The role of the ubiquitin system in human cytomegalovirus-mediated degradation of MHC class I heavy chains

The role of the ubiquitin system in human cytomegalovirus-mediated

degradation of MHC class I heavy chains

Hassink, G.C.

Citation

Hassink, G. C. (2006, May 22). The role of the ubiquitin system in human

cytomegalovirus-mediated degradation of MHC class I heavy chains. Retrieved from

https://hdl.handle.net/1887/4414

Version:

Corrected Publisher’s Version

CHAPTER

1

I

NTRODUCTI

ON

VIRAL IMMUNE EVASION AND QUALITY CONTROL

OF THE ENDOPLASMIC RETICULUM

Protein degradation and MHC class I-restricted antigen

presentation

Duri

ng

t

hei

r

l

i

fecycl

e,

prot

ei

ns

are

prone

t

o

agi

ng

as

a

resul

t

of

di

fferent

cel

l

ul

ar

st

resses

and

prot

ei

n

oxi

dat

i

on.

Cel

l

s

renew

t

hei

r

prot

ei

n

popul

at

i

on

by

means

of

const

ant

prot

ei

n

synt

hesi

s

and

degradat

i

on

_.

Al

t

hough

many

cel

l

ul

ar

organel

l

es

have

t

he

means

of

prot

ei

n

degradat

i

on,

t

he

prot

easome

pl

ays

a

domi

nant

rol

e

i

n

t

hi

s

process

_.

The

prot

easome

i

s

formed

by

a

l

arge

barrel

-shaped

20S

prot

ei

n

compl

ex

consi

st

i

ng

of

four

ri

ngs

of

t

wo

t

i

mes

seven

al

pha

and

t

wo

t

i

mes

seven

bet

a

subuni

t

s

resi

di

ng

i

n

t

he

cyt

osol

and

nucl

eus

_.

_The

_prot

_easome

_can

ei

t

her

be

i

n

compl

ex

wi

t

h

t

wo

11S

caps,

whi

ch

faci

l

i

t

at

es

t

he

accel

erat

ed

degradat

i

on

of

l

arge

pept

i

de

fragment

s

_,

_or

_i

_n

_compl

_ex

_wi

_t

_h

_t

_wo

_19S

_caps

on

bot

h

si

des,

whi

ch

faci

l

i

t

at

es

t

he

degradat

i

on

of

whol

e

prot

ei

ns

_.

_Bot

_h

_t

_he

19S

and

t

he

11S

caps

have

ATPase

act

i

vi

t

y.

The

19S

cap

t

oget

her

wi

t

h

t

he

20S

prot

easome

core

forms

a

26S

compl

ex.

Thi

s

compl

ex

recogni

zes

pol

y-ubi

qui

t

i

n

t

rees

t

hat

are

at

t

ached

t

o

prot

ei

n

subst

rat

es

(

see

l

at

er

on

i

n

t

hi

s

i

nt

roduct

i

on)

and

removes

t

hem

_.

_At

_t

_he

_same

_t

_i

_me,

_t

_he

_19S

_cap

_unfol

_ds

t

he

subst

rat

e

and

aqui

res

access

t

o

t

he

port

s

of

t

he

out

er

ri

ng

of

t

he

prot

easome

_.

_In

_t

_he

_core

_of

_t

_he

_prot

_easome,

_a

_set

_of

_t

_rypsi

_n-,

chemot

rypsi

n-

and

post

-gl

ut

amyl

pept

i

dyl

hydrol

yt

i

c-l

i

ke

prot

eases

cl

eave

t

he

unfol

ded

prot

ei

ns

i

nt

o

ol

i

gopept

i

des

wi

t

h

an

average

l

engt

h

of

4

t

o

20

ami

no

aci

ds

_.

_These

_short

_pept

_i

_de

_fragment

_s

_l

_eave

_t

_he

_prot

_easome

_and

_are

_furt

_her

processed

by

endopept

i

dase

t

o

ret

ri

eve

t

he

ori

gi

nal

ami

no

aci

ds

_.

_A

_smal

_l

number

of

pept

i

de

fragment

s,

however,

are

used

for

an

i

mport

ant

defense

syst

em

of

t

he

organi

sm.

solubilize the unfolded proteins, attract and coordinate the binding of

additional chaperones, and form disulfide bridges, respectively (figure 1).

