N La Monica

O Marin

F Zacchello

M Scarpa

A Friso

R Tomanin

A Zanetti

C Mennuni

F Calvaruso

Gene therapy of Hunter syndrome: Evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS)

Keywords: Hunter syndrome, iduronate-2-sulfatase, electro-gene-transfer, hyaluronidase, muscle, gene therapy

Gene therapy of

Hunter syndrome: Evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS)

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INTRODUCTION

Mucopolysaccharidosis type II (MPS II), Hunter syndrome, [MIM 309900] is a rare, Xlinked lysosomal storage disorder (LSD) due to the deficiency of the enzyme iduronate-2-sulfatase (IDS, EC 3.1.6.13) involved in the lysosomal catabolism of heparan-and dermatan-sulfate. Two major forms of the disease, severe and attenuated, are now recognized as having different progression and life expectancy. The pathology is mainly characterized by the accumulation of undegraded glycosaminoglycans (GAG) in spleen, liver, heart, kidney, bone and joints, and in the most severe forms it also involves the central nervous system leading to progressive neurodegeneration.

Only palliative procedures and symptomatic therapies are currently available to treat this disorder. Haematopoietic stem cell transplantation which has been successful for some other forms of MPS [START_REF] Krivit | Bone marrow transplantation for storage diseases[END_REF][START_REF] Coppa | Bone marrow transplantation in Hunter sindrome[END_REF][START_REF] Sammarco | Effects of bone marrow transplantation on the cardiovascular abnormalities in canine mucopolysaccharidosis VII[END_REF], is ineffective in Hunter disease [START_REF] Peters | Hematopoietic cell transplantation for mucopolysaccharidosis IIB (Hunter syndrome)[END_REF]. After the recent conclusion of the phase III clinical trial [START_REF] Muenzer | A phase II/III clinical study of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II (Hunter syndrome)[END_REF], enzyme replacement therapy (ERT) has become the elective therapy, although it is expensive and, in addition, details of long term efficacy are not yet available.

Alternative therapeutic strategies have been evaluated including gene transfer, mainly using viral vectors [START_REF] Braun | Metabolic correction and cross-correction of mucopolysaccharidosi type II (Hunter syndrome) by retroviralmediated gene transfer and expression of human iduronate-2-sulfatase[END_REF][START_REF] Whitley | Retroviral-mediated transfer of the iduronate-2-sulfatase gene into lymphocytes for treatment of mild Hunter syndrome (Mucopolysaccharidosis type II)[END_REF][START_REF] Di Francesco | In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer[END_REF][START_REF] Daniele | Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro[END_REF][START_REF] Hong | Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome[END_REF][START_REF] Cardone | Correction of Hunter syndrome in the MPSII mouse model by AAV2/8-mediated gene delivery[END_REF], together with a somatic cell therapy approach [START_REF] Friso | Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts[END_REF].

Electro gene transfer (EGT) is a highly efficient method of delivering exogenous molecules into cells. It has been used to introduce DNA into various mouse and rat tissues [START_REF] Suzuki | Direct genbe transfer into rat liver cells by un vivo electroporation[END_REF][START_REF] Goto | Highly Efficient electr-gene therapy of solid tumor by using an expression plasmid for the herps simplex virus thymidine kinase gene[END_REF][START_REF] Maruyama | Epidermis-targeted gene transfer using in vivo electroporation[END_REF][START_REF] Mir | Electric pulse-mediated gene delivery to various animal tissues[END_REF][START_REF] Ho | Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis[END_REF][START_REF] Prud'homme | Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases[END_REF] and it has also been successfully proposed to improve transfection of skeletal muscle with plasmid DNA [START_REF] Mir | Electric pulse-mediated gene delivery to various animal tissues[END_REF][START_REF] Rizzuto | Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation[END_REF][START_REF] Rizzuto | Electro-gene-transfer results in a high level transduction of rat skeletal muscle and corrects anaemia of renal failure[END_REF][START_REF] Fattori | Electro-gene-transfer: a new approach for muscle gene delivery[END_REF][START_REF] Tomanin | Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome)[END_REF][START_REF] Cappelletti | Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA[END_REF][START_REF] Tan | Chemokine-like factor 1, a novel cytokine,contributes to airway damage, remodeling and pulmonary fibrosis[END_REF][START_REF] Fattori | Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates[END_REF]. In fact, although muscle represents a very accessible tissue, and therefore a good gene transfer target, many drawbacks in obtaining high transfection efficiencies had been previously described by using direct injection [START_REF] Wolff | Direct gene transfer into mouse muscle in vivo[END_REF][START_REF] Acsadi | Human dystrophin expression in mdx miceafter intramuscular injection of DNA constructs[END_REF]. Attempts to further improve EGT efficiency have included pre-treatment of the muscle with hyaluronidase (HYA) [START_REF] Mcmahon | Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase --increased expression with reduced muscle damage[END_REF][START_REF] Mennuni | Hyaluronidase increases electrogene transfer efficiency in skeletal muscle[END_REF][START_REF] Molnar | Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles[END_REF].

In this paper we present our results on muscle electro gene transfer applied to the MPS II mouse model [START_REF] Garcia | The characterization of a murine model of mucopolysaccharidosis II (Hunter syndrome)[END_REF] and to control mice. The aim was to evaluate the feasibility and efficiency of this procedure when applied to the production and release of a systemic therapeutic protein, iduronate-2sulfatase. The use of the Hunter disease-mouse model, characterized by null IDS activity in all the organ systems, allowed a clearer evaluation.

MATERIALS AND METHODS

Plasmid construction

All cloning procedures were performed following standard techniques [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. Human IDS cDNA (kindly provided by J Hopwood, Children, Youth and Women's Health Service, Adelaide,

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Australia) had been previously modified by Bielicki et al in 1993 [START_REF] Bielicki | Recombinant human iduronate-2-sulphatase: correction of mucopolysaccharidosis-type II fibroblasts and characterization of the purified enzyme[END_REF] by adding the 45 bp of the rat pre-proinsulin leader sequence. The cDNA had been cloned by us in pvIJnsB [START_REF] Montgomery | Heterologous and homologous protection against influenza A by DNA vaccination[END_REF] as previously described [START_REF] Tomanin | Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome)[END_REF]. The resulting plasmid, called pvIJIDS, carries the IDS cDNA sequence under CMV trascriptional control and bovine growth hormone polyA.

Mice

C57BL6 control mice were purchased from Charles River Italia while IDS-knockout (IDS-ko) mice were obtained from heterozygous founders kindly provided by J. Muenzer (Chapel Hill, USA).

The IDS-ko mouse model was created by replacing exon 4 and part of exon 5 of the IDS gene with the neomycin resistance gene [START_REF] Muenzer | Enzyme replacement therapy in mucopolysaccharidosis type II (Hunter syndrome): a preliminary report[END_REF]. As previously reported [START_REF] Friso | Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts[END_REF], knockout mice show no detectable plasma and tissue IDS activity.

In the first experiment a total of 9 IDS-ko and 12 healthy mice were treated with EGT after pretreatment with bovine hyaluronidase. All animals were injected with 50 µg of plasmid DNA and seven further healthy mice were injected with PBS only, as controls. Animals treated at 12-16 weeks of age were euthanized at 3 time-points 1, 2, and 5 weeks post-injection and blood and tissue samples were collected and analyzed.

In a second study, a total of 44 healthy mice were treated with the same procedure. Only half of the mice were pre-treated with hyaluronidase and animals were progressively euthanized 1, 2, 5 and 8 weeks later. Moreover, 18 mice not pre-treated with hyaluronidase were injected with 1 µg of plasmid DNA and monitored for 5 weeks post-treatment. Blood and tissue samples were collected and analyzed.

All animal experimentation was performed in accordance to National and International Animal Ethic Guidelines.

