Biotech 2012 spring_8_post-trans

Post-translational Modifications:
March 28, 2006 J. R. Lingappa, Pabio 552, Lecture 1 1
1. Purposes of post-translational modifications
2. Quality control in the cytoplasm
3. Quality control in the ER
4. Selective post-translational proteolysis
5. Glycosylation in the ER and beyond: N-linked vs. O-linked
6. Other post-translational modifications
7. 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

1. Review of Translation:

1. 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

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.

Protein folding in vivo
aggregation due to exposure of
hydrophobic regions
DEAD-END PATHWAY
nascent chain
final folded structure
PRODUCTIVE PATHWAY
B. 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.

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.

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).
Hsp 70
Hsp 70 stabilizes
the nascent chain

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.
iii. Others: I.e. small heat shock proteins, hsp 90, etc.

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 70:603, 2001

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

Translocation of Secretory Protein
Translocation of Single Pass TM Protein Translocation of Double Pass TM Protein

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 Ca+2 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

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

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).

D. Mechanisms, cont.
ii. 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

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).

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 ε-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.

4. 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).

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.

4. Post-translational Quality Control: Selective Proteolysis
C. 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.
ER lumen
misfolded protein
cytoplasm
translocon
hsp 70 (BiP)
proteasome
U
U ATP
U
cytoplasm
ER lumen
ER-Associated Degradation (ERAD)
U
U ubiquitin
U
U
U
UU

5. 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-Glc3Man9GlcNAc2.
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.

5. Glycosylation in the ER and
beyond:
A. N-linked glycosylation, cont.
iii. α -Glucosyltransferase recognizes
misfolded glycoproteins and
reglycosylates them.
iv. Calreticulin and calnexin serve as
sensors by binding to mono-
glucosylated proteins, facilitating
their folding and assembly.
v. Only glycoproteins that have been
correctly folded (by calnexin and
calreticulin), get packaged into ER-to-
Golgi transport vesicles.
vi. In the cis Golgi, further processing &
addition of GlcNac's to form
branched structures
vii. 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.
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.

2. trimming
of glucose
residues
in ER
1. core sugar
added en bloc
co-translationally
to asparagine
residues
in nascent chains
(from dolichol
donor)
3. α−glucosyl
transferase
adds back
glucose
in ER to unfolded
glycoproteins
4. monoglucosylated
proteins are bound
and folded bycalnexin
and calreticulin
cis-Golgimedial-Golgi
6. in the medial and
trans-Golgi
more
N-acetylglucosamines
and fucose are added as
well as galactoses and
sialic acid (terminal
glycosylation)
using GlcNac
transferase
5. in the
Golgi,
trimming
of mannose
residues
occurs
= Sialic Acid
= GlcNac
= Mannose
= Glucose
= Galactose
Simplified view ofN-glycosylation

5. 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

6. 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.
substrate
substrate
S
S
SH
SH
PDI
S
S
PDI
SH
SH
redox
regenerator
SH
SH
S
S
redox
regenerator
Disulfide Bond Formation

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

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

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 aa’s
interact with acidic phospholipids PS and PI), or myristate plus palmitate
Myristoylation GlyMet
Gly
O
Gly
N-myristoyl transferase (NMT)
CH3
C-N-CH2-C
H
Removal of initiating methionine
Addition of myristate to N-terminal

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
Cys
SH2
C
O
CH 3
S
H
Cys

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
Cys
S
AA X
Cys
SH
AA X
Cys
S
Cys
S -O-CH3
addition offarnesyl group
proteolysis
methylation
Farnesylation

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)

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

C=O
CH2
CH2
NH
P
CH2CH2NH3
Mannose
N-Acetylgalactosamine
Inositol head of
PHOSPHATIDYLINOSITOL
ETHANOLAMINE
Protein
C-terminus
Lipid Bilayer
Glucosamine
OOLIGOSACCHARIDELIGOSACCHARIDE
P
Structure of a GPI anchor:

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
ER lumen
cytoplasm
ER
GPI
ER lumen
cytoplasm
ER
=C terminal
GPI signal
ER lumen
cytoplasm
ER
GPI
cleavage of
hydrophobic
C terminal
sequence and
transfer of
preformedGPI
moiety
GPI Anchor Formation
GPI
ER lumen
cytoplasm
ER
vesicle
formation
vesicle
transport
protein
translation
and
translocation
vesicle
fusion
cytoplasm
extracellular
space
PM
GPI
cytoplasm
extracellular
space
PM
=N terminal
signal sequence
GPIanchored
protein tethered
to outer leaflet
of PM

8. Examples from Pathobiology:
A. 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
Ploegh’s group studying a CMV protein raised the possibility of retrograde movement thru
translocon.
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.
ERAD:

8. Examples from Pathobiology:
B. 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: won’t 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).

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
Aug;7(8):736-41.
Gill, G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev. 2004 Sep
1;18(17):2046-59. Review.

Biotech 2012 spring_8_post-trans

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Andere mochten auch

Andere mochten auch (6)

Ähnlich wie Biotech 2012 spring_8_post-trans

Ähnlich wie Biotech 2012 spring_8_post-trans (20)

Mehr von BioinformaticsInstitute

Mehr von BioinformaticsInstitute (20)

Kürzlich hochgeladen

Kürzlich hochgeladen (20)

Biotech 2012 spring_8_post-trans