Purposes%20of%20post-translational%20modifications

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• Purposes of post-translational modifications
• 2. Quality control in the cytoplasm
• Quality control in the ER
• 4. Selective post-translational proteolysis
• 5. Glycosylation in the ER and beyond N-linked
  vs. O-linked
• Other post-translational modifications
• Modifications that alter location
• A. Acylation myristoylation, palmitoylation,
  prenylation
• B. GPI anchor formation
• 8. Examples from pathobiology
• A. ERAD discovered through studying CMV US 11
  protein
• B. HIV-1 envelope undergoes critical
  post-translational modifications

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• Review of Translation

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• Purposes of Post-translational Events
  Modifications
• A. Quality Control Chaperones, Glycosylation
• B. Degradation of misfolded proteins
  Ubiquitination, ERAD
• C. Proper protein function Glycosylation,
  Phosphorylation, Ubiquitination
• D. Target protein to proper locations
  Acylation, GPI anchors

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• 2. Quality Control in the Cytoplasm
• A. Anfinsen's dogma
• All information needed for folding contained in
  the amino acid sequence
• Leads to the concept of spontaneous protein
  folding.
• B. Problems with Anfinsen's dogma (and the
  notion of spontaneous folding)
• Features of cellular environments cause
  misfolding aggregation.
• 1. Some proteins take a very long time to fold
  spontaneously.
• 2. Some protein domains are prone to
  misfolding and aggregation.

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Protein folding in vivo

• 2. Quality Control in the Cytoplasm
• Problems with Anfinsen's dogma, cont.
• Folding in the cell differs from refolding of a
  denatured protein in vitro due to
• Vectorial nature of protein synthesis in vivo.
• Exposure of hydrophobic regions during synthesis.
• Translation happens more slowly than folding,
  requiring a delay mechanism to allow
  translation to "catch up".
• Highly crowded cytoplasm 300 mg/ml prot.
• Folding in vitro is inefficient (20 - 30) in
  the cell, efficiency close to 100.
• Conditions of stress found in vivo exacerbate
  misfolding and aggregation.

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• 2. Quality Control in the Cytoplasm
• C. Molecular Chaperones Proteins that mediate
  correct fate of other polypeptides but are not
  part of the final structure.
• Fate includes folding, assembly, interaction
  with other cellular components, transport, or
  degradation.
• A. History
• ?Molecular chaperones initially identified as
  heat shock proteins, i.e. proteins upregulated by
  heat shock and other stresses.
• ?Heat shock causes protein denaturation with
  exposure and aggregation of interactive surfaces.
  
• ?Heat shock proteins inhibit
  aggregation by binding to exposed surfaces during
  times of stress but also during normal protein
  synthesis
• ?Thus, the stress response is simply an
  amplification of a normal function that is used
  by cells under non-stress conditions.

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• D. Features of molecular chaperones
• i. Hsp 70 family members
• ?70 kD protein monomers.
• ? Include DnaJ (bacteria) BiP (ER)
• ?Stabilize polypeptide surfaces in an unfolded
  state.
• ?Bind transiently to newly-synthesized
  proteins paradoxically, efficient folding
  requires "antifolding".
• ?Bind permanently to misfolded protein.
• ?Have affinity for exposed hydrophobic
  peptides.
• ?Do NOT bind a specific sequence.
• ?Present in bacteria, eukaryotes all
  compartments.
• ?Regulated by ATP hydrolysis.
• ?Undergo cycles of binding and release
• ?Act with cofactors (i.e. DnaJ, GrpE, Hip, Hop,
  Bag1).

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• D. Features of molecular chaperones
• ii. Chaperonins (GroEL, Hsp 60, TCP-1)
• ?Facilitate proper folding
• ?Bind and hydrolyze ATP
• ?Bind transiently to 10-15 proteins, but
  2-3fold more w/stress
• ?60 kD proteins that form oligomeric, stacked
  double rings
• ?Bring non-native substrate protein to central
  cavity folding where protected from aggregation
  with other non-native proteins
• ?Cycles of binding and release until the protein
  is properly folded
• ?GroEL (prokaryotic hsp 60) uses a cofactor,
  GroES.
• Others I.e. small heat shock proteins, hsp 90,
  etc.