Electro Gene Transfer (EGT)

EGT was performed on anaesthesized mice by injecting 50 µg of plasmid DNA per animal diluted in 50 µl total saline into the quadricep muscles. The mouse muscle was surgically exposed, injected and then immediately electro-stimulated by inserting needle steel electrodes around the injection site. The electric field was applied in a pulsed form by using a Pulsar 6bp-a/s bipolar stimulator and pulses were monitored with a digital oscilloscope. Mice were electrically stimulated with ten trains of 1000 square bipolar pulses, maintaining current control at 75 mA, delivered every other second, for a total duration of 20,1 seconds. The frequency was 1000 Hz. Type VI-S hyaluronidase from bovine testis (36 units per animal), resuspended in 50 µl sterile saline, was injected into the muscle

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IDS enzyme assay

IDS activity was evaluated by a fluorimetric assay [START_REF] Voznyi | A fluorogenic assay for the diagnosis of Hunter disease (MPS II)[END_REF] mM EDTA pH 8.0, 0.1% Triton X-100) and, following centrifugation, the supernatants were recovered. All samples were evaluated for protein content by using the BIORAD protocol. Plasma samples were obtained from heparinized blood, microcentrifuged at 3000 rpm for 20 minutes.

Anti-iduronate-2-sulfatase antibodies determination

Anti-iduronate-2-sulfatase antibodies were measured by using an immunoprecipitation assay [START_REF] Friso | Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts[END_REF] Negative control was obtained by incubation of an equal amount of plasma from ko untreated mice.

For the positive control 80 nmol/4h of IDS were incubated with 9 µg total of a goat anti-human IDS polyclonal antibody (kindly provided by Shire-TKT, Cambridge, USA) in order to completely abolish the enzyme activity.

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Tissue glycosaminoglycan content

Tissue GAG content (expressed as µg of GAG/mg of protein) was measured as previously suggested [START_REF] Bjornsson | Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue[END_REF]. Before performing the assay, tissues were lyophilised, resuspended and homogenized in 0.9% NaCl + 0.2% Triton X-100. Samples were then shaken overnight at 4°C and microcentrifuged for 5' at 3000 rpm. Supernatants were collected and kept at -80°C until analysis.

50 µl of blank (water), calibrators (chondroitin sulfate C, Sigma-Aldrich, Milano, Italy) and samples in duplicate were incubated with 50 µl of 8 M Guanidine-hydrocloride for 15 minutes at room temperature, with 50 µl of SAT solution (0.3% sulforic acid, 0.75% Triton X-100) for 15 minutes and with a solution of Alcyan blue 0.038% overnight. Afterwards, samples were centrifuged and rinsed with a DMSO washing solution (40% DMSO, 0,05% Magnesium chloride).

After centrifugation, pellets were resuspended in Gu-Prop (4M Guanidine-hydrocloride, 33%

Isopropanol, 0.25% Titon X-100) and absorbance was measured at 620 nm.

Statistical analysis

Data represent the average of different samples, the number of which is indicated under each figure.

Bars represent SEM (mean standard error). Statistical comparisons of experimental groups were performed using a Student's t-test. p values less than 0,05 were considered statistically significant.

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RESULTS

Experimental plan. We first evaluated the EGT procedure for the release of iduronate-2-sulfatase in hyaluronidase pre-treated IDS-ko and control mice. In a second experiment, performed in healthy control mice, we compared the efficacy of the procedure in animals pre-treated and not pre-treated with hyaluronidase.

EGT in hyaluronidase pre-treated IDS-ko mice

IDS activity induced in quadricep muscles

Our first evaluation was performed in a total of 9 IDS-ko and 12 healthy mice treated with EGT (after injection of 50 µg of the plasmid DNA pvIJIDS per animal) and pre-treated with bovine HYA. Nine further healthy animals underwent EGT following injection of PBS only, and were used as controls. Mice were euthanized at 3 time-points: 1, 2 and 5 weeks post-injection. Quadriceps were removed, homogenized and evaluated for IDS activity by performing a fluorometric assay, and the results are shown in Figure 1. The basal enzyme level reported (9.8 ± 1.9 nmol/4h/mg) was calculated in quadricep muscles from PBS injected healthy animals (indicated as UT wt), while no basal IDS activity was measured in the IDS-ko mice (data not shown). The difference between electro-injected muscles and the basal level was assumed to represent enzyme activity due to the recombinant protein. In all groups, a significantly increased activity (p<0.0001) of at least six fold (63.8 ± 10.8 nmol/4h/mg) with respect to the basal value (9.8 ± 1.9 nmol/4h/mg) was detected, reaching a level 24-fold higher (234.9 ± 46.5 nmol/4h/mg) than baseline in healthy mice euthanized 5 weeks post-treatment.

IDS activity measured in plasma

In the same animals we evaluated plasma IDS activity (Figure 2). Since our previous experience

showed that IDS plasma level in adult mice mantains constant with time (laboratory data, not shown), we took as comparing level for the present experiment the mean activity detected before treatment (basal level, time 0, 74.8 ± 14.8 nmol/4h/ml). A higher IDS activity with respect to the control value was detected in the healthy mice 2 and 5 weeks post-treatment, with a maximum increment of about 30 units (+34%) (100.4 ± 32.1 nmol/4h/ml, p<0.05) at 5 weeks; in contrast no induced activity was measured in the IDS-ko animals (not shown in the figure).

Anti-IDS immune response

The amount of specific anti-IDS antibodies raised by the treatment was evaluated in the animal's

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plasma. The analysis was performed in both ko and wt mice at different time-points, by using an immunoprecipitation assay. Starting from a known IDS activity, we evaluated the residual activity following immunoprecipitation with the plasma of the treated animals. Results, given as percentage (%) of IDS activity neutralized by the plasma antibodies, are reported in Figure 3 and show an increasing amount of neutralizing antibodies from 1 to 5 weeks post-treatment. In particular, a considerably higher immune response was detected in IDS-ko mice with respect to controls at 2 weeks (53% vs 11% of IDS immunoprecipitated, p<0.001).

Tissue IDS activity and GAG content

IDS activity was also measured in liver, spleen, kidney, lung, heart and controlateral quadricep muscle of the treated IDS-ko mice. Enzyme activity detected was around 0 nmol/4h/mg of protein (data not shown). In the same organs glycosaminoglycan content measured 5 weeks post-treatment did not decrease with respect to untreated tissues (data not shown).

EGT in hyaluronidase pre-treated and not pre-treated control mice

IDS activity induced in quadricep muscles

In a second experiment forty-four wild type animals were treated with the same procedure, half of which were pre-treated with HYA while half of them were not. Animals were progressively euthanized 1, 2, 5 and 8 weeks later. Figure 4 shows the enzyme activity detected in the mice quadriceps obtained post-euthanasia. As in the previous experiment, the difference between electroinjected muscles and the basal level was assumed to represent enzyme activity due to the recombinant protein. In most animals enzyme activity appeared significantly higher than baseline (9.8 ± 1.9 nmol/4h/mg, indicated as UT in the figure). Moreover, it was significantly higher in HYA pre-treated versus not pre-treated animals (p<0.0001), reaching a 20 fold increase (187.5 ± 32.4 nmol/4h/mg), with respect to basal level at 8 weeks.

IDS activity measured in plasma

Figure 5 reports plasma IDS levels measured in the EGT-treated animals. Mice were monitored weekly by retro-orbital bleeding. T0 represents pre-treatment values. A sudden increase of the activity was measured 1 week post-injection in both groups of samples (around +40%, p<0.0001).

In the following time-points, up to 5 weeks, variable levels of activity were detected, some of which significantly increased compared to baseline, as indicated in the figure. The activities measured were quite similar between the two groups of animals. At 6, 7 and 8 weeks post-treatment samples collected from mice which were not treated with HYA showed a higher level of enzyme activity

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with respect to the group of mice pre-treated (+39%, +24.5%, +29% respectively). In particular, such increase was statistically significant 6 and 7 weeks post-treatment (p<0.01).

Anti-IDS immune response

The appearance of specific anti-IDS antibodies was monitored weekly in the plasma of both animal groups (Figure 6). Neutralizing antibodies were measurable starting 2 weeks post-treatment and progressively increasing for the entire follow-up. The amount of antibodies detected in the samples derived from mice who underwent HYA pre-treatment was higher than that detected in the plasma obtained from animals who were not pre-treated with HYA, in the first group up to 50% of the IDS activity was immunoprecipitated by the antibodies. There was up to a four fold difference between the two groups at weeks 5 and 8 post-injection. Statistical analysis showed significant differences between the 3 rd and 7 th week of treatment (p<0.005 at weeks 3 and 4, p<0.0001 at weeks 5, 6, 7).

