The possible biological and reproductive functions of ubiquitin

The possible biological and reproductive functions ofubiquitin

C.Bebington1, F.J.Doherty2 and S.D.Fleming3,4

1Shef®eld Fertility Centre, Shef®eld, UK, 2School of Biomedical Sciences, University of Nottingham Medical School, Queen's

Medical Centre, Nottingham, NG7 2UH, UK and 3Department of Obstetrics and Gynaecology, University of Sydney, Westmead

Hospital, NSW 2145, Australia

4To whom correspondence should be addressed. E-mail: [email protected]

The protein ubiquitin (Ub) appears to be present in all eukaryotic cells. Its widespread presence and extremelyconserved structure indicate that it may play a vital role in cell metabolism. The roles of Ub are mediated by itscovalent attachment to target proteins, a process known as ubiquitylation, a form of protein modi®cation which maylead to degradation of the modi®ed protein. A number of proteins with similar structure to Ub but varying infunction have been isolated. Recently, there has been much interest in the role of Ub and its related proteins inreproductive processes. Ub and Ub-related proteins may be involved in gametogenesis, modulation of steroidreceptor concentrations, placental development and endometrial modi®cation at the beginning of pregnancy. Thesewide-ranging effects have led to extensive research which will be reviewed in this article.

Key words: human/reproduction/ubiquitin/ubiquitin cross-reactive protein

TABLE OF CONTENTS

The ubiquitin pathway for protein degradation

Functions of ubiquitylation

Ubiquitin in reproduction

Ubiquitin-like proteins

Summary

References

The ubiquitin pathway for protein degradation

Ubiquitin and ubiquitylation

Ubiquitin (Ub) is a 76-residue protein (Schlesinger et al., 1975)

that possesses one of the most conserved sequences known

(Jentsch et al., 1991). The compact, globular structure of Ub may

protect it from proteolysis during the degradation of ubiquitylated

proteins, while its carboxyl terminus (which is essential for Ub

function) is extended from the body of the protein, allowing

interaction with target proteins (Viijay-Kumar et al., 1987).

The attachment of a multi-Ub chain to a protein is necessary for

proteolysis of the target protein by the 26S proteasome.

Ubiquitylation is a multi-enzyme process that generates an

isopeptide bond between the a-carboxyl group of the C-terminal

glycine of Ub and the e-amino group of a lysine side-chain in the

target protein (Chau et al., 1989; Ciechanover et al., 2000).

Additional Ub±Ub isopeptide bonds may be formed between the

C-terminal of an incoming Ub and an amino group (usually within

Lys48) of the bound Ub. This may continue until a chain of up to

20 units is formed (Hershko and Ciechanover, 1998). The process

of ubiquitylation is summarized in Figure 1.

Ub is coded by a multigene family (Jentsch et al., 1991). No

gene produces Ub as a mature protein; instead, it is translated as a

fusion protein with a C-terminal extension consisting of either a

ribosomal protein (Finley et al., 1989; Redman and Rechsteiner,

1989) or further copies of Ub to generate a poly-Ub protein

(OÈ zkaynak et al., 1984; Wiborg et al., 1985). Monomeric Ub is

generated from precursors by the action of Ub C-terminal

hydrolases which cleave after the C-terminal glycine of mature

Ub (see Figure 1). Poly-Ub genes often carry a heat shock promoter

element and may be involved in the response to environmental

stress (Bond and Schlesinger, 1985; Finley et al., 1987).

E1 enzymes

Ubiquitylation begins with the ATP-dependent activation of Ub to

form a Ub-adenylate-E1 complex, where E1 is a Ùb-activating'

(UbA) enzyme (Ciechanover et al., 1982). The Ub is then

transferred to a cysteine residue in E1 to form an enzyme-Ub thiol

ester (Haas and Rose, 1982). E1 enzymes from different species

appear highly conserved and often possess a gly-X-gly-X-X-gly

motif (where `X' indicates any amino acid) (McGrath et al., 1991).

