INTRODUCTION INTRODUCTION 1 INTRODUCTION

Preamble

In high-income countries, cancer is the leading cause of death, and it is the second cause worldwide [START_REF] Dagenais | Variations in common diseases, hospital admissions, and deaths in middle-aged adults in 21 countries from five continents (PURE): a prospective cohort study[END_REF]. Furthermore, as CVD (CardioVascular Diseases) rates continue to fall, cancer could likely become the leading cause of death worldwide, within just a few decades. It is a major cause of morbidity in France, where the estimated annual number of cancer-related deaths is more than 150 000 (INCa report, 2019).

Cancer is one of the most important health problems with various mechanisms of pathogenesis, some of them remaining unknown. In addition, its capacity to produce temporary or permanent damage, besides death, is very high: for patients who survived 5 years after diagnosis, more than 60% suffer from after effects (INCa report, 2019). Although many anticancer therapies are available, the 2009-2015 five years survival rate in USA is around 70 % overall, and for some localisations by far lower: pancreas 10 %, lung 21% (American Cancer Society website, 2020).

Cancers have a high requirement for methionine in comparison with non-cancerous cells, and its deprivation in cancer cells induces growth arrest and cell death, whereas normal cells are much more resistant [START_REF] Sato | Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers[END_REF].

As methionine is an essential aminoacid, a permanent diet without methionine is not practically possible. Methionine depletion can be achieved using the methionine gamma-lyase INTRODUCTION 2

I. Methionine deprivation as an anticancer strategy

Normal cells can synthesize sufficient methionine endogenously for growth requirements from homocysteine through methionine synthase [START_REF] Parkhitko | Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species[END_REF], which renders normal cells largely resistant to methionine depletion. Conversely, most cancer-cell types require exogenous methionine for survival and therefore methionine restriction is a promising avenue for treatment. One of the early studies conducted in diet restriction evaluated several outcomes produced from regimens lacking specific amino acids in rats with Walker-256 carcinosarcoma tumor [START_REF] Sugimura | Quantitative nutritional studies with watersoluble, chemically defined diets. VIII. The forced feeding of diets each lacking in one essential amino acid[END_REF]. Diets lacking valine, isoleucine or methionine showed decreased tumor growth. These results have promoted the discovery of methionine gamma lyase (MGL), an enzyme that metabolizes methionine, as a more effective and rapid method to deplete serum methionine.

II. Methionine Metabolism and cancer therapy

Methionine is an essential amino acid; it is involved in many biological processes. There are at least two major functions of methionine with relevance to cancer biology: 1) polyamine synthesis, and 2) methyl group donation (Figure 1).

Polyamine synthesis

Methionine is a precursor for the polyamines. These organic compounds with two or more amine functions are recognized as having substantial and fundamental roles in various biological systems, including their involvement in growth and proliferation of mammalian cells.

Studies have shown that elevated levels of polyamines are associated with increased tumour growth, so methionine depletion provokes a disruption in the cell cycle, inducing apoptosis [START_REF] Morgan | Polyamines. An overview[END_REF][START_REF] Thomas | Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications[END_REF].

DNA methylation

DNA methylation is one of the most intensely studied epigenetic modifications in mammals.

In normal cells, it assures the proper regulation of gene expression and stable gene silencing.

Methionine is used for the biosynthesis of S-adenosylmethionine (SAM), the universal methyl donor for the methylation of DNA, RNA, histones, phospholipids, catecholamines, and proteins. These epigenetic modifications are crucial to regulate the functioning of the genome by changing chromatin architecture. Methionine restriction has the potential to affect cancer development and progression through modifying DNA methylation patterns.

III. Methionine Gamma Lyase (MGL), a tool for Methionine restriction

The traditional chemotherapy treatment has been the primary one for cancer based on its ability to inhibit rapidly dividing cancer cells (Khamisipour et al., 2016), but it has toxic effects on normal cells. It induces side effects in a high percent of patients, and some cancers resist to this treatment. Further studies explored the use of MGL for reducing methionine as an antitumor strategy. This enzyme has shown efficacy against human tumours propagated in nude mice, either administered alone or in combination with chemotherapy [START_REF] Yoshioka | Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase[END_REF]. The advantage of anticancer therapy using MGL is its wide range of target tumours, including those resistant to the conventional chemotherapies and radiation. Methioninase treatment provides a novel paradigm for cancer therapy.

MGL is an enzyme present in bacterial species, in plants, and absent in mammals. It directly degrades sulfur-containing amino acids (Met, Cys) into an α-keto butyrate and a volatile methanethiol (Figure 2). Its active site is formed at the interface of two neighboring subunits.

It forms a homotetramer; each subunit of which contains one pyridoxal 5'phosphate (PLP) cofactor (Figure 3).