Finally, a further decrease of the immune response was observed in a subsequent experiment, in which we injected 18 animals with 1 µg of DNA. Results showed, 5 weeks post-treatment, a reduction from 10 to 5% of the IDS immunoprecipitated, compared to injection of 50 µg of plasmid DNA without HYA pre-treatment. However, in this experiment we did not register a significant increase of plasma IDS activity with respect to basal level.

IDS activity measured in tissues

IDS activity was also measured in liver, spleen and controlateral quadricep muscles of the treated healthy mice. Data shown in Table 1 present the comparison between the two groups of animals euthanized at 5 and 8 weeks post-injection. An increased enzyme activity was seen in the spleen of mice not pre-treated with hyaluronidase at 5 and 8 weeks post-treatment, in the spleen of HYA pretreated animals at 5 weeks and in the liver of not-pre-treated mice at the same time. None of the increased values were statistically significant with respect to basal level.

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DISCUSSION

EGT is a common procedure for efficient targeted transduction of genes of interest. It has been applied to numerous tissues and organs in several animal models [START_REF] Suzuki | Direct genbe transfer into rat liver cells by un vivo electroporation[END_REF][START_REF] Goto | Highly Efficient electr-gene therapy of solid tumor by using an expression plasmid for the herps simplex virus thymidine kinase gene[END_REF][START_REF] Maruyama | Epidermis-targeted gene transfer using in vivo electroporation[END_REF][START_REF] Mir | Electric pulse-mediated gene delivery to various animal tissues[END_REF][START_REF] Ho | Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis[END_REF][START_REF] Prud'homme | Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases[END_REF] and, in particular, it has been proposed to improve transfection of skeletal muscle with plasmid DNA [START_REF] Mir | Electric pulse-mediated gene delivery to various animal tissues[END_REF][START_REF] Rizzuto | Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation[END_REF][START_REF] Rizzuto | Electro-gene-transfer results in a high level transduction of rat skeletal muscle and corrects anaemia of renal failure[END_REF][START_REF] Fattori | Electro-gene-transfer: a new approach for muscle gene delivery[END_REF][START_REF] Tomanin | Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome)[END_REF][START_REF] Cappelletti | Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA[END_REF][START_REF] Tan | Chemokine-like factor 1, a novel cytokine,contributes to airway damage, remodeling and pulmonary fibrosis[END_REF][START_REF] Fattori | Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates[END_REF]. EGT has significantly improved muscle gene transduction, allowing a several fold increase in the amount of recombinant protein produced. Moreover, the procedure has been more recently optimized by pretreatment of the muscle with hyaluronidase [START_REF] Mcmahon | Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase --increased expression with reduced muscle damage[END_REF][START_REF] Mennuni | Hyaluronidase increases electrogene transfer efficiency in skeletal muscle[END_REF], which has further increased its performance.

We here evaluated the feasibility of utilizing muscle EGT for the efficient production and release of iduronate-2-sulfatase in the IDS-ko mouse model. IDS is a lysosomal enzyme involved in the catabolism of the glycosaminoglycans heparan and dermatan-sulfate. A deficiency in this enzyme activity is responsible for a genetic disease called Hunter syndrome or mucopolysaccharidosis type II (MPS II). The intravenous infusion of the recombinant enzyme has shown to improve the pathological phenotype; however, it remains extremely expensive (around 300.000

Euros/patient/year) and the search for new therapies is thus of primary importance.

It has recently been suggested that very small amounts of a lysosomal protein (3-5% of normal)

would be sufficient for the effective treatment of a specific LSD [START_REF] Muenzer | Advances in the treatment of mucopolysaccharidosis type I[END_REF][START_REF] Desnick | Enzyme replacement and enhancement therapies for lysosomal diseases[END_REF]. These data strongly encourages a gene therapy approach, albeit the amount of recombinant protein produced, released and taken up by the different tissues is very difficult to control.

Muscle EGT has never been assayed so far in animal models as a potential tool for gene therapy of lysosomal disorders. Previous works proposing muscle transduction for the treatment of these pathologies, had been performed using viral vectors, either adenovirus or adeno-associated virus, obtaining contradictory results. Enzyme release, uptake by the peripheral organs and a therapeutic effect were observed for Pompe [START_REF] Martin-Touaux | Muscle as a putative producer of acid alpha-glucosidase for lycogenosis type II gene therapy[END_REF] and Fabry diseases [START_REF] Takahashi | Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer[END_REF], while no release from the muscle was described for MPS VII [START_REF] Daly | Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged bglucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice[END_REF] and MPS VI [START_REF] Tessitore | Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle[END_REF].

We here reported the experiments performed in the IDS-ko and wild type animal muscles, both pretreated with hyaluronidase, to evaluate the efficacy of the procedure for IDS muscle production and its release into the plasma. The results showed an elevated production of the protein inside the muscle, but a limited release into the bloodstream and a strong anti-IDS immune response, which likely cleared the plasmatic fraction of the recombinant protein. To elucidate this sudden and strong immune reaction we performed an additional experiment on healthy control mice, half of which were pre-treated with hyaluronidase and half were not.

In the first experiment healthy and IDS-ko mice showed a very high induced enzyme activity in the muscle, up to 24 fold and 14 fold higher respectively than baseline wild type level. Since the

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pathology presents a systemic involvement, to obtain therapeutic efficacy the recombinant protein needs to escape the muscle, enter the blood stream and reach the organs. Our experiments suggest that a small part of the large amount of IDS produced was released by the quadriceps since an increased IDS activity with respect to baseline level was detected in the plasma of the control animals. However, such activity showed values hardly detectable in the knock out mice plasma, where conversely an anticipated and stronger immune response with respect to the healthy animals was detected. This immune reaction started 2 weeks post-treatment and reached levels 5 fold higher than that measured in the control mice. In the latter an important immune response was detected at 5

weeks post-treatment. As a consequence, no induced activity was found in the organs analyzed from ko mice: liver, spleen, kidney, lung, heart and contralateral muscle.

Could this immune reaction be due to the procedure in itself? It is, in fact, well known that muscle per se is an important route of immunization: might hyaluronidase represent an adjuvant in such an immune reaction?

To address these questions we performed a second set of injections in healthy control mice, either pre-treated or not pre-treated with hyaluronidase. For this analysis we treated healthy animals because the reduced and slower anti-IDS antibodies production expected in these animals versus IDS-ko mice would have allowed a clearer evaluation of the immunogenicity of the procedure per se.

Comparison of the results obtained in the control animals pre-treated with hyaluronidase in the two experiments performed, showed a very good reproducibility of the procedure although carried on at two different times in two different batches of mice.

As previous papers had already shown [START_REF] Mcmahon | Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase --increased expression with reduced muscle damage[END_REF][START_REF] Mennuni | Hyaluronidase increases electrogene transfer efficiency in skeletal muscle[END_REF], hyaluronidase pre-treatment of the muscle significantly improves the outcome of EGT, with notable increases in the amount of protein produced inside the muscle. In our experiment, we observed an IDS level enhanced up to six fold in HYA pre-treated muscle versus not pre-treated. However, the present data suggest that the amount of recombinant protein released in the blood stream represents only a small amount of the IDS produced by the treated muscle, and we did not detect any significant differences between hyaluronidase pre-treated and not pre-treated animals up to 5 weeks post-injection. Starting at 6

weeks the IDS activity measured in the plasma derived from HYA-treated animals went back to baseline level, whilst the activity measured in not pre-treated mice remained at significantly higher levels with respect to baseline, until the end of the follow-up. This was shown to be related to the immune response against IDS: in pre-treated animals we detected a quicker and stronger immune reaction, which, starting at 5 weeks post-treatment, consistently maintained about four fold higher than that measured in the animals not pre-treated.

