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Proteasome inhibitors are effective in mammalian eggs

In order to draw any conclusions from experiments using proteasome inhibitors, it was first necessary to prove that these inhibitors were active in mouse oocytes. It is well known that release from meiotic arrest is stimulated by destmction of cyclin B, the regulatory subunit of MPF. This is thought to be mediated by the proteasome (Glotzer et at., 1991) and therefore provided an ideal assay for the effectiveness of the proteasome inhibitors. As predicted, the proteasome inhibitors inhibited oocyte activation. The inhibitors had no inhibitory effect on the abihty of sperm to generate the Ca^^ transients responsible for stimulating cyclin B degradation and egg activation (see 5.3.3). The presence of normal oscillations indicates that the proteasome inhibitors prevented egg activation by specifically inhibiting the proteasome. Since these inhibitors were effective in mouse oocytes in blocking egg activation, I used them to investigate the role of the proteasome in InsP^R degradation.

The role o f the proteasom e in InsP^R degradation a t fertilisation

The results of my experiments with protease inhibitors have not conclusively identified any one protease responsible for InsP^R degradation. ALLN is the only inhibitor to have shown any significant inhibition of InsP^R downregulation (t-test: P<0.01), with the specific proteasome inhibitors, lactacystin and MG 132, having no significant effect. The inability of all three proteasome inhibitors to completely block downregulation means that it is not possible to conclude decisively from my experiments that it is the proteasome that causes the degradation of InsP^Rs at fertilisation. Although ALLN is known to inhibit both the proteasome and cysteine proteases (Groll et al., 1997; Mellgren, 1997; Rock et al,,

1994), Ca^^-dependent cysteine proteases such as calpain are unlikely to be responsible for InsPjR degradation since I have demonstrated that degradation is independent of Ca^^ increases (Chapter 4). Preliminary experiments have been carried out to examine the role of ubiquitin-dependent proteolysis by the proteasome, by trying to detect ubiquitination of InsPgRs at fertilisation. Experiments were performed (in collaboration with Richard Wojcddewicz, Department of Pharmacology, College of Medicine, SUNY Health Science Center at Syracuse, Syracuse, New York.) to try to immunoprecipitate InsP^Rs from fertilised mouse oocytes using an anti-ubiquitin antibody. However, no signal has been detected using protein from 500 mouse oocytes. The transient nature of ubiquitination means that many more oocytes may be required.

Inconclusive results using proteasome inhibitors necessitated the investigation of a role for caspases and lysosomal proteolysis in InsP^R degradation. Caspases are a family of cysteine proteases which are involved in programmed cell death (Cryns and Yuan, 1998). Lysosomal proteolysis (in addition to proteasomal degradation) is a major pathway of cellular protein degradation, particularly involved in the destruction of transmembrane proteins. It has been shown that chloroquine and NH4CI (inhibitors of lysosomal proteases) increase the basal levels of InsP^Rs in WB rat liver epithehal cells by 2-fold, which indicates that basal turnover of InsP^Rs occurs via lysosomal proteolysis (Bokkala and Joseph, 1997). However, the same study found that stimulated degradation of InsP^Rs (by angiotensin II) occurs via a non-lysosomal pathway, since it was not affected by these inhibitors. My results show that alternative pathways such as those involving caspases and lysosomal degradation of InsP^Rs, do not appear to be necessary for stimulated InsP^R downregulation at fertilisation.

The results obtained using proteasome inhibitors at fertilisation are inconsistent with those of studies on InsP^R degradation in cell lines. There is convincing data in somatic cells that InsPjR downregulation is carried out by the 26S proteasome. InsP^Rs undergo ubiquitination and downregulation is inhibited by the specific proteasome inhibitor lactacystin and by ALLN, which inhibits both cysteine proteases and the proteasome (Bokkala and Joseph, 1997; Oberdorf et aL, 1999). Furthermore, secretagogues such as CCK stimulate InsP^R ubiquitination and downregulation in rat pancreatic acinar cells, and proteasome inhibitors have been shown to cause the accumulation of ubiquitinated species in these cells (Wojcikiewicz et aL, 1999). This indicates that the ubiquitin-proteasome pathway is involved in InsP^R downregulation in a physiological signaling pathway.