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• iv. Cytosolic chaperone co-ordination
• Chaperones act in tandem. Stabilization by Hsp
  70 plus cofactors) may be followed by use of Hsp
  60 for proper folding.

From Frydman, J. Annual Rev. of Biochemistry
70603, 2001
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• 3. Quality control in the ER
• A. Translation and translocation of proteins
  into the ER
• ? Proteins that translocate into ER of
  mammalian cells include secretory proteins, TM
  proteins, or residents of a membranous
  compartment.
• ? These are targeted to the ER
  CO-TRANSLATIONALLY by an N-terminal signal
  sequence that generally gets cleaved during
  translocation across the ER membrane.

The Signal Hypothesis
SRP and SRP Receptor
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Translocation of Secretory Protein
Translocation of Single Pass TM Protein
Translocation of Double Pass TM Protein
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• 3. Quality Control in the ER
• B. Features of the ER
• ?Proteins need to be unfolded to translocate
• ?Until signal sequence cleaved, N terminus of
  protein is constrained "incorrectly
• ?ER lumen is topologically equivalent to
  extracellular space
• ?High oxidizing potential (unlike cytoplasm
  which is highly reduced)
• ?High Ca2 concentration unlike cytoplasm
• ?Many sugars present along with machinery for
  glycosylation
• ?As in cytoplasm high protein conc. (100
  mg/ml) promotes aggregation
• ?As in cytoplasm delay between translation/
  translocation vs. folding
• ?Site of specific post-translational events
  signal cleavage, S-S bond formation, N-linked
  glycosylation and GPI anchor addition

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• 3. Quality Control in the ER
• C. Specific ER chaperones
• i. HSP 70 family members BiP/GRP78
• ?Recognize hydrophobic sequences in nascent
  chains.
• ?Undergo successive rounds of ATP-dependent
  binding and release.
• ?Essential for translocation of
  newly-synthesized proteins across the ER lumen
  and for retrograde transport into the cytosol
  (see ERAD, below).
• ii. Immunophilins/ FKBP - peptidyl prolyl
  isomerases.
• iii. Thiol-disulfide isomerases - PDI and
  ERp57
• iv. Calnexin and Calreticulin
• ?Unique to the ER
• ?Are lectins (carbohydrate binding proteins)
• ?Calreticulin - lumenal Calnexin - integral
  membrane protein

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• 3. Quality Control in the ER
• D. Mechanisms
• To pass QC checkpoints, protein must be
  correctly folded (most energetically favorable,
  native state)
• If protein fails to fold properly it must be
  degraded
• I. Example 1 BiP
• BiP (Hsp70 in ER) binds to newly-synthesized and
  unfolded chains.
• BiP stays associated with misfolded (but not
  properly folded) proteins.
• Retention by BiP leads to degradation (see
  proteolysis below).

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3. Quality Control in the ER

• D. Mechanisms, cont.
• Example 2 Calnexin/calreticulin bind to
  incompletely folded monoglucosylated glycans
• Cycles of binding/release controlled by
• Glucosidase II cleaves glucose from core glycan
• UDP-glucose glucosyltransferase (GT)
  reglucosylates incompletely-folded proteins so
  that they bind lectins again
• Thus GT acts as a folding sensor proteins exit
  the cycle when GT fails to re-glucosylate.
  Glucose is a tag that signifies incomplete
  folding

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• 3. Quality Control in the ER
• D. Mechanisms, cont.
• iii. Example 3 Trimming of a single mannose is
  a signal for degradation.
• Causes association with ER degradation-enhancing
  mannosidase like protein (EDEM), which is a link
  to ER-associated degradation (see proteolysis
  below)

Tsai, B. et al. Nature Rev. Mol. Cell Bio. 3 246
(2002).
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• 4. Selective post-translational proteolysis.
• Selective proteolysis is critical for cellular
  regulation.
• 3 steps for proteolysis in the cytoplasm
• identify protein to be degraded
• mark it by ubiquitination
• deliver it to the proteasome, a protease complex
  that degrades it
• A. The Ubiquitin/Proteasome system
• Ubiquitin
• A highly-conserved 76 aa protein present in all
  eukaryotes.
• Covalently attached to e-amino groups in lysine
  side chains,
• Can be a single ubiquitin or multiple branched
  ubiquitins.
• Signal for ubiquitination can be
• 1. Programmed via hydrophobic sequence or
  other motif
• 2. Acquired by 1) phosphorylation, 2) binding
  to adaptor protein, or 3) protein damage due to
  fragmentation, oxidation or aging.