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Previous experience by using recombinant IDS in preclinical experiments [START_REF] Friso | Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts[END_REF][START_REF] Garcia | Preclinical dose ranging studies for enzyme replacement therapy with idursulfase in a knock-out mouse model of MPS II[END_REF] did not support for the protein an important immunogenic role in the short period. Therefore, present results suggest that most of the immune response here observed might have been caused by the administration route in itself, and that hyaluronidase might act as an adjuvant. In any case, analysis of the organs (liver, spleen, contralateral muscle) obtained from the euthanized animals at different time-points post-treatment, did not detect significant increases of IDS activity with respect to baseline levels,

nor showed any differences between the two groups of animals.

Our data suggest that the muscle is a closed system in which only small amounts of the recombinant IDS produced can escape and reach the blood stream, and this seems to be independent from the amount of protein produced. This would seem to be different from what happens to proteins specifically directed or targeted to the blood stream, such as erythropoietin, interferon, interleukins and VEGF [START_REF] Fattori | Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates[END_REF][START_REF] Aurisicchio | Tamarin alpha-interferon is active in mouse liver upon intramuscular gene delivery[END_REF][START_REF] Jeong | Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis[END_REF][START_REF] Baumgartner | Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia[END_REF] for which a good release had been described.

As for lysosomal enzymes in general, the results published so far seem to be quite disappointing and contradictory. The transgene was always delivered to the muscle by a viral vector, and only few authors demonstrate a release of the enzyme into the blood stream [START_REF] Desnick | Enzyme replacement and enhancement therapies for lysosomal diseases[END_REF][START_REF] Takahashi | Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer[END_REF]. Other authors rather suggest that the enzyme activity observed outside of the target muscle might be due to a spill over of the virus at the time of injection [START_REF] Daly | Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged bglucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice[END_REF][START_REF] Tessitore | Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle[END_REF].

In conclusion, we here confirmed that muscle electro gene transfer can very efficiently produce recombinant proteins inside the target muscle, in this case the iduronate-2-sulfatase; hyaluronidase pre-treatment significantly improved the outcome of the procedure.

However, we could not detect a high release of the protein from the producing muscle, either HYA pre-treated or not. Instead we observed an important anti-IDS immune reaction, which was increased by HYA pre-treatment.

Although EGT might be proposed as a tool to efficiently produce proteins used by the muscle, such as dystrophin for the treatment of muscular dystrophy, or for the efficient release of proteins specifically targeted to the circulation, such as erythropoietin or factor VIII or IX for the treatment of blood disorders, our study shows that EGT performed in the described conditions cannot successfully determine an efficient release of iduronate-2-sulfatase from the injected muscle, necessary for the treatment of Hunter disease.

The application of this procedure to pathologies caused by housekeeping genes, which involve most of the organ systems, whose treatment thus requires a body-wide distribution of the protein is an area in need of improvement,which might take advantage by technical modifications such as the use of external plate electrodes and/or the high voltage-low voltage combination, providing a more homogenous distribution of the electric field and a reduction of the tissue damage. a Tissue IDS activity in not treated healthy mice.

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b Tissue IDS activity in treated healthy mice.

: 5 µl

 5 of mouse plasma was diluted in 40 mM Tris-HCl, pH 8.0 to a final volume of 60 µl, mixed with 80 nmol/4h of iduronate-2-sulfatase collected from over-expressing C2C12 cell medium and incubated at 4° C overnight. The day after, samples were incubated with 20 µl of Pansorbin protein A (200 mg/ml) (Calbiochem, INALCO, Milano, Italy) and centrifuged to pellet iduronate-2sulfatase bound to Pansorbin in the presence of anti-iduronate-2-sulfatase antibodies. The unbound enzyme in the supernatant was measured with the fluorometric assay. Antibody titre was expressed as percentage of IDS immunoprecipitated by the antibodies, with respect to the initial IDS content.

5 w and 8

 8 Fig 1

Table 1

 1 IDS activity detected in mice tissues: comparison between hyaluronidase pre-treated and not pre-treated animals.

	Basal level
	a
				5 w b	8 w b
	Liver	16,5±0,6	-HYA	22,7±4,9	17,6±2,2
			+ HYA	17,9±1,3	16,9±0,8
	Spleen	21,1±1,7	-HYA	25,3±1,8	25,5±1,9
			+ HYA	26,8±3,3	21,8±1,5
	Cl Muscle	9,8±1,9	-HYA	10,8±3,7	7,3±2,7
			+ HYA	8,4±1,3	8,8±1,2

ACKNOWLEDGEMENTS

This work was partly supported by the Italian MPS Society.

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Mariola J Edelmann

Benedikt M Kessler

Ubiquitin and Ubiquitin-Like Specific Proteases Targeted by Infectious Pathogens: Emerging Patterns and Molecular Principles

Keywords: bacteria, hydrolase, ISG15, Nedd8, protease, SUMO, ubiquitin, viruses ovarian tumor domain, UBP: ubiquitin processing protease

Attachment of ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers is a reversible posttranslational modification that regulates the fate and function of proteins. In particular, proteolytic enzymes with Ub/Ubl processing activity appear to be more widespread than originally anticipated. It is therefore not surprising that bacterial and viral pathogens have exploited many ways to interfere with Ub/Ubl conjugation, but also de-conjugation. On one hand, pathogens were shown to manipulate host encoded enzymes. On the other hand, pathogen derived sequences of proteases specific for Ub/Ubls are emerging as a common feature shared by many viruses, bacteria and protozoa, and we are at an early stage of understanding how these proteases contribute to the pathogenesis of infection. Whereas some of these proteases share a common origin with mammalian cell encoded hydrolases with specific properties towards Ub/Ubls, most of them have ancient intrinsic functions, such as processing pathogen protein components, and may have acquired the specificity for Ub/Ubls by interacting with mammalian hosts and their immune system throughout evolution. Since many of these proteases are clearly distinct from their mammalian counterparts, they represent attractive targets for drug design against infectious diseases.

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Introduction

In the course of evolution viral and bacterial pathogens have acquired a series of molecular strategies to promote infection, proliferation and survival during host-pathogen interactions. This aspect has been of considerable interest over many years, since much can be learned about cell biological processes in general by observing how pathogens are capable of manipulating them to their advantage. In addition, a greater understanding of the underlying molecular mechanisms of these processes will continue to provide entry points for drug targets and therapeutic treatment strategies, for which there is a great demand, since many infectious diseases are still not curable.

Pathogens are able to corrupt many of the cell biochemical pathways of the host, one of which is the ubiquitin system that has attracted significant attention in recent years. A reason for this is the fact that to date, over a thousand proteins have been associated with this pathway (reviewed in [START_REF] Kerscher | Modification of proteins by ubiquitin and ubiquitin-like proteins[END_REF], [START_REF] Welchman | Ubiquitin and ubiquitin-like proteins as multifunctional signals[END_REF]). Modification by ubiquitin and ubiquitin like proteins is now recognized to be essential for the fate and function of the majority of proteins and can regulate biological processes including protein turnover, receptor internalisation, protein targeting to different cellular compartments, regulation of cell function and cell signalling. There are more than 500 proteins that are known to recognize ubiquitin and attach it to specific substrates (referred to as E2 and E3s), and over 90 deubiquitylating enzymes that can reverse this reaction [START_REF] Nijman | A genomic and functional inventory of deubiquitinating enzymes[END_REF]. This extraordinary diversity reflects the fact that ubiquitin conjugation and deconjugation is a very general regulatory process, which requires to be tightly controlled. Furthermore, an additional complexity of this pathway is based on the existence of many ubiquitin-like molecules (Ubls), such the as small ubiquitin-like modifiers (SUMO1/2/3), neuronal precursor cell expressed, developmentally down-regulated 8 (NEDD8), ubiquitin cross-reactive protein (UCRP, also known as interferon stimulated gene ISG15), ubiquitin-like protein-5 (UBL-5, also known as homologous to ubiquitin-1, Hub1), Ubiquitin D (also known as F-adjacent transcript 10, FAT10/UBD), Fau ubiquitin-like protein (Fau/Fub1), ubiquitin-related modifier 1 (URM1), and Atg8 (GABARAP / GATE16 / MAP-LC3) (reviewed in [START_REF] Herrmann | Ubiquitin and ubiquitin-like proteins in protein regulation[END_REF], [START_REF] Kerscher | Modification of proteins by ubiquitin and ubiquitin-like proteins[END_REF]). Targeting Ub/Ubl conjugation via the E1, E2 and E3 enzyme cascade by viral and bacterial pathogens has been a well recognized theme and the subject of recent reviews [START_REF] Chen | Viral hijacking of cellular ubiquitination pathways as an anti-innate immunity strategy[END_REF], [START_REF] Munro | Bacteria and the ubiquitin pathway[END_REF], [START_REF] Rytkonen | Bacterial interference of ubiquitination and deubiquitination[END_REF]. However, removal of ubiquitin or Ubls from substrates by deconjugating enzymes appears to be another biochemical step that viruses and bacteria can interfere with [START_REF] Lindner | Deubiquitination in virus infection[END_REF], [START_REF] Rytkonen | Bacterial interference of ubiquitination and deubiquitination[END_REF]. This is accomplished by proteolytic enzymes, predominantly by cysteine proteases and a small number of metalloproteases. It has become evident that a vast number of proteases have acquired the ability to process Ub/Ubls, probably far beyond the currently known classifications, as novel enzymes are being discovered as well as new predictions made by recent bioinformatics studies [START_REF] Hotson | Cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity[END_REF], [START_REF] Iyer | Novel predicted peptidases with a potential role in the ubiquitin signaling pathway[END_REF], [START_REF] Makarova | A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae[END_REF]. We shall therefore refer to the classification of proteases into clans and families as proposed by the MEROPS database (http://merops.sanger.ac.uk/index.htm) for a more detailed discussion of these enzymes.