The lack of a general effect of proteasome inhibitors is even more surprising given that the mechanism for stimulating downregulation appears to be similar in oocytes and somatic cells. Work on somatic cells has suggested a hypothesis in which InsP^R activation signals InsPgR degradation via the ubiquitin-proteasome pathway. Thus the proposal (in Chapter 4) that prolonged activation of InsP^Rs stimulates receptor degradation at fertihsation as well, is consistent with this degradation also being carried out by the ubiquitin-dependent proteasome. The most likely hypothesis for how InsP^R activation may signal recruitment of the proteasome at fertilisation is that a conformational change in the InsP^R induced by InsPj binding (Mignery and Sudhof, 1990) facihtates ubiquitination (Oberdorf et aL,

1999). This is supported by studies showing that mutant InsP^Rs that are unable to bind InsPj are not downregulated (Zhu et aL, 1999) or ubiquitinated (Zhu and Wojcikiewicz, 2000). It would also explain why InsP^Rs are downregulated in a specific manner, at least in cell lines (Bokkala and Joseph, 1997; Sipma et aL, 1998; Wojcikiewicz, 1995; Wojcikiewicz et aL, 1994; Wojcikiewicz and Oberdorf, 1996). It is not clear how a conformational change might lead to ubiquitination, as it is mostly unknown how ubiquitin- conjugating enzymes select their targets (Haas and Siepmann, 1997; Varshavsky, 1997). In

any case, it appears that if InsP^R activation is indeed responsible for stimulating degradation at fertilisation, a conformational change and recruitment of ubiquitin followed by proteasomal proteolysis is a likely sequence of events.

To summarise, InsP^R degradation (1) appears to be stimulated by InsP^R activation and executed by the proteasome in cell lines and mammalian tissues and (2) is stimulated at fertilisation of mouse oocytes by persistent InsP^R activation. Hence it seems reasonable that the proteasome is also involved in InsP^R degradation at fertilisation of mouse oocytes and that, for one or more of the reasons laid out below, the proteasome inhibitors employed in this study were not completely effective in blocking InsP^R degradation, despite successfully blocking egg activation.

(1) The proteasom e may only be partially inhibited

If I assume that the proteasome is solely responsible for InsP^R degradation at fertilisation, I need to explain how proteasome inhibitors could inhibit the proteasome sufficiently to prevent cyclin B degradation (and thus egg activation) but not InsP^R degradation. One possibility is that, in the presence of inhibitors, a low residual activity of the proteasome in oocytes may have different effects on the levels of proteins with different turnover rates. Cyclin B turns over at a high rate (t 1/2= 1.9 hours in the unactivated MU oocyte (Winston, 1997)) and there is a large maternal store of cyclin B in mouse oocytes (Kanatsu-Shinohara

et ah, 2000). As such, a low residual proteasome activity is unlikely to suppress cyclin B levels below the threshold that permits egg activation. In contrast, InsP^Rs are turning over at a much slower rate (tl/2 « ll hours in other cell types (Joseph, 1994)) and are therefore not rapidly replaced, after stimulated degradation, by protein synthesis or from maternal stores. It is possible then that low residual activity of the proteasome is sufficient to degrade and lower the level of InsP^Rs while not having any impact on the role of cyclin B in maintaining meiotic arrest.

It is not known whether the inhibitors completely or partially blocked the proteasome. In the case of lactacystin, evidence in the literature suggests that it is possible that inhibition was not complete. Lactacystin itself does not react with the proteasome but undergoes a spontaneous conversion in aqueous solutions to the active proteasome inhibitor, clasto-

lactacystin 6-lactone. However, the active compound can react with glutathione, a compound present at lOmM in mature oocytes (Perreault et aL, 1988), to form a second product which is inactive (Dick et aL, 1997). It may therefore be difficult to completely inhibit the proteasome with lactacystin. This may also be true of ALLN and MG 132 which act as competitive inhibitors of the proteasome. The concentrations used were effective at inhibiting InsP^R downregulation in other cell types (Bokkala and Joseph, 1997; Oberdorf

et aL, 1999; Wojcikiewicz and Oberdorf, 1996). However, the large cytoplasmic volume and low surface area/volume ratio of the mouse oocyte means that higher concentrations of inhibitors may be necessary to fully inhibit the proteasome and block InsP^R

downregulation, than in the smaller somatic cells with much higher surface area/volume ratios.