E2 enzymes

Activated Ub is next transferred to an àctive site' cysteine residue

within an E2 enzyme (also known as a Ub-carrier/conjugating or

Human Reproduction Update, Vol.7, No.1 pp. 102±111, 2001

102 Ó European Society of Human Reproduction and Embryology

UbC enzyme: Hershko et al., 1983; Haas and Bright, 1988; Jentsch

et al., 1990). In contrast to the E1 enzymes there appear to be a

variety of E2 enzymes within each cell (Pickart and Rose, 1985).

Again, Ub attachment is via a thiol ester linkage (Hershko et al.,

1983; Pickart and Rose, 1985; Sung et al., 1990). All known E2

enzymes share a homologous sequence known as the UbC domain,

which contains the cysteine residue essential for activity (Jentsch et

al., 1990; Sung et al., 1990), while others possess additional

sequences which may be necessary for substrate speci®city

(Morrison et al., 1988). Ub passes from the E2 enzyme to the

target protein either directly or via a third enzyme.

E3 enzymes

The E3 (Ub ligation/recognition) enzymes are not always essential

for ubiquitylation, at least in vitro, especially if the E2 enzyme

shows substrate speci®city (Ciechanover and Schwartz, 1989), and

it is postulated that a wide range of E3 enzymes may exist, each

with a unique substrate speci®city. Some E3 enzymes can bind

both the E2 enzyme and the target protein and promote transfer of

Ub to form an isopeptide bond with the target protein. However,

some E3 enzymes are directly involved in the transfer of Ub,

accepting Ub from an E2 enzyme to form a thiol ester and being the

proximal donor to the target protein (reviewed in Hershko and

Ciechanover, 1998). Recently, a class of additional factors

involved in the formation of multi-Ub chains on target proteins

have been discovered, dubbed, unsurprisingly E4 (Koegl et al.,

1999).

Substrate recognition

The presence of a lysine residue is necessary, but not suf®cient, for

ubiquitylation of target proteins. In addition, it appears that

proteins must contain a `degron'Ða motif which is recognized by

elements of the Ub system (reviewed in Varshavsky, 1991) for

ubiquitylation to occur. One such degron may be the nature of the

N-terminal residue of a protein (Hershko et al., 1984; Bachmair

et al., 1986).

Other degrons include the `destruction box' found in the

cyclins (Glotzer et al., 1991) whose concentration during progress

through the cell cycle is controlled, at least in part, by Ub-

mediated proteolysis. Indeed, Ub-mediated proteolysis plays a

very important role in control of the cell cycle (reviewed in

Yamao, 1999).

The proteasome

The 26S proteasome is a complex cytosolic and nuclear protease

that degrades ubiquitylated proteins in an ATP-dependent fashion

(Hough et al., 1987; Ganoth et al., 1988). The 26S proteasome

consists of a core particle, the 20S proteasome comprising four

stacked rings of seven subunits each, capped at one or both ends

by a 19S regulatory complex. The 19S regulatory complex

confers speci®city for ubiquitylated substrates on the proteasome,

while the 20S proteasome is involved in degradation of non-

ubiquitylated proteins (reviewed in Voges et al., 1999). The 26S

proteasome degrades proteins tagged with multi-Ub chains.

C-terminal hydrolases

Following proteolysis of the target protein, monomeric Ub is

released by the action of speci®c isopeptidases known as the C-

terminal hydrolases (Matsui et al., 1982; Wilkinson et al., 1989).

Different members of this class of enzymes generate monomeric

Ub from precursors, and may also be important in de-ubiquitylat-

ing proteins that have been mistakenly targeted, or in removing

single Ub molecules from stable substrates at times when

modi®cation is not required (Hershko and Ciechanover, 1998).

Functions of ubiquitylation

There is evidence that ubiquitylation is important in processes as

diverse as antigen presentation (reviewed in Goldberg and Rock,

1992) and apoptosis (reviewed in Drexler, 1998).