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• Post-translational Quality Control Selective
  proteolysis.
• B. Ubiquitination requires 3 enzymes
• E1 (ubiquitin-activating enzyme) activates
  ubiquitin (U)
• E2 (ubiquitin-conjugating enzyme) acquires U
  via high-energy thioester
• E3 (ubiquitin ligase) transfers U to target
  proteins
• Hierarchical organization one or few E1s exist,
  more E2s, many E3s.
• Other functions for ubiquitination (to be
  discussed in plasma membrane lecture).

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• 4. Post-translational Quality Control
  Selective proteolysis
• B. The Proteasome - high molecular weight (28S)
  protease complex that degrades ubiquitinated
  proteins in the cytoplasm
• Present in cytoplasm and nucleus, not ER
• Uses ATP
• Contains a 700 kD protease core and two 900 kD
  regulatory domains.
• Highly conserved and similar to proteases found
  in bacteria.
• Shaped like a cylinder.
• Proteins enter the cavity, and are cleaved into
  small peptides.
• Most but not all proteasome substrates are
  ubiqutinated.

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• 4. Post-translational Quality Control
  Selective Proteolysis
• Misfolding in the ER results in
• ER-associated degradation (see below)
• Lysosomal degradation (next lecture)
• ER-Associated Protein Degradation (ERAD)
• Earlier notion was that ER had proteases.
• However, in fact most ER proteins targeted for
  degradation undergo retrograde translocation
  into cytosol and delivery to the proteasome.

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• Glycosylation in the ER and beyond
• Role of sugars in the ER bulky hydrophilic
  groups that maintain proteins in solution, affect
  protein conformation, and allow lectins to
  facilitate folding and exert quality control.
• A. N-linked glycosylation - co-translational
  recognizes Asn-x-Ser/Thr on nascent chain
• Catalyzed by oligosaccharyltransferases -
  integral membrane proteins with active site in
  the lumen. Transfers a preformed "high mannose"
  14-residue sugar(Glc3Man9GlcNAc2) en bloc to
  asparagine residues on the acceptor nascent
  polypeptide chains. Highly conserved in
  mammals, plants, fungi.
• i. Donor molecule is dolichol-P-P-Glc3Man9GlcNAc
  2. Dolichol is a very long lipid carrier.
• ii. Subsequent trimming of residues (also
  called processing) off core sugar attached to
  protein occurs in the ER via glucosidases and
  mannosidases.
• N glycosylation can be prevented using
• Tunicamycin inhibits formation of the
  dolichol-P-P precursor.

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Bacteria no N-glycosylation via dolichol Yeast
have only oligomannose type N-glycans, because
they don't have the ability to add GlcNac in the
trans Golgi Since bacteria yeast lack
Glc-Nac transferase enzyme, this enzyme
demarcates a fundamental evolutionary boundary
between uni- and multicellular organisms.

• Glycosylation in the ER and beyond
• A. N-linked glycosylation, cont.
• iii. ? -Glucosyltransferase recognizes misfolded
  glycoproteins and reglycosylates them.
• Calreticulin and calnexin serve as sensors by
  binding to mono-glucosylated proteins,
  facilitating their folding and assembly.
• Only glycoproteins that have been correctly
  folded (by calnexin and calreticulin), get
  packaged into ER-to-Golgi transport vesicles.
• In the cis Golgi, further processing addition
  of GlcNac's to form branched structures
• Addition of more sugar residues in the
  trans-Golgi (I.e. fucose and sialic acid) to
  produce the diversity that is seen in mature
  glycans.

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• Glycosylation in the ER and beyond
• B. O-linked glycosylation
• Many different types of sugars are added onto
  -OH of serine or threonine residues.
• Most of these are added in ER or Golgi
• However, addition of N-acetylglucosamine
  (GlcNac) can occur in cytoplasm on many different
  types of proteins
• May play an important role in signaling, much
  like phosphorylation
• May act in signaling to oppose phosphorylation

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• Other post-translational modifications
• A. Disulfide bond formation in the ER
• Protein disulfide isomerase (PDI) in the ER
  catalyzes oxidation of disulfide bonds
• in the cytosol and at the plasma membrane
  reduces disulfide bonds
• Other proteins that act like PDI may be even
  more important in disulfide bond formation
• Requires action of a regenerating molecule (i.e.
  glutathione) NADPH is the source of redox
  equivalents.