It should also be pointed out that, perhaps for the reasons speculated above, inhibitors of the proteasome have been shown to vary in their ability to block protein degradation in different cell types. For example, degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) in a mammalian cell-free system has been shown to be significantly inhibited by proteasome inhibitors hemin and MG 132, partially inhibited by c/fl^ro-lactacystin B-lactone and ALLN and relatively unaffected by lactacystin (Xiong et aL,

1999). Like the InsP^R, the CFTR is a transmembrane polytopic protein. Although residing in the plasma membrane, 80% of CFTR proteins are degraded while associated with the ER. Despite the variable effects of proteasome inhibitors, degradation of this protein requires an intact ubiquitin-conjugation pathway and is thought to involve the 26S proteasome (Jensen et a l, 1995; Ward et a l, 1995).

There is another case in the literature of BR-membrane protein degradation which, like InsPgRs at fertilisation, appears resistant to a range of inhibitors. The bioluminescent protein apoaequorin can be targeted to the ER in HeLa cells where it undergoes degradation in response to release of Ca^^ from the ER (the ER stress response) (Jeffery et aL, 2000). The study found that degradation could not be inhibited by the proteasomal inhibitor lactacystin, serine protease inhibitors or zinc, a known caspase-3 inhibitor. There is another potential similarity between the degradation of apoaequorin and InsP^Rs. Aequorin undergoes a conformational change in the absence of Ca^^ (Ray et aL, 1985) and it has been suggested that this may reveal a structural site sensitive to attack by proteases (Jeffery

et aL, 2000). As described in Chapter 4, the binding of InsP^ to its receptor induces a conformational change (Mignery and Sudhof, 1990) which may be sufficient to induce downregulation. The similarities between the aequorin case and degradation of InsP^Rs at fertilisation points to the mechanisms of degradation being similar. However, in contrast to the degradation of InsP^Rs in mouse eggs and cultured cells, loss of apoaequorin is stimulated by thapsigargin and ionomycin, which cause only a single, sustained release of ER Ca^^ (Jeffery et aL, 2000). Thus the mechanisms causing degradation of apoaequorin and InsPgRs are probably not identical.

(2) Degradation at fertilisation may involve m ultiple p r o te o ly tic m ech an ism s

Another explanation for why proteasome inhibitors fail to block InsP^R degradation is that multiple proteolytic pathways are involved. An interesting case in the hterature suggests that it may be possible for more than one proteolytic mechanism to function simultaneously and result in the downregulation of InsP^Rs. Secretagogues, which activate PLC-linked cell surface receptors (e.g. CCK), acting on rat pancreatic acinar cells, stimulate InsP^R ubiquitination and downregulation that is not inhibited by lactacystin or ALLN

(Wojcikiewicz et aL, 1999). Receptor ubiquitination and the finding that proteasome inhibitors cause the accumulation of ubiquitinated species (Wojcikiewicz et aL, 1999) indicate that the ubiquitin-proteasome proteolytic pathway contributes to the downregulation. However, to explain why proteasome inhibitors have no effect on downregulation, it has been suggested that an additional proteolytic mechanism functions in parallel in these cells. Indeed, such a system has previously been described in acinar cells. It may be that fertilisation stimulates more than one mechanism of proteolysis for the degradation of InsP^Rs, as is proposed for secretagogues. This may explain why the use of proteasome inhibitors alone appears to have a variable effect in blocking InsP^R degradation.

It is not unreasonable to make a comparison between the downregulation of InsP^Rs in pancreatic acinar cells and oocytes. Secretagogue-stimulated degradation {in vitro and in vivo) is as yet the only example, besides fertilisation, of InsP^R degradation as part of a physiological pathway. In acinar cells, InsP^R downregulation takes place as a way of regulating the cellular response to CCK exposure. In the case of fertilisation as well, downregulation may modulate the sperm-induced Ca^"^ oscillations, although it is probable that entry into interphase has a more dominant effect on oscillations (see General Discussion). Therefore, in both physiological instances of InsP^R degradation, it is conceivable that Ca^^ release is accompanied by the activation of more than one proteolytic pathway. For example, fertilisation may stimulate activation of both the ubiquitin- proteasome pathway as well as an additional, as yet unspecified protease. There are, however, differences between InsP^R degradation in these two cell types. Degradation is more rapid in acinar cells than in oocytes, with a half-life of 30 and 15 minutes in vitro and