IV. Previous studies on bacterial MGL from Pseudomonas putida for cancer treatment

MGL is widely distributed in bacteria, for instance in Pseudomonas aeruginosa and in other bacteria like Brevibacterium aurantiacum, Clostridium sporogenes or Citrobacter freundii, in parasitic protozoa like Trichomonas vaginalis and in some plants like Arabidopsis thaliana [START_REF] Sato | Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers[END_REF]. It is induced by the addition of L-methionine to the culture medium.

MGL from Pseudomonas putida

Amongst the cited MGL sources, P. putida is reported to be the best source for MGL production, so a lot of work has been performed on this MGL (Pp-MGL) to prepare possible therapeutic use. Previous experiments on Pp-MGL showed that it induced growth arrest and death of cancer cells. It has an anticancer cytotoxicity that has been demonstrated in a mouse model. However, during the transition to the monkey, this MGL caused anaphylactic shocks.

Pegylation (conjugation to polyethylene glycol) trials of the enzyme in order to reduce allergenicity have altered its stability [START_REF] Machover | Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination[END_REF]. Therefore, the development of

INTRODUCTION 4

In addition to this limitation, the therapeutic exploitation of MGL from Pseudomonas putida showed two other limitations: i) it is rapidly inactivated in human serum, and ii) it is not specific for methionine, but also degrades cysteine with possible undesirable effects.

Undesirable effects of Cys degradation

Cysteine is an aminoacid that plays several roles in the human body, so its degradation may cause undesirable effects:

-The alteration of endogenous GSH synthesis: cysteine is responsible for the synthesis and maintenance in adequate cell quantity of glutathione. It is an important antioxidant and its degradation induces oxidative stress.

-The decrease in CoA synthesis, the first step of which is the addition of an L-Cys to phosphopantothenate. So the deficiency in CoA has important consequences on the Krebs cycle, lipid synthesis, neurotransmission, histone acetylation, etc.

-The decrease in the incorporation of L-Cys into proteins, altering specificities of the proteins, such as the binding to various metals, some post transcriptional modifications, etc. ,and under three PLP concentrations (i.e.,20,100,and 200 mM). Bars are the mean of experiments done in quadruplicate [START_REF] Machover | Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination[END_REF].

V. MGL from Brevibacterium aurantiacum, a new approach for cancer therapy

The use of the MGL from Pseudomonas putida being a dead end, another MGL, produced by the cheese bacterium Brevibacterium aurantiacum, has been identified at INRA Grignon, in collaboration with Dr Machover's team. The idea was to use this MGL from a non-pathogenic bacterium for cancer therapy (INRA / INSERM patents 2012 and[START_REF] Marant-Micallef | Approche et méthodologie générale pour l'estimation des cancers attribuables au mode de vie et à l'environnement en france métropolitaine en 2015 / overall approach and methodology to estimate cancers attributable to lifestyle and environmental risk factors in metropolitan france in[END_REF]. Given that it is abundantly present in food, the risk of allergy is minimized. In addition, it is specific of the degradation of L-methionine and its precursor, L-homocysteine (Table1), like MGL from Arabidopsis thaliana [START_REF] Goyer | Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway[END_REF]. Conversely, other bacterial MGLs also degrade cysteine.

Dr. Machover's team tested the cytotoxicity of Brevibacterium aurantiacum MGL on many tumor lines in vitro. They found that Ba-MGL was cytotoxic on 33 of the 35 human cancer cell lines tested (the two that resist, SW480 and SW620, are from the same clone: primary tumor and metastasis). On the other hand, it was not toxic to normal cells (Table 2).

However, under conditions that are increasingly approaching in vivo conditions, Ba-MGL loses its activity in plasma or in PBS supplemented with human serum albumin (HSA) after 8 hours of incubation. Its activity is restored when adding PLP in PBS supplemented with human serum albumin (Figure 4). Therefore, Dr. Machover's team supposed that this loss of activity was due to the leakage of PLP and binding to other proteins (in particular HSA). This problem was less important with Pp-MGL whose activity in the human serum lasts longer [START_REF] Machover | Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination[END_REF].

VI. Previous results on Ba-MGL variants

To improve the affinity of MGL from Brevibacterium aurantiacum, it was suggested to imitate the sequence and the structure of Pp-MGL which retains PLP better than Ba-MGL.

Based on the sequences alignment (BLASTp, NCBI) we identified the conserved positions close to PLP (Annex 1). The superposition of the 3D structures of Ba-MGL and Pp-MGL indicated 4 different residues: (Thr92, Met 97, Phe 187 and Met 218) in Ba-MGL, corresponding respectively to Ser, Ile, Tyr and Thr in Pp-MGL (Figure 5). Four mutants were designed by single point mutation replacing Ba-MGL residues by the corresponding amino acids that interact with PLP in the crystallographic structure of Pp-MGL.