Shortly after this process has been completed, the specialized

chaperone tapasin mediates the binding of the newly synthesized MHC class I

complexes to the transporter associated with antigen presentation (TAP). In

addition, the luminal lectin calreticulin

_{and Ƣ2m bind the heavy chain,}

together forming the MHC class I peptide-loading complex

_{. The}

proteasome-derived peptides are transported into the ER via the TAP

complex

_{. In the ER, the N-terminal ends of the peptides are trimmed to}

8-10 residues

_{, in order to fit into the newly synthesized MHC class I}

molecules

_{. Recently the ER localized exopeptidase ERAAP was found to}

be required for this trimming

_{. Only high affinity binding peptides allow}

further maturation and transport of MHC class I molecules to the cell surface

_.

At the cell surface, the protein complex is screened by another

specialized polymorphic molecule, the T-cell receptor, which is present on

cytotoxic T-lymphocytes, among others. The polymorphic TCRs are required

to recognize all the possible peptides presented by the MHC class I molecules.

A process of clonal selection ensures that only T-cells that fail to recognize

peptides derived from “self-proteins” are able to survive their maturation into

T-cells. W hen in addition to the MHC class I-peptide-TCR interaction, the

appropriate co-stimulatory signals are present, a signaling cascade is induced

in the T-cell resulting in the release of cytotoxic granules, which will ultimately

result in the death of the target cell in a process called apoptosis. Viruses have,

however, developed counter measures against this immune strategy.

Figure 1. M odel for translocation and formation of the M H C class I complex.

Life-long CMV infection and its risks

Human cytomegalovirus belongs to the herpes viridae, which are

common among all vertebrates. Cytomegaloviruses have a very large double

stranded DNA genome of 125-235 kbp. An important feature of these viruses

is that they are able to produce a life-long infection. The infection is usually

without symptoms as a long co-evolution of host and virus has resulted in a

delicate balance;

the host’

s immune system constantly represses upsurges of

viral expansion, while the virus continuously avoids detection by the host.

Yet CMV is a well-known pathogen. It is associated with acute and

chronic rejection of transplanted organs

_{and has also been implicated as a}

causative agent of atherosclerosis

_{. Furthermore, it is known to cause}

congenital defects in fetuses of pregnant women who have had a primary

CMV infection

_{. Finally, CMV frequently causes serious disease in}

immuno-compromised individuals. When the immune system of the host is

suppressed in periods of stress or fatigue, the virus may activate and use the

metabolism of the cells to proliferate. During sustained immuno-suppression,

e.g. in HIV-infected individuals or individuals using immuno-suppressive

agents to prevent rejection of transplanted tissue, CMV can cause hepatitis,

encephalitis, or pneumonia, all of which may be fatal

_.

To study the mechanisms of the HCMV-induced pathology, rat and

mouse CMV are often used as models. Mouse CMV (MCMV) is used to study

retinitis

_{, vasculitis}

_{, pneumonitis}

_{, and the role of the immune system in}

the pathogenesis of human CMV in general

_{. Rat CMV (RCMV), on the}

other hand, is used particularly in models of hart transplant-induced vascular

scleroses

_{, lung transplant-induced obliterative bronchiolitis}

_{, and in the}

study of diabetes

_.

Viral immune escape by targeting of MHC class I molecules is not

restricted to human CMV; cytomegaloviruses infecting other mammalians

also interfere with MHC class I expression. Mouse CMV encodes three

proteins affecting MHC class I expression at the cell surface. The m152 gene

product prevents export of class I complexes from the post-ER/early Golgi

_{. A second protein, the m06 gene product, induces lysosomal degradation}

of the MHC complex after a transient interaction in the ER

_{. A third gene}

product, the m04-encoded gp34, associates with properly folded MHC class I

molecules in the ER. The resulting complex is transported to the cell

membrane where it may modulate the function of NK cells

_{. Together,}

these MCMV-encoded immune evasion genes appear to counteract MHC

class I restricted T cell activation

_{. Although RCMV also establishes life}

long infection, the genes facilitating this persistence are yet to be identified.

HCMV, MCMV and RCMV all encode homologues of MHC class I heavy

chains

_{, which may deviate NK cell}

_{, and B-Cell- and}

macrophage-responses

_{. Although RCMV has been found to downregulate MHC class I}

expression

_{, the viral genes underlying this phenotype remain to be}

identified.