Protein degradation by the 26S proteasome

The Ub system is implicated in the degradation of a large and

growing number of regulatory proteins. These include p53

(Chowdary et al., 1994), phytochrome (Shanklin et al., 1987),

the yeast MAT-a2 repressor (Hochstrasser et al., 1991),

transcription factors and their regulators (Alkalay et al., 1995;

Figure 1. The ubiquitin (Ub) system. Ub is generated as a poly-Ub chain or asa fusion protein with a C-terminal extension (1) and is cleaved to formmonomeric Ub (2). Ub is activated by E1 enzymes in a process requiring ATPto form Ub-adenylate, and then transferred to a cysteine on E1 to form a thiolester (3), followed by transfer to one of many E2 enzymes (4). Some E2enzymes are capable of direct ubiquitylation of target proteins (5), whileothers transfer Ub to an E3 enzyme that acts as the proximal donor to theprotein target (6 and 7). Additional Ub-Ub isopeptide bonds may be formed tomake a multi-ubiquitin chain. Ubiquitylated proteins may later have the Ub tagremoved by isopeptidases (8), or they may be targeted to the 26S proteasomefor degradation (9). Monomeric Ub is regenerated by the action of furtherisopeptidases (10).

Biological and reproductive functions of ubiquitin

103

Stancovski et al., 1995; Traenckner and Baeuerle, 1995) and

cyclins (Glotzer et al., 1991). Multi-ubiquitylation of these

proteins targets them for degradation by the 26S proteasome.

These proteins are often short-lived, and control a wide range of

physiological processes including signal transduction. It is now

clear that the Ub-mediated degradation of intracellular proteins is

an extremely important factor in the regulation of cellular

function. Ub-mediated degradation may also be important in the

removal of mis-folded, denatured or mis-compartmentalized

proteins that may otherwise damage the cell (Seufert et al.,

1990; Rechsteiner, 1991; Figueiredo-Pereira et al., 1997),

including some endoplasmic reticulum-resident proteins that are

translocated back into the cytosol and degraded by the Ub/

proteasome pathway (reviewed in Plemper and Wolf, 1999).

Ubiquitin-mediated receptor down-regulation

Ub is involved in the degradation of several membrane-bound

proteins. Ligand-induced degradation of several receptors appears

to require ubiquitylation (Cenciarelli et al., 1992; Miyazawa et

al., 1994; Hicke and Riezman, 1996; Strous et al., 1996). Ligand-

induced mono-ubiquitylation of the cytoplasmic tail of cell-

surface receptors may serve as an internalization signal. The

receptor may subsequently be degraded, at least in part, in the

lysosome (reviewed in Strous and Govers, 1999) although there is

evidence for proteasomal degradation of the platelet-derived

growth factor (PDGF) receptor (Mori et al., 1995). Ubiquitylation

of cell-surface molecules with relevance to reproductive pro-

cesses, such as receptors for prolactin (PRL) (Cahoreau et al.,

1994), PDGF (Mori et al., 1993), tumour necrosis factor (TNF)

(Loetscher et al., 1990), growth hormone (GH) (Leung et al.,

1987; Spencer et al., 1988), ®broblast growth factor (FGF),

colony-stimulating factor-1 (CSF-1) (Mori et al., 1995) and

epidermal growth factor (EGF) (Yee et al., 1994; Galcheva-

Gargova et al., 1995) have also been reported. Ub-mediated

ligand-induced degradation has also been observed in some

soluble steroid hormone receptors, including the rat uterine

oestrogen receptor (ER; Nirmala and Thampan, 1995), the human

ER (Nawaz et al., 1999), the chicken oviduct progesterone

receptor (PR, SyvaÈlaÈ et al., 1998) and the human breast PR

(Lange et al., 2000).