In the case of ubiquitin, five classes of enzyme families have been defined to date, including ubiquitin C-terminal hydrolases (UCH -C12 family), ubiquitin processing proteases (UBP, also referred to as ubiquitin specific proteases, USP -C19 family), Josephin domain containin proteases (JD), ovarian tumor domain containing proteases (OTU -C64, C85, C87, C88 families) and metalloproteases (MM) (Figure 1) [START_REF] Amerik | Mechanism and function of deubiquitinating enzymes[END_REF], [3] [13]. The UCH class contains only three members that are small proteins and that were originally identified to process Ub precursors and hydrolyse small chemical groups at the C-terminus of ubiquitin. UBPs (USPs) form the largest enzyme family, and all members share the conserved cysteine and histidine boxes as short motives, but otherwise can be very diverse. Another family of proteases sharing the ovarian tumor domain (OTU) as a common feature is conserved throughout evolution, and is found in ca 80 genes derived from viruses, bacteria, plants and mammals, suggesting their involvement in fundamental biological processes. Deubiquitylating activity was also assigned to a small number of Josephin-domain and JAB metalloprotease domain containing proteases. It is anticipated that many more enzymes may be added to this list, since bioinformatics based prediction algorithms suggest larger families of cysteine proteases including a Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes (PPPDE) and Wss1p-like metalloproteases (WLM) domains to be potential DUB candidates [START_REF] Iyer | Novel predicted peptidases with a potential role in the ubiquitin signaling pathway[END_REF]. Proteolytic enzymes with specificities towards Ubls are classified as ubiquitin-like specific proteases (ULPs) and small ubiquitin-like modifier specific proteases (SENPs), both of which belong to the cysteine protease superfamily (reviewed in [13] [14] ). In addition, experimental evidence using activity-based profiling strategies based on Ub/Ubl scaffolds and tandem mass spectrometry retrieved a number of novel gene products (including pathogen derived ones) with Ub/Ubl-specific protease activities, which previously escaped to be predicted by any sequence or structure base alignment algorithms (reviewed in [START_REF] Kessler | Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes[END_REF], [START_REF] Love | Mechanisms, biology and inhibitors of deubiquitinating enzymes[END_REF], [START_REF] Ovaa | Active-site directed probes to report enzymatic action in the ubiquitin proteasome system[END_REF]). Such probes mainly detect the ability of proteins to interact with Ub/Ubls with sufficient affinity that suicide inhibition of the catalytic site can occur, thereby providing a list of enzyme candidates. A number of these were subsequently confirmed to exert Ub/Ublspecific processing activities. Clearly, these findings provide the framework for the biochemical and functional characterisation of novel proteolytic enzymes with Ub/Ublspecific processing activity.

In this review, we first discuss examples where host cell encoded Ub/Ubl-specific proteases are manipulated by pathogens during the infection process (Table 1). Second, we review pathogen encoded proteases that have Ub/Ubl specific activities and what is known about their function (Table 2), and finally argue that some of them have a common origin with mammalian counterparts, whereas others may have evolved completely independently for different functions within pathogen biogenesis, and may have adapted to host-pathogen interactions throughout evolution.

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Host encoded UB/Ubl specific proteases modulated by pathogens

Proteases specific for ubiquitin-like proteins stimulated by interferons

Infection of most cell types by viruses or bacteria leads to expression of interferon stimulated genes, which include the ubiquitin-like protein ISG15 and one of its specific proteases UBP43 (USP18) (reviewed in [START_REF] Kim | Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling[END_REF]). This suggests that they are components of the innate immune system and can contribute to an inherent antimicrobial response.

During infection, ISG15 is conjugated to substrate proteins by UBE1L, an ISG15activating enzyme. This process can be specifically blocked by the influenza B virus encoded protein NS1 [START_REF] Yuan | Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein[END_REF]. Furthermore, ISG15-/-mice have an increased susceptibility to influenza A/B, herpes simplex virus type 1, murine gammaherpesvirus 68 and sindbis virus [START_REF] Lenschow | Virgin, IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses[END_REF].

Interference with deISG15ylation also affects susceptibility to viral infection as demonstrated in USP18-/-mice. Cells derived from these animals did not support replication of a number of viruses, and bacteria infected animals succumbed to an overshooting immune reaction. This was accompanied by increased levels of ISGylation, suggesting a possible involvement of USP18 mediated isopeptidase activity in this process [START_REF] Ritchie | Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection[END_REF]. However, increased susceptibility to infection may be explained through multiple mechanisms, since USP18 was also reported to regulate interferon signaling independently of its ISG15 isopeptidase activity [START_REF] Malakhova | UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity[END_REF]. No example has yet been reported in which USP18 is targeted by pathogens directly, but a number of viral gene products have recently been described to have ISG15 deconjugating activity, therefore dampening the IFN response (discussed below).

In addition, several DUBs encoded by mammalian cells can cross-react with ISG15specific active site probe, such as USP2, IsoT1 (USP5), IsoT3 (USP13) and USP14, suggesting potential specificity for Ub and ISG15 [START_REF] Catic | Screen for ISG15-crossreactive deubiquitinases[END_REF]. However, in the case of USP2 and USP5, no significant ISGylating activity was detected using fluorescence based substrates [START_REF] Nicholson | Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities[END_REF].The functional relevance of the dual specificity of these enzymes still awaits confirmation with protein based substrates, and has not yet been determined.

A second ubiquitin-like protein, FAT10, is also upregulated upon exposure to IFN or TNFα [START_REF] Groettrup | Activating the ubiquitin family: UBA6 challenges the field[END_REF]. Overexpression of FAT10 can induce apoptosis, and was reported to target substrates for proteasomal proteolysis as an alternative pathway to ubiquitin [START_REF] Hipp | FAT10, a ubiquitin-independent signal for proteasomal degradation[END_REF]. The IFN dependence of FAT10 expression suggest that this molecule also represents a component of the innate immune defence against pathogens, but its specific role in this process as well as FAT10-specific proteases remain to be examined.

