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 pathobiologyA. ERAD discovered through studying CMV US 11 proteinB. 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 ofhydrophobic regions

DEAD-END PATHWAY

nascent chainfinal folded structure

PRODUCTIVE PATHWAY

2. Quality Control in the Cytoplasm: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.



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.



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 stabilizesthe 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

3. Quality Control in the ER



3. Quality Control in the ERD. 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 degradedmark it by ubiquitinationdeliver 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 -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 motif2. 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 lumenmisfolded protein

cytoplasm

translocon

hsp 70 (BiP)

proteasome

U

U ATP

Ucytoplasm

ER lumen

ER-Associated Degradation (ERAD)

U

U ubiquitin

UU

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

chainCatalyzed 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.




A. N-linked glycosylation, cont.iii. -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. trimmingof glucose

residues in ER

1. core sugar added en bloc

co-translationally to asparagine

residues in nascent chains

(from dolicholdonor)

3 glucosyltransferase adds back

glucose in ER to unfolded

glycoproteins

4. monoglucosylated proteins are bound

and folded by calnexinand calreticulin

cis-Golgimedial-Golgi

6. in the medial andtrans-Golgi

moreN-acetylglucosamines

and fucose are added aswell as galactoses and

sialic acid (terminalglycosylation)using GlcNac

transferase

5. in theGolgi,

trimming of mannose

residuesoccurs

= Sialic Acid

= GlcNac

= Mannose

= Glucose

= Galactose

Simplified view of N-glycosylation




B. O-linked glycosylationMany 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 ERProtein 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

redoxregenerator

SH

SH

S

S

redoxregenerator

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 CleavageFurin - 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



7. Post-translational Modifications that Alter Location: i. Myristoylation: addition of C-14 FA myristate to N-terminus in cytoplasm

Donor is myristoyl CoAOccurs 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 aconformational change (myristoyl switch).Promotes weak but typically irreversible interaction with cytosolic membrane faceMyristoylated proteins traffic through the cytoplasmMyristoylation necessary but not sufficient for membrane bindingSecond 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



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 membranePalmitoylated proteins traffic to membrane via cytoplasm or via secretory pathwayEnzymes not well understood

Myristoylated and palmitoylated proteins are enriched in caveolae and rafts

Palmitoylation

Cys

SH2

C

O

CH 3SH

Cys



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

Cys

S

AA X

Cys

SH

AA X

Cys

S

Cys

S -O-CH3

addition of farnesyl group

proteolysis

methylation

Farnesylation



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)



7. Post-translational Modifications that Alter Location:B. GPI anchors - Glycophosphatidyl inositol (GPI) attached to the C terminusComposed of oligosaccharides and inositol phospholipidsProvides 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 ERTargets to PM via secretory pathwayUnlike with N- or O-glycosylation, no more than ONE GPI anchor per proteinUnlike 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 yeastsConcentrated in lipid rafts



C=O

CH2

CH2

NH

P

CH2CH2NH3

MannoseN-Acetylgalactosamine

Inositol head ofPHOSPHATIDYLINOSITOL

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 acylationSome 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 terminalGPI signal

ER lumen

cytoplasm

ER

GPI

cleavage of hydrophobic

C terminalsequence and

transfer ofpreformed GPI

moiety

GPI Anchor Formation

GPI

ER lumen

cytoplasm

ER

vesicleformation

vesicletransport

proteintranslation

and translocation

vesiclefusion

cytoplasm

extracellular space

PM

GPI

cytoplasm

extracellular space

PM

=N terminal signal sequence

GPI anchored 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.

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