Ubiquitin in disease

Given the role for Ub in removal of damaged proteins it is perhaps

not surprising that ubiquitylation has been implicated in a number

of pathological processes. Ub has been identi®ed within inclusion

bodies in a variety of chronic neurodegenerative disorders (for

example, see Lowe et al., 1988). The function of ubiquitylation in

these diseases is not known, but the wide-ranging appearance of

Ub within these bodies indicates an important role (reviewed in

Schwartz and Ciechanover, 1999).

Ubiquitin in development

The Ub-mediated degradation of proteins may play an important

role in tissue remodelling and development. Several developmen-

tally regulated genes of Drosophila melanogaster play a role in

ubiquitylation, including the fat facets gene which is involved in

eye development and encodes a de-ubiquitylating enzyme (Huang

et al., 1995), and the hyperplastic disk gene, encoding an E3

enzyme, which appears to play a major role in development and

reproductive viability (Mans®eld et al., 1994). The Ub system has

also been implicated in developmental processes in Dictyostelium

discoides (Clark et al., 1997; Lindsey et al., 1998),

Caenorhabditis elegans (Zhen et al., 1997), during chick

embryogenesis (Smith-Thomas et al., 1994), in human myogen-

esis (Bornman et al., 1996), the post-natal development of the rat

testis (Rajapurohitam et al., 1999) and of the human brain

(Kishino et al., 1997).

Developmental processes requiring wholesale destruction of

organelles are often accompanied by increased protein ubiquityla-

tion, for example the development of the lens (Scotting et al.,

1991; Cai et al., 1998; Yang et al., 1999) and the plant vascular

system (Bachmair et al., 1990).

Ubiquitin in reproduction

With the important role of the Ub system in a growing number of

examples of tissue remodelling and development and signal

transduction, it is perhaps unsurprising that there has been recent

interest concerning its potential role in reproduction.

Ubiquitin in spermatogenesis

The Ub system is implicated in different aspects of gametogenesis

(reviewed in Baarends et al., 1999). Post-natal development of the

rat testis requires a Ub-conjugating (E2) enzyme, UBC4

(Rajapurohitam et al., 1999). Sertoli cells express the Ub-C-

terminal hydrolase PGP9.5 (Kon et al., 1999) and germline

differentiation in mouse testis is accompanied by increased

expression of a multi-Ub chain binding protein (Pusch et al.,

Figure 2. Anti-Ub immunostaining in human uterine tissues. Photomicrographs of paraf®n sections through human uterine tissues probed with polyclonal antibodyto Ub, except panels (C) and (E), which were probed with antibody to Ub pre-incubated with Ub overnight at 4°C. Panel A: human endometrium obtained at theproliferative phase of the menstrual cycle. Panels B and C: serial sections of endometrial tissue obtained at the late secretory phase of the menstrual cycle, stainedwith antibody to Ub (panel B) or negative control (panel C). Panels D and E: sections of human ®rst-trimester decidua labelled with antibody to Ub (panel D) ornegative control as described previously (panel E). Panel F: section of human ®rst-trimester placental tissue probed with antibody to Ub. ctb, cytotrophoblast; dc,decidua compacta; ds, decidua spongiosa; g, endometrial gland lumen; hb, Hofbauer cell; s, endometrial stroma; stb, syncytiotrophoblast. Scale bars indicate 50 mmand bar in panel (B) applies to panels (C±F).

Figure 3. Anti-ubiquitin cross-reactive protein (UCRP) immunostaining in human uterine tissues. Photomicrographs of paraf®n sections through human uterinetissues. Panels (A±C) show the result of probing human non-pregnant endometrium (panel A), decidua (panel B) or placenta (panel C) with af®nity-puri®edantibody to human UCRP. Panels (D±F) show the result of hybridizing paraf®n sections with probe for human UCRP mRNA (panels D and E) and a negativecontrol; hybridization following RNase treatment of tissues (panel F). ctb, cytotrophoblast; dc, decidua compacta; ds, decidua spongiosa; g, endometrial glandlumen; s, endometrial stroma; stb, syncytiotrophoblast. Scale bars indicate 50 mm, and the bar in panel (A) also applies to panels (B) and (E±F). (Figures 3D±Fpreviously published in Bebington et al., 1999c).