USP7 targeted by Herpesviruses

The ubiquitin specific protease 7 (USP7) was initially described as herpes associated ubiquitin specific protease (HAUSP) based on the initial observation that it interacted with the Epstein-Barr nuclear antigen 1 (EBNA1) and the herpes simplex virus type 1 regulatory protein ICP0 [START_REF] Everett | A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein[END_REF]. However, despite that these interactions have been studied in detail [START_REF] Holowaty | Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7[END_REF], their functional significance is not yet completely understood. In the case of EBNA1, the USP7 interaction domain overlaps with the one of the tumour suppressor gene P53, suggesting that EBNA1 may interfere with p53 mediated effects, such as premature apoptosis of infected cells [START_REF] Saridakis | Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization[END_REF]. USP7 also contributes to ICP0 induced

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reactivation of gene expression from integrated adeno-associated virus AAV [START_REF] Geoffroy | Impact of the interaction between herpes simplex virus type 1 regulatory protein ICP0 and ubiquitinspecific protease USP7 on activation of adeno-associated virus type 2 rep gene expression[END_REF]. USP7 is generally involved in processes such as transcriptional regulation, DNA replication, apoptosis, and possible endosomal organisation [START_REF] Kessler | Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7[END_REF]. It appears that multiple functions of USP7 are exploited by those viruses, and further experimental work is required to shed more light on these mechanisms.

CYLD and bacterial infections

Cylindromatosis is a benign skin tumour also referred to as turban tumour, and is originated by mutations in the CYLD gene [START_REF] Bignell | Identification of the familial cylindromatosis tumour-suppressor gene[END_REF]. Biochemical studies revealed that CYLD is a deubiquitylating enzyme acting on TRAF2 and TRAF6, which results in a negative regulation of NF-κB by tumour necrosis factor (TNFR) and toll like receptors (TLRs), suggesting that CYLD plays an important role in controlling inflammation [START_REF] Kovalenko | The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination[END_REF], [START_REF] Brummelkamp | Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB[END_REF], [START_REF] Trompouki | CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB activation by TNFR family members[END_REF]. Recent work uncovered that CYLD-/-mice are hypersensitive to infection by Nontypeable Haemophilus influenza (NTHi) [START_REF] Lim | Tumor suppressor CYLD acts as a negative regulator for nontypeable Haemophilus influenza-induced inflammation in the middle ear and lung of mice[END_REF], [START_REF] Sakai | The bacterium, nontypeable Haemophilus influenzae, enhances host antiviral response by inducing Toll-like receptor 7 expression: evidence for negative regulation of host anti-viral response by CYLD[END_REF]. CYLD expression is induced in cells infected by this pathogen, and it was suggested to down-regulate NF-κB activation through deubiquitylation of TRAF6 and 7, therefore preventing the formation of an antiviral response of the host. A similar observation was made in an infection model using Escherichia coli pneumonia, in which an enhanced innate immune response was observed in CYLD-/-mice [START_REF] Lim | CYLD is a crucial negative regulator of innate immune response in Escherichia coli pneumonia[END_REF]. An adverse effect was observed when CYLD-/-mice were challenged with Streptococcus pneumoniae [START_REF] Lim | Tumor suppressor CYLD regulates acute lung injury in lethal Streptococcus pneumoniae infections[END_REF]. In this case, CYLD was shown to provoke acute lung injury in the context of infection and therefore contributing to lethality. CYLD was suggested to interfere with the NFAT signalling pathway by deubiquitylating transforming growth factor-β-activated kinase 1 (TAK1), and to negatively regulate KK3-p38 kinase-dependent expression of plasminogen activator inhibitor-1 [40] [39]. The example of CYLD having opposing effects in three different infection models in particular highlights the potential challenge that will be faced upon the development of novel therapeutic approaches, which may interfere with one infectious agent, but may promote sensibility to another. Since there are other Ub/Ubl processing enzymes that are involved in regulating inflammation and the function of immune cells, such as A20 [START_REF] Boone | The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses[END_REF], it is expected that additional pathogen derived molecular mechanisms might exist that can interfere with ubiquitin mediated activation of immune responses.

Ub/Ubl proteases in viral pathogens

Coronaviruses (SARS)

An example of a viral DUB that was discovered through structural bioinformatics is the papain-like protease (PLpro) encoded by Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). Following this prediction, it has been demonstrated that PLpro can indeed cleave polyUbK 48 -chains as well as ISG15 and Nedd8 in vitro, but with a preference for ISG15 cleavage [START_REF] Barretto | The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity[END_REF] [43] [START_REF] Lindner | Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease[END_REF]. Crystallographic studies confirmed that PLpro shares considerable similarities to the catalytic core domain of the herpesivirusassociated ubiquitin-specific protease (HAUSP) and other already characterized DUBs like USP14 (Figure 2) [45] [43]. It was therefore suggested to belong to the USP family [START_REF] Ratia | Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme[END_REF]. The role of this enzyme is to process nsp1 to nsp3 from the viral replicase polyprotein [START_REF] Harcourt | Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity[END_REF]. Additionally, PLpro has been shown to block IRF3-dependent antiviral responses through inhibition of phosphorylation and translocation of IRF3. These two actions were suggested to not be attributable to the protease domain of PLpro, although the results obtained with the catalytic mutants used in this study were inconclusive [START_REF] Devaraj | Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus[END_REF]. Interestingly, these events disturb activation of the type I interferon (IFN) responses, which suggests that PLpro is an important molecule in the infection process. It still remains a challenge to functionally distinguish between the general proteolytic activity and specific isopeptidase activity of PLpro, although more recent protease protection experiments suggest that the putative cytoplasmic domain of nsp3, which includes the PLpro domain, might be exposed and therefore potentially capable of processing ubiquitylated and/or ISGylated host cell substrates [START_REF] Van Hemert | SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro[END_REF].

In another human coronavirus, NL63, two papain-like proteases (PLPs), PLP1 and PLP2, have been characterised, which process viral replicase polyproteins essential for RNA synthesis and viral replication [START_REF] Chen | Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63[END_REF]. PLP2 shares some similarity with PLpro encoded by SARS-CoV, and has been shown to cleave poly-UbK 48 -chains in vitro [START_REF] Chen | Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63[END_REF]. Moreover, PLP2 has deISGenylation activity, although it has a preference for Ub over ISG15 [START_REF] Nicholson | Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities[END_REF]. In this case, bioinformatics studies revealed that all coronaviruses encode for at least one papain like protease with deubiquitylating activity [START_REF] Sulea | Binding site-based classification of coronaviral papain-like proteases[END_REF].

Adenoviruses

Biochemical experiment on adenovirus-infected cells performed using ubiquitin aldehyde (Ubal) as the bait suggested deubiquitylating properties of Avp (23K proteinase, Ad L3, adenain). During infection, Avp processes viral proteins and appears to deubiquitylate some cellular proteins [START_REF] Balakirev | Deubiquitinating function of adenovirus proteinase[END_REF]. In order to perform this task, it requires activation through viral DNA, an 11-amino acid viral peptide and actin as cofactors [START_REF] Mangel | Specific interactions of the adenovirus proteinase with the viral DNA, an 11-amino-acid viral peptide, and the cellular protein actin[END_REF]. Additionally, Avp has been shown to cleave the peptide bond of an ISG15 precursor protein. Despite its structural similarity to Ulp1, a protease that cleaves SUMO, Avp does not have SUMO deconjugating properties [START_REF] Balakirev | Deubiquitinating function of adenovirus proteinase[END_REF]. This viral proteinase plays a crucial role in the viral infection process by cleaving virion precursor proteins essential for the infectivity of the virus. In addition, Avp disrupts the cytoskeleton by cleavage of cytokeratin 18 in the cytosol, thereby provoking more efficient cell lysis [START_REF] Mangel | Specific interactions of the adenovirus proteinase with the viral DNA, an 11-amino-acid viral peptide, and the cellular protein actin[END_REF]. Avp is also targeted by nitric oxide (NO), and its inhibition via NO blocks replication of the virus [START_REF] Cao | Nitric oxide inhibits the adenovirus proteinase in vitro and viral infectivity in vivo[END_REF]. Nevertheless, the deubiquitylating function of Avp has not been implicated in any physiological processes.

Viruses encoding for ovarian tumour (OTU)-like proteases

In the family of nairoviruses that includes Crimean-Congo Hemorrhagic Fever (CCHF), Dugbe (DUG) and Nairobi Sheep Disease (NSD) viruses, an OTU-like domain is present within their large (L) protein that also contains the RNA polymerase domain [START_REF] Frias-Staheli | Ovarian tumor domain-containing viral proteases evade ubiquitin-and ISG15-dependent innate immune responses[END_REF], [START_REF] Honig | Crimean-Congo hemorrhagic fever virus genome L RNA segment and encoded protein[END_REF].

