaX box is

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Prenylation Sequence of Reactions
Fig. 18-17
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Palmitoylation Reactions (16C)

• K-ras, one type of ras, is both farnesylated and
  palmitoylated
• A protein cysteine is modified as a thioester
• Palmitoyl-CoA protein CysSH ? protein
  CysSpalmitate CoA
• This concludes the Cytosolic Pathway
• Next, the Secretory pathway

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Secretory Pathway

• Products for secretion, transmembrane proteins,
  and import into Golgi/ER/Secretory granules
• Preproinsulin (secreted)
• Prealbumin (secreted)
• Preproinsulin receptor beta subunit
  (transmembrane)
• The pre refers to the signal peptide
• A signal peptide pre sequence at the amino
  terminus of a protein targets polypeptide/ribosome
  to the ER
• 6-13 hydrophobic proteins near the N-terminus
• Usually a positively charged residue nearby
• This is the second sequence besides CaaX that you
  should learn
• Some proteins are cleaved by signal peptidases
  and are found entirely within the lumen
• Some proteins contain stop transfer sequences
• These proteins become membrane spanning portions
  of integral membrane proteins

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Protein Synthesis

• Well see this again when we cover amelogenin
  biosynthesis in enamel formation
• Fig. 18-4

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Role of the Signal Sequence and Signal
Recognition Particle in Directing a Peptide to
the ER (Fig. 18-2)
Fig. 18-2
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Insulin Biosynthesis

• Synthesized as preproinsulin, the first such
  discovered protein
• The presequence is cleaved to yield proinsulin
  (signal peptidase)
• Disulfide bonds form, and the connecting peptide
  is cleaved by prohormone convertase
• Carboxypeptidase H finishes the job by cleaving
  the basic residues
• This yields mature insulin with its A and B
  chains
• Learn this process its an important prototype
• Fig. 18-3

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GPI Anchor Biosynthesis

• GPI GlycoPhosphatidyl-Inositol anchor
• Fatty acids in membrane
• Polar groups in lumen
• GPI transamidase catalyzes the reaction of the
  amino group with a protein carboxylate to give a
  new amide bond
• C(O)-N(H)-.. H2N- ? C(O)-N(H)-.. H2N-

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Cotranslational and Posttranslational
Modifications in the ER and Golgi

• Most of the modifications produced in the ER are
  constitutive (remain until the protein is
  degraded)
• These modifications take advantage of the
  unfolded nature of the polypeptide as it enters
  the lumen of the ER
• Glycosylation Reactions attach carbohydrate
  O-linked and N-linked (Fig. 18-7)
• O-linked refers refers to the attachment of
  sugars to serine or threonine (simple)
• N-linked refers to the attachment of sugars to
  asparagine (difficult)
• High mannose
• Complex
• Hybrid

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Endoplasmic Reticulum and Golgi

• Endoplasmic (inside the cell) reticulum a
  network
• ER, a network inside the cell
• Disulfide bond formation occurs in the ER
• N-linked oligosaccharide synthesis is initiated
  in the ER trimming and completion occurs in the
  Golgi
• Most O-glycosylation occurs in the Golgi
• Attachment of mannose 6-phosphate occurs in the
  Golgi
• Sulfation of secreted proteins occurs in the
  Golgi
• Proline and lysine hydroxylation, alpha
  amidation, and vitamin K-dependent carboxylation
  reactions occur in the Golgi

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N-Linked Oligosaccharides (Fig. 18-8)
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Activated Carbohydrates

• These serve as carbohydrate donors
• As activated sugars, a high-energy bond is used
  for the synthesis of a low-energy compound
• UDP-Glu, UDP-Gal, UDP-GlcUA, UDP-Xyl, UDP-GlcNAc,
  UPD-GalNAc, GDP-Man
• CMP-NeuNAc
• Dolichol phosphates
• It is not necessary to memorize the following
  pathways, but you should remember the identity of
  the activated sugars