C.Bebington, F.J.Doherty and S.D.Fleming

104

Figure 2.

Figure 3.


105

1998). Mammalian spermatogenesis requires the removal of

DNA-bound histones to allow chromosome packaging within the

sperm head, a process which may involve the Ub-system

(Lanneau and Loir, 1982; Agell and Mezquita, 1988; Baarends

et al., 1999). Certain E1 and E2 enzymes are clearly implicated in

spermatogenesis (Mitchell et al., 1991; Roest et al., 1996;

Grootegoed et al., 1998). Development of mature spermatozoa

involves the loss of sperm mitochondria. It appears that sperm

mitochondria are marked for degradation by ubiquitylation during

spermatogenesis, and are destroyed by the proteasomal machinery

of the fertilized egg (Sutovsky et al., 1999). Testis-speci®c

proteasomal subunits have been identi®ed in Drosophila (Belote

et al., 1998), and active proteasome has been isolated from human

spermatozoa (Tipler et al., 1997). Interestingly, one of the few

demonstrated locations of extracellular Ub in high concentration

is within human seminal ¯uid (Lippert et al., 1993), although the

function of the protein in this situation is not known.

Ubiquitin in oogenesis and follicle growth

Gametogenesis in female Drosophila requires a speci®c E2

enzyme, UbcD1 (Lilly et al., 2000). Polyubiquitin appears to be

produced in both human granulosa and porcine luteal cells

(Einspanier et al., 1987a,b), and extracellular Ub has been

detected in bovine follicular ¯uid (Einspanier et al., 1993), but the

possible function of Ub in these tissues is not known.

Interestingly, the use of prostaglandins to induce bovine luteolysis

leads to a rapid induction of Ub expression, and accumulation of

Ub is associated with apoptosis in marmoset corpus luteum

(Young et al., 1998), indicating that ubiquitylation may be

involved in this example of tissue remodelling (Murdoch et al.,

1996).

Ubiquitin in the menstrual cycle

High concentrations of Ub and Ub±protein conjugates have been

detected in endometrial glandular epithelium (Bebington et al.,

1999a, 2000b) during the menstrual cycle (Figure 2, panels A and

B). The protein appears in both cytoplasmic and nuclear locations,

and the intensity of nuclear anti-Ub staining varies throughout the

cycle, being most intense in the late secretory phase (Bebington et

al., 2000b; also see Figure 2, panel B). Similarly, concentrations

of total endometrial PR and relative concentrations of PR

isoforms vary during the menstrual cycle (Mangal et al., 1997).

The relative abundance of nuclear Ub and the PR in endometrial

glandular epithelium appears to be inversely correlated through

the menstrual cycle and in pregnancy (Bebington et al., 2000b),

and Ub is involved in down-regulation of the chicken (SyvaÈlaÈ et

al., 1998) and human breast PR (Lange et al., 2000). So far,

however, there is no direct evidence for the Ub-mediated

degradation of the human endometrial PR.

Ubiquitin in early pregnancy

Ruminant pregnancy

Recent work has focused on the possible functions of Ub in

ruminant reproduction. Ub has been detected within uterine

¯ushings from pregnant and non-pregnant cows (Austin et al.,

1996b). This is one of the few demonstrations of signi®cant

concentrations of extracellular Ub, though its function in this

situation is unknown.