The L protein of CCHFV has been shown to posses a deubiquitylating and deISGylating activity. It has been demonstrated that this protein directly deconjugates poly-Ub chains and ISG15-conjugated proteins in vitro. Moreover, it also decreases levels of ISGylated and ubiquitinated host proteins, when expressed in mammalian cells. All these properties have been observed in L proteins of NSDV and DUGV, when these were expressed in mammalian cells. Moreover, Sindbid viruses over-expressing the OTU-like domain of CCHFV-L showed an increase in infectious pathogenicity in mice as compared to controls. This result has been attributed to neutralisation of the protective effect of ISG15

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ACCEPTED MANUSCRIPT 8 synthesis in infected cells [START_REF] Frias-Staheli | Ovarian tumor domain-containing viral proteases evade ubiquitin-and ISG15-dependent innate immune responses[END_REF]. Another family of viruses, arteriviruses, including Equine Arteritis (EAV) as well as Porcine Respiratory and Reproductive Syndrome (PRRSV) viruses, encode for an OTU-like domain within the nonstructural protein 2 (nsp2) [START_REF] Frias-Staheli | Ovarian tumor domain-containing viral proteases evade ubiquitin-and ISG15-dependent innate immune responses[END_REF], [START_REF] Honig | Crimean-Congo hemorrhagic fever virus genome L RNA segment and encoded protein[END_REF]. Nsp2 cleaves the large viral polyprotein in the nsp2/nsp3 site [START_REF] Snijder | The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases[END_REF]. When overexpressed in mammalian cells, nsp2 decreases the level of Ub and ISG15 conjugates [START_REF] Frias-Staheli | Ovarian tumor domain-containing viral proteases evade ubiquitin-and ISG15-dependent innate immune responses[END_REF]. In the case of the family of Furoviruses, the OTU-like domain was found in the Rice Stripe Virus (RSV) Zhejiang isolate as well as other RSV isolates, and it is present in the pc1 protein encoded by RNA1 [START_REF] Zhang | Genomic analysis of rice stripe virus Zhejiang isolate shows the presence of an OTU-like domain in the RNA1 protein and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses[END_REF]. It has been suggested that a putative role of this domain is the auto-proteolytical cleavage of pc1, which leads to activation of the viral polymerase [START_REF] Zhang | Genomic analysis of rice stripe virus Zhejiang isolate shows the presence of an OTU-like domain in the RNA1 protein and a novel sequence motif conserved within the intergenic regions of ambisense segments of tenuiviruses[END_REF]. Additionally, the deubiquitylating function of pc1 would provide a way to interference with the proteolytic pathway in the host.

Herpesviruses

Activity-based profiling of herpes simplex virus-1 (HSV-1) infected cells using active site-directed probes with a ubiquitin scaffold combined with tandem mass spectrometry has revealed a virus-encoded USP, UL36 USP , that corresponds to the N-terminal fragment of the large viral tegument protein UL36 [START_REF] Kattenhorn | A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae[END_REF]. UL36 USP cleaves ubiquitin, but not SUMO, ISG15 or Nedd8, and it has specificity towards K 48 linkages of polyubiquitin [START_REF] Kattenhorn | A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae[END_REF]. The catalytic site of UL36 USP (Cys65) is conserved throughout the whole family of Herpesviridae [START_REF] Kattenhorn | A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae[END_REF], but there are no homologues of UL36 USP in eukaryotic cells. Other members of the Herpesviridae family such as human cytomegalovirus (CMV), murine cytomegalovirus (MCMV), murine Gammaherpesvirus 68 (MHV), Marek's disease virus (MDV) and pseudorabies virus (PrV) contain UL36 USP homologues. In most of these viruses it has been shown that the UL36 USP homologues have deubiquitylating activity in vitro [START_REF] Schlieker | A deubiquitinating activity is conserved in the large tegument protein of the herpesviridae[END_REF], [START_REF] Wang | Highmolecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable[END_REF], [START_REF] Bottcher | Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo[END_REF], [START_REF] Jarosinski | A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation[END_REF]. In CMV, the whole 235-kDa high-molecular-weight protein (HMWP) displays a deubiquitylating activity [START_REF] Wang | Highmolecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable[END_REF], similarly to MHV USP , where the polypeptide that is reactive to the ubiquitin active probe is as large as ~250 kDa [START_REF] Gredmark | A functional ubiquitin-specific protease embedded in the large tegument protein (ORF64) of murine gammaherpesvirus 68 is active during the course of infection[END_REF]. However, it is not the case for the HSV-1 UL36 USP , where the active UL36 USP is only ~47 kDa. The physiological relevance of these viral DUBs is not yet fully understood, although for two herpesvirus tegument USPs (MDV and PrV), mutation of the catalytic site cysteine affected the rate of viral replication. The expression of MDV USP is additionally associated with lymphoma formation and metastasis in chicken [START_REF] Jarosinski | A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation[END_REF], whereas mutagenesis of the active site of PrV USP inhibits viral neuroinvasion [START_REF] Bottcher | Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo[END_REF], which may be linked to a possible defect in transporting membrane associated virus particles along microtubules [START_REF] Shanda | UL36p is required for efficient transport of membrane-associated herpes simplex virus type 1 along microtubules[END_REF].

Ub/Ubl proteases in bacteria and protozoa

Bacteria

Although bacteria do not have an intrinsic ubiquitin system, it was found that many bacterial strains contain Ub/Ubl specific proteases. For instance, the Salmonella genome encodes for two different proteins with deubiquitylating activities, AvrA and SseL. Both are effector proteins translocated through type three secretion system (TTSS) of Salmonella (reviewed in [START_REF] Munro | Bacteria and the ubiquitin pathway[END_REF]). AvrA deubiquitylates IκBα and β-catenin as demonstrated with GST-tag purified enzyme, thus stabilizing IκBα and β-catenin and inhibiting the NF-κB inflammatory responses of the host [START_REF] Ye | Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination[END_REF]. Similarly, SseL through its deubiquitilating activity suppresses IκBα ubiquitination and thereby prevents NF-κB activation [START_REF] Le Negrate | Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses[END_REF]. SseL can deubiquitylate mono-and poly-ubiquitylated substrates and has affinity towards K 63 poly-Ub chains [START_REF] Rytkonen | SseL, a Salmonella deubiquitinase required for macrophage killing and virulence[END_REF], suggesting that it may play a role in disruption of cellular signaling. In addition, SseL may be involved in delayed cytotoxicity of macrophages that constitutes an important mechanism of Salmonella virulence [START_REF] Le Negrate | Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses[END_REF] During infection with Yersinia, another enterobacterium that uses a type three secretion system, a virulence factor, referred to as YopJ promotes intracellular survival of the bacterium via inhibition of NF-κB and MAPK, as well as IRF3 signaling pathways [START_REF] Sweet | YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction[END_REF].

YopJ was first described to act as a deubiquitinase cleaving the K 63 -poly-Ub chains of TRAF2, TRAF3 and TRAF6 [START_REF] Zhou | Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kappa B activation[END_REF], [START_REF] Sweet | YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction[END_REF]. YopJ can also deubiquitylate K 48 -poly-Ub chains of IκBα, thereby preventing its degradation and translocation of NF-κB [START_REF] Orth | Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease[END_REF]. It has been proposed that YopJ may also cleave sumoylated proteins [START_REF] Orth | Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease[END_REF], but more recent studies [START_REF] Mukherjee | Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation[END_REF] also revealed that it also acts as an acetyltransferase to modify serine and threonine residues in the activation loop of MAPKK6, thereby blocking its phosphorylation and activation. In this way, YopJ interferes with the host proinflammatory responses most likely via both, its Ub/Ubl processing properties, but also via its acetylating activity [START_REF] Orth | Function of the Yersinia effector YopJ[END_REF], [START_REF] Mukherjee | Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation[END_REF]. This finding may represent a common feature that can be found in other proteolytic enzymes, such as the E. coli derived TAP protein [START_REF] Lee | Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1[END_REF]. Activity-based profiling studies in the Chlamydia proteome using ubiquitin based probes identified two active deubiquitylating enzymes of Chlamydia trachomatis, ChlaDub1 and ChlaDub2. They have been reported to cleave Ub as well as Nedd8 conjugates [START_REF] Misaghi | Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection[END_REF], but neither their physiological role nor any substrate candidates have yet been discovered. The Escherichia coli encoded protease ElaD, has also been identified as a deubiquitylating enzyme specific for ubiquitin, but not for Ubls, like ISG15, Nedd8 or SUMO. ElaD is found in all intestinal strains, but absent from extraintestinal strains of E.coli, an observation that may point towards the significance of ElaD in the infection process [START_REF] Catic | ElaD, a Deubiquitinating protease expressed by E. coli[END_REF].