ER protein quality control and degradation of ER proteins

The discovery that US2 and US11-dependent degradation of MHC

class I heavy chains takes place at the level of the ER, suggested that US2 and

US11 used a pre-existing ER degradation pathway to downregulate MHC

class I molecules at the cell’s surface

_{. This pathway was shown to dispose}

of misfolded ER proteins like CFTR

_{, CPY*}

_{, and ste6}

_{. Since the}

discovery that ER proteins can in fact be retro-translocated into the cytosol

where they are degraded by the proteasome, substantial effort has been made

to elucidate the precise mechanism behind this process.

in the nucleus

_{. Therefore, protein expression is tightly controlled in}

eukaryotic cells. Prolonged production of misfolded proteins in the ER will

induce the translation of chaperones

_{, reduce translation of mRNA’s to}

relieve protein folding machineries

_{, and may eventually even induce}

apoptosis

_{. This process has been called the unfolded protein response}

(UPR).

Amino acid chains produced by the ribosome are recognized as

nascent ER proteins when they contain either one or more transmembrane

domains, a signal sequence, or a signal anchor. As soon as the ribosome

recognizes one of those domains in the newly synthesized amino acid chain,

further translation is halted until the ribosome has bound to the signal

recognition particle adjacent to a conduit for translocation in the ER which is

called the translocon (figure 2) (reviewed in

_{). The core of the translocon}

consists of a complex of the subunits Sec61ơ, Ƣ, and ƣ, which together form a

50 Ångström pore. The translocon can be equipped with a translocation-

associated membrane protein (TRAM), which is thought to facilitate a lateral

opening in the wall of this pore through which newly-translated

transmembrane-regions (TM) can move laterally into the lipid bi-layer

_.

Domain structure and polartity of TM-adjacent residues ultimately determine

the orientation of TM regions in the membrane

_{. Adjacent to the cytosolic}

side of the complex is the oligosaccharyltransferase (OST), which provides the

core glycan to be attached to asparagine residues present in NXT/S motifs

(where X is any amino acid except proline). Besides these complexes, a fourth

comlex, the translocon-associated complex (TRAP) can be found in complex

with the translocon and enhances translocation of proteins that have

prolonged acces to the cytoplasm

_{. Recently, a closer analysis using electron}

micrograph analysis (EMAN), shows that the ribosome actually binds four

Sec61 complexes and two TRAP complexes at the same time

_{. Only one of}

the Sec61 complexes, however, seems to be active in translocation

_.

In the ER, the N-linked glycans play an important role in protein

folding and quality control, as has been shown for yeast alpha1-antitrypsin

and mammalian IgM heavy chains, and many others

_{. Glucosidases I and}

II remove glucose moieties from GlcNAc2Man9Glc3-containing

glycoproteins to form GlcNAc2Man9 proteins. The membrane-bound

calnexin and soluble calreticulin recognize GlcNAc2Man9Glc1 sugar moieties

on the unfolded proteins and allow protein disulfide isomerase (PDI) ERP57

to bind these complexes. The PDI can now establish disulfide bridges within

the newly synthesized molecule and between subunits of protein complexes

_{. When all the glucose residues have been removed and the protein has}

folded correctly, it is promoted further into the secretion pathway, the Golgi

network, where the N-linked glycans are processed to larger structures. If the

complex has not acquired the correct conformation, however, it is recognized

by UDP-glucose:

glycoprotein glucosyltransferase (UGTR) which reattaches a

glucose residue to the N-linked glycan, thus enhancing the affinity for

calnexin and calreticulin to allow another round of folding

_{. At the same}

time, there is a chance that ER manosidase I will remove a mannose residue,

forming GlcNAc2Man8Glc1

_{. The GlcNAc2Man8Glc1 glycan is a poor}

substrate for glucosidase II. Therefore, it does not remove the last glucose

residue, and the protein is not released to the Golgi

_{. Prolonged ER}

retention eventually leads to the degradation of ER proteins.

This mannose clock is just one of the many checkpoints in the

maturation of proteins. A variety of specialized ER-associated compartments

are implied in quality control

_{. Furthermore, en route to the cell surface,}

misfolded or mal-complexed proteins are degraded in a pathway involving

lysomes

_{, the membrane-enveloped organelles responsible for}

degradation of the bulk of endocytosed material.

Retro-translocation of proteins from the ER to the cytosol (‘Dislocation’)

ER-resident proteins that do not pass the ER quality control are

destined for degradation by the proteasome which resides in the cytosol. How

exactly proteins are transported from the ER to the cytosol, a mechanism

termed retro-translocation or dislocation, is an intriguing question which is

not well understood (figure 3). The first step for ER proteins destined for

degradation, is the transport of these substrates through a conduit which may

be formed by components of the Sec61 complex

_{. Once in the cytosol,}

any N-linked glycans are removed by a recently identified N-glycanase

_.