Human pregnancy

Although endometrial stromal cells contain apparently low

concentrations of Ub in the non-pregnant uterus, its abundance

may increase during pregnancy, with decidualized stromal cells

containing high concentrations of both nuclear and cytoplasmic

Ub (Bebington et al., 1999a) (see Figure 2, panel D). The function

of this protein in these cells is not clear, but it is possible that Ub

is exported since human term decidual cells appear to secrete Ub

in vitro (Ren and Braunstein, 1995) and Ub has been detected in

bovine uterine ¯ushings (Austin et al., 1996b). Given the

involvement of ubiquitylation in many examples of tissue

remodelling and development, it is possible that this protein

modi®cation is involved in the development of the decidua from

the cycling endometrium.

Both monomeric and conjugated Ub protein have been detected

in human cytotrophoblast throughout gestation, but appear absent

from the syncytiotrophoblast layer (Bebington et al., 2000a). The

importance of ubiquitylation during placental development is

indicated by the multiple developmental abnormalities observed

in mice lacking the UbcM4 gene (encoding an E2 enzyme), such

as intrauterine growth retardation and perinatal death (Harbers et

al., 1996). This enzyme is homologous with the human gene

UbcH7, which also encodes a functional E2 enzyme (Nuber et al.,

1996). The only organ to show consistent pathological defects in

homozygous mutant animals lacking the UbCM4 gene was the

placenta, speci®cally the fetal mesenchyme in the labyrinth layer

and the placental vasculature (Harbers et al., 1996). A number of

homozygous mutant embryos died around day 11.5 of gestation, a

time when the placenta becomes vital for intrauterine survival.

This evidence suggests that activity of the Ub system is necessary

for normal placental growth and development.

Ubiquitin-like proteins

In addition to Ub itself, there are a number of related proteins that

in recent years have been the subject of much investigation. These

proteins are termed Ùb-like proteins' due to their structural

similarities to Ub, but they often possess quite distinct roles and

mechanisms of action. One group of Ub-like proteins apparently

contains a region of homology to Ub within a larger protein but do

not conjugate to target proteins. Others [e.g. small ubiquitin-like

modi®er (SUMO), neural precursor cell-expressed developmen-

tally downregulated (NEDD8), ubiquitin cross-reactive protein

(UCRP) and ubiquitin-like protein (Ubi-L)] retain the C-terminal

region of Ub and are capable of conjugation to target proteins

(reviewed in Tanaka et al., 1998). In this review, we shall discuss

only those Ub-like proteins known to be involved in reproductive

processes.

Ubiquitin cross-reactive protein

UCRP is so-called due to its immunoreactivity with antibodies

raised against Ub (Haas et al., 1987), and is also known as

interferon (IFN)-stimulated gene product 15 (ISG-15). ISG-15

mRNA and protein accumulate in Erlich ascites tumour cells

treated with exogenous IFN (Farrell et al., 1979). The protein has

been puri®ed, characterized (Korant et al., 1984; Blomstrom et


106

al., 1986), and found to be a 15 kDa peptide which is synthesized

as a 17 kDa precursor in response to IFN. Processing is necessary

to reveal the gly-gly at the C-terminus, which is required to enable

conjugation of the 15 kDa mature protein to target proteins

(Knight et al., 1988).

Up-regulation of UCRP

UCRP may be induced by treatment with either IFN-a or -b (type

I IFN). IFN-g was also seen to promote synthesis in some studies,

but over a longer time period, and to a lesser extent (Korant et al.,

1984; Haas and Bright, 1987; Knight and Cordova, 1991; Taylor

et al., 1996). The induction of UCRP within the host cell

coincides with generation of the anti-viral state, and occurs only

in cells sensitive to the action of speci®c IFN (Korant et al.,

1984). Non-IFN-based mechanisms (heat shock, exposure to

heavy metals, etc.), which are known to promote synthesis of Ub

(Bond and Schlesinger, 1985), are ineffective at UCRP induction

(Korant et al., 1984), but TNF-a may indirectly induce UCRP

through up-regulation of type I IFN (Ahrens et al., 1990).