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• 6. Other post-translational modifications, cont.
• B. Phosphorylation
• Kinases phosphorylate proteins at the hydroxyl
  groups of serine, threonine, and tyrosine
• Occurs in cytoplasm and nucleus
• C. Intracellular Proteolytic Cleavage
• Furin - protease that cleaves specific sites,
  located in the trans-Golgi network and in
  endosomes.
• D. Modified amino acids
• hydroxyproline, hydroxylysine,
  3-methylhistidine
• E. Lipidation

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• 7. Post-translational Modifications that Alter
  Location
• A. Acylation - Lipid attachments that anchor
  proteins to the membranes
• Include myristoylation, palmitoylation,
  prenylation
• Involves addition to protein of fatty acids
  (long hydrocarbon ending in COOH)
• Allows proteins to target to the cytoplasmic
  faces of membrane compartments

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• 7. Post-translational Modifications that Alter
  Location
• i. Myristoylation addition of C-14 FA
  myristate to N-terminus in cytoplasm
• Donor is myristoyl CoA
• Occurs co-translationally in the cytoplasm can
  occur post-translationally when hidden motif is
  revealed by protein cleavage (i.e. pro-apoptotic
  protein BID)
• Enzyme NMT recognizes consensus sequence at
  N-terminus often revealed by a
• conformational change (myristoyl switch).
• Promotes weak but typically irreversible
  interaction with cytosolic membrane face
• Myristoylated proteins traffic through the
  cytoplasm
• Myristoylation necessary but not sufficient for
  membrane binding
• Second signal needed for membrane binding
  myristate plus basic (basic aas interact with
  acidic phospholipids PS and PI), or myristate
  plus palmitate

Myristoylation
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• 7. Post-translational Modifications that Alter
  Location
• ii. Palmitoylation - addition of a C-16 fatty
  acid to the thiol side chain of an internal
  cysteine residue.
• Promotes a reversible interaction with membrane
• Palmitoylated proteins traffic to membrane via
  cytoplasm or via secretory pathway
• Enzymes not well understood
• Myristoylated and palmitoylated proteins
  are enriched in caveolae and rafts

Palmitoylation
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• 7. Post-translational Modifications that Alter
  Location
• iii. Prenylation - addition of prenyl groups
  (two types) to S in internal cysteine
• a. Farnesylation - C15 fatty acid to C
  terminus by thioester linkage
• Occurs at CAAX sequences cys, 2 aliphatic
  residues and C-terminal residue
• After attachment, last 3 residues are removed
  and new C terminal methylated
• Creates a highly hydrophobic C terminus
• b. Geranylgeranylation - similar to above but
  addition of C-20 to C terminal Cys

Farnesylation
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• 7. Post-translational Modifications that Alter
  Location
• iii. Examples of acylated proteins important
  for pathogenesis
• Myristoylated proteins HIV-1 Gag, HIV-1 Nef
  which target to the PM Arfs involved in coat
  protein binding to vesicles (see ER-Golgi
  lecture)
• Palmitoylated proteins caveolin (see PM
  lecture)
• Dual acylated proteins (myr plus palm) found
  in Src tyrosine kinases, i.e. Lyn, Fyn, Hck, etc.
  (see Signaling overview lecture)
• Met-Gly-Cys signal for dual acylation
• Farnesylation Ras, does not insert into the
  membrane or act in signal transduction unless
  farnesylated.
• Geranylgeranylation Rab GTP-binding proteins
  that mediate initial vesicle targeting events
  (see PM lecture)

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• 7. Post-translational Modifications that Alter
  Location
• B. GPI anchors - Glycophosphatidyl inositol (GPI)
  attached to the C terminus
• ?Composed of oligosaccharides and inositol
  phospholipids
• ?Provides a mechanism for anchoring cell-surface
  proteins to the membrane
• as a flexible leash that allows the entire
  protein (except for anchor) to be in
  extracellular space (unlike a transmembrane
  protein)
• ?Added to translocated proteins in ER
• ?Targets to PM via secretory pathway
• ?Unlike with N- or O-glycosylation, no more than
  ONE GPI anchor per protein
• ?Unlike acylation, targets proteins to outer
  leaflet of plasma membrane
• ?Can be cleaved by PI-phospholipase C (PI-PLC)
• ?Are minor components on mammalian cells but
  abundant on surfaces of parasitic protozoa (i.e.
  trypanosomes and Leishmania) and yeasts
• ?Concentrated in lipid rafts