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Hexosamine Metabolism (Fig. 18-9)
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Dolichol phosphate (Fig. 18-10)
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Dolichol Phosphate Metabolism (Fig. 18-11)
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First Stage of N-Linked Oligosaccharide
Synthetsis (Fig. 18-12) Occurs in the ER
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Second and Third Stages of N-Linked
Oligosaccharide Synthesis (Fig. 18-13)

• The hydrolysis reactions are unidirectional
• Donation of activated sugars is energetically
  favorable
• Each reaction is catalyzed by an enzyme that
  determines the sequence of sugars and the
  configuration of the glycosidic linkages
• Occurs in Golgi

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O-Linked Blood Group BiosynthesisTable 18-2
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Blood Group Glycoproteins (Fig. 18-15)
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Blood Group Biosynthesis

• Fig. 18-16
• Golgi reactions
• People with type O blood groups lack functional A
  and B genes
• Genes A and B differ by 4 nucleotides which
  alters the substrate specificity
• A GalNAc transferase
• B Gal transferase

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Targeting Enzymes to LysosomesA Golgi Process

• Lysosomal proteins contain N-linked
  oligosaccharides with terminal mannose
  6-phosphates
• The addition of phosphate occurs by an unusual
  mechanism or pathway
• There is a mannose 6-phosphate receptor that
  recycles between the Golgi and lysosome and
  participates in the translocation of lysosomal
  enzymes

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Mannose 6-Phosphate Synthesis (Fig. 18-14)
37
Protein Sulfation Reactions (Fig. 12-12)
Active sulfate PAPS, phosphoadenosylphosphosulfat
e
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Protein Sulfation

• A Golgi pathway modification
• Fig. 18-6

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Procollagen Hydroxylation

• Fig. 18-18
• Golgi reactions
• Proline and lysine hydroxylation reactions
• Requires vitamin C
• These two hydroxylases
• Dopamine beta-hydroxylase
• Peptidyl amidating mono-oxygenase
• The lysyl oxidase Rxn (next slide) inititates
  collagen cross linking

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Lysyl Oxidase Reaction (Fig. 23-12)
Golgi reactions
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Protein Amidation

• The amide group is derived from a carboxyterminal
  glycine
• Ascorbate and oxygen are required
• Golgi reactions
• Fig. 18-19

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Vitamin K-Dependent Carboxylation Reactions

• Protein-glutamate is carboxylated
• Carbon dioxide, Vitamin K, and oxygen are
  required
• Several blood clotting factors and other proteins
  that bind calcium ions contain gamma
  carboxylation of protein-glutamates
• Golgi reactions

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Vitamin K Carboxylation/Oxidation (Fig. 18-20)
44
Thyroid Hormone Biosynthesis (Fig. 18-21)
45
Plasma Membrane Topology

• It is not necessary to remember which protein has
  which type of membrane topology except for GPCRs

46
Topological Classes

• Topological classes I-III have a single pass
  through the membrane
• I N-terminus is intraluminal and C-terminus is
  extraluminal
• II C-terminus is extra, N-terminus is intra no
  cleaved sequence
• III Same as I, but no cleavable sequence
• Class IV has multiple passes
• Type I, without a signal peptide, contains a 22
  aa hydrophobic stop transfer sequence
• Type II and III
• Lack a N-terminal signal peptide
• Contain a signal-anchor sequence that functions
  as an ER signal sequence and membrane anchor
  sequence

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Type II Membrane Protein Biosynthesis

• Stop transfer anchor sequence
• C-terminus in the ER lumen or cell exterior

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Protein Targeting
Nascent polypeptide/ribosome
- Signal Seq
Endoplasmic Reticulum
Cytosol
Plasma Membrane Lipidation with
myristate, palmitate, farnesylate, or
geranylgeranylate
Golgi Glycosylation, Amidation, Sulfation, K
carbox, Hydroxylation
Mitochondria Basic aa at N-ter
Nucleus Basic amino acids
Secretory Vesicles
Lysosomes Mannose 6-P
Plasma Membrane Stop transfer sequences