Ultimately, the dislocated substrates are provided with polyubiquitin chains

and degraded by the proteasome. The energy that drives dislocation may

come from several sides. The translocon itself is a passive conduit.

Dislocation, however, involves ER luminal ATP

_{and several cytolic factors,}

namely the proteasome, the p97-ufd-npl4 AAA ATPase complex, and the

ubiquitin system.

Figure 3. Translocation of a protein from the ER into the cytosol.

The role of the proteasome in dislocation of proteins from the ER

A proportion of the 20S and 26S proteasomes have been found to be

associated with the ER membrane

_{. Involvement of the proteasome in}

the extraction of proteins from the ER membrane is suggested because the

ER-to-cytosol dislocation of many ER substrates can be prevented by the

removal or inhibition of proteasomes

_{. On the other hand, US2 and}

US11-dependent dislocation of MHC class I heavy chains

_{, and the}

dislocation of TCR-ơ

_{, have been clearly shown to occur in spite of}

proteasomal inhibition.

The Role of the p97 complex

The AAA ATPases form an abundant family, of which the members

are involved in various cellular functions, such as membrane trafficking,

proteasome function, organelle biogenesis, and microtubule regulation

(reviewed in

_{). Their modes of action are not well understood}

_{, but one}

of their functions is the ability to unfold molecules. For example, a ring of six

AAA ATPases comprise the regulatory lid in of the 20S proteasome in

eukaryotes

_{where they might drive unfolding of substrates before entrance}

of the proteolitic core

_{. Two other AAA ATPase rings, N-ethylmaleimide}

sensitive factor (NSF) and the p97-p47 complex, drive vesicle fusion and

transport by dissociating soluble NSF attachment protein receptor (SNARE)

complexes which are otherwise stable up to temperatures of 90 ºC

_{. Yet}

another function may be that they act as microtubule-based motors

_.

A role for AAA ATPase p97 in the degradadation of ER proteins was

established when Ye et al. discovered that MHC class I heavy chain

dislocation in yeast and US11-dependent dislocation of MHC class I heavy

chains was dependent on the vasolin-containing protein(VCP)/p97 in

complex with Ufd1 and Npl4

_{. The P97 protein constitutes 1% of the}

cytosol and it is, therefore, one of the most abundant proteins in the cell

_.

Every p97 molecule harbors two ATPase domains which form two hexameric

rings stacked on top of one another

_{. Exactly how p97-Ufd1-Npl4}

extracts the polypeptides from the ER membrane during dislocation is unclear

_.

has remained unclear, however, whether the actual dislocation from the ER is

also dependent on ubiquitination

_.

Attaching ubiquitin to a protein involves three steps, each providing

the versatility required to regulate the cellular process in question (Figure 4).

The first step is to activate the ubiquitin itself in order to prepare it for

attachment. This is accomplished by the E1 ubiquitin activating enzyme. The

amount of activated ubiquitin that is present in the cell ultimately limits the

many processes that depend on ubiquitination. E2 conjugating enzymes

subsequently bind the activated ubiquitin on a conserved cysteine residue in

the second step. It is thought that an E2 enzyme, probably in association with

an E3 enzyme, determines how the ubiquitin molecules will be attached to

one another and the substrate. A single, mono-ubiquitin attachment, or a

poly-ubiquitin chain linked at a certain lysine in the ubiquitin molecule is thus

formed on the target protein. Ubiquitin molecules can form isopeptide

linkages between the C-terminal glycine residue and one of the lysines at

positions 6, 11, 29, 48 or 63 on the next ubiquitin molecule. K63-based

ubiquitin linkages have been associated with DNA repair, INB kinase

activation and endocytosis, whereas K48 is mostly involved in proteasomal

degradation. K11 and K29 have also been associated with protein degradation

by the proteasome (reviewed in

_{). The E3 ubiquitin protein ligase}

enzymes accomplish the third step in the process. They bind the substrate and

facilitate the actual transfer of one or more ubiquitin molecules from the E2

enzyme to a lysine or the N-terminus of the selected protein.