Effects of UCRP

Although IFN-g was seen to have little or no effect on UCRP

production (Knight and Cordova, 1991), exogenous UCRP may

induce the secretion of IFN-g by T cells (Recht et al., 1991). Since

the two branches of the IFN family (types I and II) are

evolutionarily and structurally unrelated, any factor that links

the two is of particular interest. UCRP increases the production of

interleukin (IL)-2 from T cells and, in the presence of T cells, may

induce proliferation and activation of natural killer (NK) cells

(perhaps due to the UCRP-dependent synthesis of IFN-g by T

cells: D'Cunha et al., 1996a). Given the important role of uterine

NK cells at the initiation of pregnancy (Christiansen, 1996), this

may be a vital function of UCRP in the early decidua.

UCRP and ubiquitin

In 1987, Haas and co-workers discovered the structural similarity

between ISG-15 and Ub (Haas et al., 1987). Antibodies raised

against Ub were used to demonstrate the up-regulation of UCRP

in IFN-treated cells. UCRP is comprised of two domains, each

showing homology with Ub and predicted to retain the basic Ub

folding motif, while the mature protein possesses the C-terminal

gly-gly (GG) domain necessary for Ub activation and conjugation

to target proteins (Reich et al., 1987). Later studies (Loeb and

Haas, 1992) demonstrated that the structural similarity between

Ub and UCRP was mirrored, at least in part, by their mechanisms

of action. Conjugated UCRP is seen on immunoblots of control

cells and at a higher concentration in IFN-treated cells (Loeb and

Haas, 1992). The mechanism of conjugation is distinct from that

of the Ub pathway, at least in terms of the initial activation step

(Narasimhan et al., 1996). The function of UCRP conjugation is

not yet known, but it has been suggested that it may promote the

degradation of viral proteins within the host cell in a manner

similar to the Ub-mediated proteolysis of host cell proteins (Haas

et al., 1987).

Localization of UCRP

With the development of UCRP-speci®c antisera (Loeb and Haas,

1992), UCRP has been detected in lymphoid cells, striated and

smooth muscle, several epithelia and neurones in a punctuate

pattern suggestive of a vacuolar compartment (Lowe et al., 1995).

However, UCRP has been found associated with intermediate

®laments in cultured cells (Loeb and Haas, 1994). The co-

localization of UCRP with cytokeratin was not destroyed by

treatment with non-ionic detergents, suggesting adsorption of the

protein to this network (Loeb and Haas, 1994).

Secretion of UCRP

In contrast to Ub, which is rarely seen extracellularly, UCRP

appears to be secreted by a variety of cell types, including human

lymphocytes, monocytes and ®broblasts (Knight and Cordova,

1991; Taylor et al., 1996). Unconjugated UCRP is detectable in

the serum of human volunteers after the administration of IFN-aor IFN-b (D'Cunha et al., 1996b). The mechanism of UCRP

secretion has not yet been elucidated. The protein does not

contain a recognisable signal for secretion, and brefeldin-1 (an

inhibitor of the classical secretion pathway) does not affect its

secretion (D'Cunha et al., 1996b).

UCRP in pregnancy

Ruminant pregnancy: IFN-t is an anti-luteolytic factor, secreted

by the conceptus at the time of initiation of pregnancy in the cow

(Imakawa et al., 1987, Hansen et al., 1999), which up-regulates a

number of bovine uterine proteins (Godkin et al., 1984; Rueda et

al., 1993). A 16 kDa protein was found to be secreted from bovine

endometrial cells in vitro (Naivar et al., 1995). The connection

between this molecule and the 15 kDa UCRP protein was ®rst

suggested in 1996 (Austin et al., 1996a), and it was named

bUCRP to distinguish it from the similar, but not identical, human

UCRP (hUCRP).

bUCRP is produced by the bovine endometrium at times

coincident with maximal IFN-t production by the conceptus

(Austin et al., 1996a; Hansen et al., 1997). bUCRP mRNA and

protein (in both free and conjugated forms) are increased with

pregnancy or with in-vitro stimulation with IFN-t (Hansen et al.,

1997; Johnson et al., 1998). Recent work indicates that bUCRP

mRNA and protein are also up-regulated in the ovine uterus in

pregnancy (Johnson et al., 1999).