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• 7. Post-translational Modifications that Alter
  Location
• B. GPI anchors - Functions
• Stronger anchoring to PM than acylation
• Some GPI anchors can be replaced with TM anchors
  and be functional others cannot
• Crosslinking results in signal transdcution
  across bilayer, including Ca influx, tyrosine
  phosphorylation, cytokine secretion, etc.
• Can interact with TM proteins capable of
  intracellular signaling
• Can indirectly modulate activity of cytosolic
  signaling molecules assoc. w/ lipid rafts

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• 8. Examples from Pathobiology
• ERAD discovered through study of CMV US11 (Wiertz
  et al., Cell 84 769, 1996).
• 1. MHC class I, a TM protein, binds viral
  peptides produced in cells and presents them at
  the cell surface to cytotoxic T cells.
• 2. CMV evades the immune system by targeting
  MHC class I for destruction soon after it is
  synthesized and translocated into the ER. How
  does it do this?
• 3. CMV US11 protein expressed alone causes MHC
  class I destruction.
• 4. US 11 effect is sensitive to proteasome
  inhibitors and involves MHC class I localization
  to cytoplasm, implying movemnt of US 11 out of ER
  into cytoplasm for degradation.
• 5. Before this paper, only forward movement
  thru translocon was thought to occur this paper
  by Ploeghs group studying a CMV protein raised
  the possibility of retrograde movement thru
  translocon.

ERAD
6. Subsequently, retrograde movement thru
translocon for degradation (ERAD) was shown to be
a common in non-infected cells. 7. Note that MHC
class I needs to be poly-ubiquitinated for
retrograde transport to occur, implying a role
for ubiqutination in retrolocation, not just in
targeting for degradation. 8. Additional studies
reveal that other pathogens use this mechanism
I.e. HIV-1 accessory protein Vpu promotes
degradation of CD4 by ERAD.
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• 8. Examples from Pathobiology
• HIV-1 envelope protein undergoes many critical
  post-translational modifications
• 1. HIV env consists of gp120 soluble portion
  bound non-covalently to TM gp41.
• Role is to bind CD4 and chemokine receptors
  during HIV-1 entry.
• 2. Co-translationally translocated into ER as
  gp160.
• 3. Has 30 potential sites for N-linked
  glycosylation in ER.
• If non-glycosylated wont bind CD4.
• Some glycosylations are dispensible for proper
  folding others are needed.
• 4. Forms 10 disulfide bonds in ER (9 are in
  gp120 portion).
• 5. Trimerization of HIV-1 env in ER
• 6. Proper folding/trimerization equires BiP,
  calnexin, calreticulin, and PDI.
• 7. In Golgi protease-mediated cleavage of
  gp160 to gp120 and gp41.

Land, A. and I. Braakman, Biochimie 83 783
(2001).
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• Additional Reading
• Tsai, B. et al. Retro-translocation of proteins
  from the endoplasmic reticulum into the cytosol.
  Nature Rev. Mol. Cell Bio. 3 246 (2002).
• Freiman, R. N. and R. Tijan. Regulating the
  regulators Lysine modifications make their
  mark. Cell 112 11 - 17 (2003).
• Resh, M. Fatty acylation of proteins new
  insights into membrane targeting of myristoylated
  and palmitoylated proteins. BBA 1451 1 (1999).
• Land, A. and I. Braakman. Folding of the human
  immunodeficiency virus type I envelope
  glycoprotein in the endoplasmic reticulum.
  Biochimie 83 783 (2001).
• Chatterjee, S. and S. Mayor. The GPI-anchor and
  protein sorting. Cell Mol. Life Sci 58 1969
  (2001).
• McClellan A et al. Protein quality control
  chaperones culling corrupt conformations. Nat
  Cell Biol. 2005 Aug7(8)736-41.
• Gill, G. SUMO and ubiquitin in the nucleus
  different functions, similar mechanisms? Genes
  Dev. 2004 Sep 118(17)2046-59. Review.