Ubiquitin does not only have a role in proteasomal degradation but

also in a large number of other cellular functions. DNA repair in the nucleus,

regulation of translation, activation of transcription factors and kinases, and

proteasomal degradation all require ubiquitination. Protein sorting and

trafficking within the secretory system of the cell is also regulated by

Moreover, it has become clear in the past few years that ubiquitin also

plays a role in the degradation of ER proteins (See also chapter 3). Hrd1p of

S. cerevisiae was the first ER-resident E3 ubiquitin ligase to be identified as

having a direct role in the degradation of ER proteins

_{. Hrd1p/Der3p of}

S. cerevisiae was identified independently by Hampton and co-workers

_and

by Wolf and co-workers

_{. Hrd1p/Der3p functions as a ubiquitin ligase in}

the degradation of S. cerevisiae Hmg2p, one of the yeast isozymes of

3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR)

_.

Hrd1p/Der3p is also involved in degradation of other ER proteins,

including CPY* and sec61-2p

_{. Hrd1p/Der3p is a multispanning}

membrane protein with six transmembrane domains, and its C-terminal

RING-H2 domain is located in the cytosol

_{. In yeast, Hrd1p is found in a}

stoichiometric complex with Hrd3p, a lumen-oriented ER membrane protein

that stabilizes Hrd1p and modulates its ligase activity

_{. Hrd3p is, therefore,}

essential for a functional Hrd1p E3 ligase in yeast. Hrd1p acts with the E2

enzymes Ubc1p and Ubc7p, of which Ubc7p is more important in the

degradation of ER proteins

_{. Initially, it was thought that Hrd1p might be}

the central E3 ligase that served the degradation of yeast ER proteins in

general, together with Hrd1 associates Hrd3p and Der1p, and the E2’s Ubc1p

and Ubc7p

_{. However, while a number of substrates depend on Hrd1p for}

their degradation from the ER, a number of others do not

_.

Besides Hrd1p, two other E3 ligases dedicated to degradation of ER

proteins have been identified in yeast to date, Rsp5p and Doa10. Human

CFTR degradation has recently been shown to depend on Hrd1p in yeast, as

well as on Doa10, illustrating that the different E3 ligases active in

degradation of ER proteins, may cooperate to create a versatile degradation

machinery

_{. In mammalians, two homologues of Hrd1p have been}

identified: AMFR/gp78

_{and HRD1}

_{. AMFR facilitates the degradation}

of apoliprotein B100, whereas HRD1 facilitates the degradation of TCR-ơ

_{. Additionally, HRD1 and AMFR both facilitate the degradation of}

CD3-Ƥ

_{, stressing the versatility of the system.}

Outline of this thesis

It is clear from the above that the degradation of ER (glyco-)proteins is

a tightly regulated and complicated process involving a diversity of players not

necessarily dedicated to ER-associated degradation alone. The discovery that

viruses may co-opt this process to evade detection by the immune system has

significantly boosted research in this direction and forms the basis for this

thesis.

In this thesis we explored the mechanism by which the

HCMV-encoded proteins US2 and US11 alter the expression of MHC class I

molecules in order to evade MHC class I-dependent recognition by T-cells. In

the second chapter it was investigated whether, like human CMV and mouse

CMV, rat CMV also evades the host’s immune system using cell surface

downregulation of the MHC class I molecules.

In the third chapter we studied the role of the ubiquitin system in the

CMV US11-dependent dislocation of the MHC class I heavy chains. As

US11-mediated dislocation was indeed found to depend on a functional

ubiquitin system, a number of E3 ligases potentially involved in the

degradation of ER proteins were characterized. One of these potential E3

ligases is HRD1, a yet uncharacterized human homologue of the yeast Hrd1p.

In chapter four we characterized this homologue as an ER-resident ubiquitin

ligase involved in the degradation of TCR-ơ and CD3-Ƥ. Another potential E3

ligase is TEB4, a yet uncharacterized human homologue of the yeast Doa10.

This E3 was characterized in chapter five as an ER-resident ubiquitin ligase.

The crucial role of the ubiquitin system in protein dislocation suggested

that ubiquitination of lysines within the target proteins would be required to

facilitate dislocation. In chapter six we investigated whether lysines in the

MHC class I molecules themselves were indeed required for the dislocation of

these proteins in the presence of US2 and US11. Interestingly, this was not

the case. Nevertheless, a functional ubiquitin system was required for

on the role of the ubiquitin system in the dislocation process.

In chapter seven, we investigated whether Ƣ2m association and folding

of MHC class I heavy chains influenced US2 and US11-dependent

dislocation. Finally, in chapter eight we studied the class I dislocation process

in more detail. In particular, we investigated the order in which the amino acid

chains of MHC class I molecules were transported through the dislocon,

another important but hitherto unexplored aspect of the dislocation pathway.

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