The sequence of bUCRP has been determined (Austin et al.,

1996a), and despite sequence homology between hUCRP and

bUCRP, several potentially important differences are apparent. In

the cow, UCRP is synthesized in a mature 17.3 kDa form,

requiring no C-terminal processing, and the protein contains three

cysteine residues (the human protein contains only one: Reich et

al., 1987), which may have profound structural or functional

implications. Like hUCRP, bUCRP also undergoes conjugation to

target proteins (Johnson et al., 1998, 1999).

The functional signi®cance of the up-regulation of bUCRP at

the initiation of pregnancy is not clear, but it is postulated that

bUCRP may coordinate IFN activity within the bovine uterus, or

it may be involved in maternal recognition of pregnancy.

Alternatively, given the immunoregulatory properties of UCRP,

bUCRP may act on the bone marrow-derived cell populations

within the implantation site and control invasion of the

trophoblast in some manner (Austin et al., 1996b).

Human pregnancy: The human decidua also contains high

concentrations of hUCRP mRNA (Bebington et al., 1999c) and


107

conjugated protein (Bebington et al., 1999a; see also Figure 3).

This protein appears to be localized to membrane-bound vesicles

within the decidualized stromal cell (Bebington et al., 1999a), but

it is not known whether it is a secretory product of these cells. As

in ruminant species, concentrations of hUCRP protein and mRNA

appear low during the menstrual cycle (Bebington et al., 1999a,c;

see also Figure 3). However, unlike the ruminant, there is no well-

de®ned signal demonstrating the initiation of pregnancy in the

human, and the mechanism by which uterine hUCRP is up-

regulated during gestation is not known.

Monoclonal non-speci®c suppressor factor

Monoclonal non-speci®c suppressor factor (MNSF) appears to be a

complex of proteins, including one which has signi®cant structural

similarity to Ub, known as Ubi-L, which is released from T cells

(Nakamura et al., 1995b). MNSF from the mouse contains Ubi-L

conjugated to the T-cell receptor alpha chain, and a putative

receptor for Ubi-L has been found in a murine T-cell helper cell

line (Nakamura and Tanigawa, 1999). MNSF, containing con-

jugated Ubi-L, inhibits lipopolysaccharide (LPS) -stimulated

TNFa release by macrophages (Suzuki et al., 1996), the IgE

response of LPS-activated B-cells (Nakamura et al., 1996) and IL-

4 secretion by T-helper cells (Nakamura et al., 1995a). Ubi-L is

apparently present within the human endometrium, particularly the

luminal epithelium, endothelial cells and the decidualized stromal

cell (Bebington et al., 1999b). Interestingly, MNSF mRNA was

among a small number of mRNAs that were differentially

regulated within murine implantation sites and adjacent uterine

regions (Nie et al., 2000). However, the function of this

immunosuppressive protein within the uterus is as yet unknown.

Summary

Ub and two Ub-like proteins, UCRP and MNSF, have been

studied within the human uterus. These, and the related protein

bUCRP, may play a vital function at the initiation of pregnancy in

the human and ruminant species respectively. The function of

these proteins is as yet unknown, but the use of knock-out animal

models and the observation of human cells in vitro may lead to a

greater understanding of these systems in the human situation.

Although the study of Ub and the Ub-like proteins within

reproductive processes is a new ®eld, the wide-ranging actions of

these proteins during the normal functioning of the cell, during

conditions of cell stress, cell division and apoptosis and during

tissue remodelling suggest that there may be several functions of

these proteins that are of reproductive importance.

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Received on April 7, 2000; accepted on September 7, 2000


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The possible biological and reproductive functions of ubiquitin