in vivo, respectively (Wojcikiewicz et aL, 1999). Also, downregulation of some other signaling proteins (a PLC and a PKC) occurs simultaneously with InsP^R degradation in acinar cells (Wojcikiewicz et aL, 1999). Thus the rapid downregulation of both InsP^Rs and other proteins in acinar cells would appear to have a greater requirement for multiple simultaneous proteolytic mechanisms. In cultured cells, similar doses of ALLN and lactacystin completely blocked InsP^R downregulation (Bokkala and Joseph, 1997; Oberdorf et aL, 1999), implying that in these cultured cell types, non-physiological stimulation with agonists of PLC-linked cell surface receptors activates only the proteasomal pathway of degradation.

The idea of multiple proteolytic pathways being activated at fertilisation appears to be consistent with another study using mouse oocytes. Evidence was found for the role of the proteasome in InsP^R degradation after injection of sperm factor into mouse oocytes (Jellerette et aL, 2000). The proteasome inhibitor lactacystin was found to inhibit the degradation of type I InsP^R. However, there are differences between sperm factor- induced and fertilisation-induced degradation in the amount of InsP^ generated and in the extent to which InsP^Rs are downregulated. Sperm factor (SF) preparation was

microinjected into oocytes to produce an intracellular concentration of 2.5-5 sperm equivalents (Wu et aL, 1998) and this produced more rapid and extensive degradation than seen at fertilisation (Jellerette et aL, 2000). One explanation for why lactacystin appears to inhibit InsP^R degradation induced by SF but not by a single sperm at fertihsation may be related to the more rapid time-course of degradation induced by SF. This explanation also relies on the notion of simultaneous activation of more than one proteolytic pathway, as proposed above. At fertilisation, the rate of receptor degradation may be sufficiently slow for inhibition of proteasomal degradation to be compensated for by a second parallel pathway of degradation. However, after the more potent stimulus of SF, the putative second pathway of degradation may be unable to compensate for the proportion of degradation usually carried out by proteasomes, and this may bring about the observed delay in downregulation two hours after SF injection (Jellerette et aL, 2000). In the latter study, no data was presented on the final InsP^R levels, which may have been unaffected by lactacystin, if a second pathway eventually compensated for ah proteasomal InsP^R degradation. An altemahve explanation for how lactacystin inhibits SF-induced InsP^R degradation (Jellerette et aL, 2000) is that the high concentration of lactacystin which was used (lOOpM) may have had a non-specific effect on non-proteasomal degradation pathways, which may be operating in parallel to proteasomal degradation. A final consideration is that lOOpM, but not 50pM lactacystin (highest concentration used in my experiments) may be sufficiently high for some to remain after inactivation by glutathione (see above). In this case, the proteasome may only be inhibited and its role made apparent, at the higher concentration.

Proteasome distribution is consistent with a role in InsP^R degradation Clearly, further work is necessary to clarify the mechanism of InsP^R degradation at fertilisation. Ubiquitin-mediated degradation by the proteasome appears to be involved, whether alone or in conjunction with other degradation pathways. In addition to the evidence from other ceU types, described above, the subcellular distribution of the proteasome appears to be consistent with a role in InsP^R degradation. In mammals, 20S proteasomes are localised in the nucleus and in the cytoplasm with a percentage of the total proteasomes (<20%) loosely associated with the cytoplasmic surface of the ER (Palmer et aL, 1996; Rivett et aL, 1992), which is the location of InsP^Rs. In Chapter 4 it was described that rat oocytes arrested at M il exhibit a colocalisation of the proteasome with the M il spindle apparatus (Josefsberg et aL, 2000). However, proteasomes can diffuse rapidly throughout the cytoplasm in mammalian cells (Reits et aL, 1997) and could be conceived to redistribute from the spindle to the ER upon meiotic resumption at fertilisation. Further work is required to determine the distribution of the proteasomes after fertilisation.