Parasitic protozoa

Although there have been predictions of many putative deubiquitylating enzymes (see review [START_REF] Ponder | Ubiquitin-like modifiers and their deconjugating enzymes in medically important parasitic protozoa[END_REF]) encoded by medically relevant parasitic Protozoa using bioinformatics studies, only three deubiquitylating enzymes have been reported to date on the basis of their binding to the Ub/Ubl active site probes [78] [79]. All of them have been found via an activity-based Ub/Ubl probe approach and no alternative validation of their DUB activity has been yet carried out. Plasmodium falciparum encodes for two deubiquitylating enzymes, PfUCHL3 and PfUCH54, the latter being a homolog of human deubiquitylating enzymes UCH37, UCH-L1 and UCH-L3 (Figure 2). Although the sequence did not reveal a high sequence identity between PfUCH54 and human DUBs, all active site residues have been preserved [START_REF] Artavanis-Tsakonas | Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum[END_REF]. Interestingly, PfUCHL3 and PfUCH54 have both deubiquitylating and deNeddylating activity [START_REF] Artavanis-Tsakonas | Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum[END_REF] [79], as shown using activity based Ub/Ubl probes. Because PfUCH54 does not have a specific N-terminal sequence that allows it to be secreted, it has been hypothesized that this protein has an important function within the parasite itself, and is not host-targeted [START_REF] Artavanis-Tsakonas | Identification by functional proteomics of a deubiquitinating/deNeddylating enzyme in Plasmodium falciparum[END_REF].

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Another protozoa-encoded deubiquitylating enzyme is TgUCHL3 that is expressed by Toxoplasma gondii. It is also a homologue of UCH-L3 and was found to be specific to Nedd8 and ubiquitin active site probes, but not SUMO [START_REF] Frickel | Apicomplexan UCHL3 retains dual specificity for ubiquitin and Nedd8 throughout evolution[END_REF]. However, the probes that were used for studying TgUCHL3 and PfUCHL3 reactivity to Nedd8 and SUMO were prepared using Ubl proteins that are more closely related to the human sequences than to their parasitic homologues, therefore the observed reactivities towards the different Ubls should be interpreted with caution. In all these cases, the precise role of these proteases within the context of pathogenicity has not yet been elucidated.

Common ancestry patterns of UB/Ubl specific proteases involved in host-cell interactions

Although there is no evidence that bacterial organisms do have a ubiquitin system, many pathogens of prokaryotic origin seem to encode for proteins with Ub and Ubl recognition and processing properties. This appears to be the case also for viral pathogens, in which the presence of Ub/Ubl-specific proteases appears to be more widespread than previously thought. Pathogens with larger genomes contain most likely more Ub/Ubl specific proteases, and more advanced bioinformatics approaches are beginning to reveal novel members, as exemplified for the recent discovery of novel bona fide DUBs encoded by EBV [START_REF] Sompallae | Epstein-Barr Virus Encodes Three Bona Fide Ubiquitin-Specific Proteases[END_REF]. For many of these, we are at an early stage of deciphering their precise functions, but sequence and structure based comparisons with mammalian Ub/Ublspecific protease families may provide some clues about aspects of their biology. Based on this, pathogen encoded Ub/Ubl proteases would appear to fall into two general categories:

The first one may consist of Ub/Ubl proteases that can be added to the list of already characterised superfamilies (Figure 2). For instance, the family of ubiquitin C-terminal hydrolases (UCHs) contains three mammalian hydrolases, and the more recently characterised proteases encoded by the parasites Plasmodium falciparum, Toxoplasma gondii and related organisms may form an extended UCH superfamily. A second example may consist of the adenovirus encoded protease adenain, the Chlamydia derived ChlaDUB1/2 proteases, E. coli derived ElaD, Salmonella encoded SseL, Yersinia encoded YopJ and similar sequences observed in other pathogens, all of which share some similarities with ULP/SENP-related proteases that are specific for SUMO and Nedd8 (Figure 2). This superfamily of cysteine proteases has been classified as the CE clan, which targets Ubiquitin and Ubls (MEROPS database [START_REF] Catic | ElaD, a Deubiquitinating protease expressed by E. coli[END_REF], but potentially harbours other enzymatic functions as illustrated in the case of YopJ [START_REF] Trosky | Yersinia outer proteins: Yops[END_REF] and adenain [START_REF] Balakirev | Deubiquitinating function of adenovirus proteinase[END_REF]. A third example is the SARS coronavirus encoded protein PLpro that contains a catalytic core that is structurally related to the ones observed in USP7 and USP14 [START_REF] Sulea | Deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease?[END_REF]. A fourth example is the protein family sharing the ovarian tumour domain (OTU), which contains ca 80 members derived from plants, drosophila, bacteria, viruses, yeast and higher eukaryotes ( [START_REF] Makarova | A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae[END_REF], [START_REF] Nijman | A genomic and functional inventory of deubiquitinating enzymes[END_REF]). In all these cases, this may point towards common fundamental ancestry functions that are essential within these organisms. Adaptation mechanisms in the context of host-pathogen interactions may have further diversified their functions, as exemplified in the case of viral OTU containing proteases that have acquired specificity for ISG15, a trait that is not observed for their mammalian counterparts [START_REF] Frias-Staheli | Ovarian tumor domain-containing viral proteases evade ubiquitin-and ISG15-dependent innate immune responses[END_REF].

The second category is represented by novel and unique classes of pathogen encoded Ub/Ubl specific proteases (Figure 2). This is most likely the case for herpes virus encoded tegument derived DUBs (reviewed in [START_REF] Sulea | Binding site-based classification of coronaviral papain-like proteases[END_REF]). The primary function of these proteases may involve the processing of viral gene products necessary for virus life cycle, and adaptation to Ub specificity as a dual function may have evolved in an evolutionary process to increase the pathogenicity of host-pathogen interactions. Unique structural features of pathogen encoded Ub/Ubl-specific proteases remain attractive entry points for the development of novel antimicrobial drugs, and research efforts in this direction may soon provide novel alternatives to combat a variety of infectious diseases.

Figure 1 :

 1 Figure1: Scheme of Ub/Ubl-specific proteases. Proteases with specificity to Ubiquitin (Ub) or Ubiquitin-like proteins (Ubls) are currently classified into the following protein families: UCHs: Ubiquitin C-terminal hydrolases; USPs: Ubiquitin specific proteases (also referred to as UBPs: Ubiquitin processing proteases); OTUs: ovarian tumour domain containing proteases; MMs: metalloproteases; JDs: Josephin domain containing proteases; SENPs: small ubiquitin-like modifier specific proteases; ULPs: Ubiquitin-like protein specific proteases. See text for a more detailed discussion.

Figure 2 :

 2 Figure 2: Pathogen encoded Ub/Ubl proteases and their relation to mammalian enzymes. Although many proteolytic enzymes derived from pathogens such as viruses (red), bacteria and protozoa (green) have unique properties, most of them are related to mammalian Ub/Ubl protease families (white) based on sequence or structure based similarities. Proteins with deubiquitylating enzyme activity encoded by Herpesviridae appear to be unique with little or no resemblance to any other Ub/Ubl protease families. See text for a more detailed discussion.