These mutants were characterized by dialysis in order to identify those that best retain PLP (Figure 6). It was found that the Pp-MGL retains the PLP much better during dialysis than Ba-MGL, which is in accordance with previous studies of Dr Machover's team (unpublished) indicating that this difference was observable in vivo or in plasma.

The Ba-MGL produced by GTP society (external provider, France) and that produced by UMR DYSCOL, as shown in Figure 6, are not significantly different as regards to dialysis. That means that the presence of the Histag, the cleavage site for the TEV protease, and additional hinge residues added by the construction of GenScript do not influence the ability of MGL to retain the PLP.

A little improvement of PLP retention was obtained for only one variant: M97I. However, dialysis of this variant was carried out only once and it would be necessary to estimate the measurement error before being able to affirm that this variant retains the PLP better than the MGL WT of Brevibacterium aurantiacum.

These tested point mutants of Brevibacterium aurantiacum have not shown improvement in the retention of PLP. For a better result, it was proposed to test variants carrying several point mutations, namely the four mutations: T92S+M97I+M218T+F187Y and the double mutations:

M97I+M218T (the latter instead of M218I that decreases significantly the PLP retention).

OBJECTIVES

Figure7: Ribbon representation of the crystal structure of the Ba-MGL tetramer at very low resolution (3.6 Å)

OBJECTIVES

The main objective of this project is to improve by enzymatic engineering the affinity of the Ba-MGL for its cofactor pyridoxal phosphate (PLP) in order to use the variant enzyme in anticancer therapy. For this, the method used was the imitation of the active site of the MGL from Pseudomonas putida for two reasons: i) its crystal structure is solved and ii) its activity lasts longer than that of Ba-MGL in the human plasma. In this context, the objectives of my internship were as follows:

 Cloning, expression in E. coli and purification of new mutants. They were chosen and guided by bioinformatics partners.

 Dialysis to search the best Ba-MGL variants that stronger retain the PLP cofactor.

To significantly improve Ba-MGL, bioinformatics modelling appears necessary. For this, a high-resolution crystallographic structure of Ba-MGL is extremely useful. The Dyscol group solved a crystallographic structure of Ba-MGL but with very low resolution (3.6 Å) (Figure 7).

Therefore, the second objective was to crystallize:

 Ba-MGL: alone or with PLP;

 Ba-MGL variants: alone or with PLP;

 At-MGL: except Ba-MGL, the only MGL specific of methionine degradation. This strain is used to express certain recombinant proteins. It has in its genome the sequence encoding the RNA polymerase of phage T7 under the control of an inducible promotor with IPTG. This allows the expression of proteins whose gene is inserted into a plasmid of pET type, and placed under the control of the T7 promoter. In addition, this strain is deficient for certain proteases Lon and OmpT, which increases the yield of the overexpressed protein.

MATERIAL AND METHODS

Plasmid used

pET-28a (+) -TEV : a commissioned construction from GenScript, which includes a gene for resistance to the antibiotic Kanamycin, a NheI / SacI cloning site and a histidine tag in the Nterminal position followed by a cleavage site by the TEV protease to remove the Histag after the affinity step (figure 8).

Culture media

Each medium was autoclaved at 120 °C for 20 min under a pressure of 1 bar.

-LB Rich medium (Luria Bertani): this rich medium is composed of tryptone (20 g / L), yeast extract (5 g / L), NaCl (5 g / L). The pH is adjusted to 7.5 by adding NaOH. For agar media, agar is added at the right rate 17 g / L of culture medium before sterilization. In order to select the recombinant clones, the appropriate antibiotic (kanamycin) is added after sterilization. This medium is used in precultures for the production of different proteins.

-TB medium (Terrific Broth) is a proprietary medium formulation used for the cultivation of recombinant strains of E. coli. This medium is supposed to improve the yield of recombinant proteins, it uses glycerol as a carbon source and has a rich nutrient base of amino acids, vitamins, inorganic minerals and trace minerals. The medium is buffered at pH 7.2 ± 0.2.

-SOC rich medium: tryptone (20 g / L), yeast extract (5 g / L), NaCl (9 mM), KCl (2.5 mM), MgCl2 (10 mM), MgSO4 (10 mM), glucose (20 mM). This medium is used for regeneration of competent bacteria after transformation.

Table 3: Composition of the buffer solutions used

Buffer Composition

Washing buffer A for HisTrap (500 ml)

NaCl (100 mM), HEPES (50 mM), TCEP (0.5 mM), Imidazole (10 mM for A), PLP (20 µM), pH 7.5

Elution buffer B for HisTrap (100 ml)

NaCl (100 mM), HEPES (50 mM), TCEP (0.5 mM), Imidazole (500 mM ), PLP (20 µM), pH 7.5 S200 buffer (500 ml) NaCl (100 mM), HEPES (50 mM), TCEP (0.5 mM), PLP (20 µM), pH 7.5 SDS-PAGE migration buffer (600 ml)

MOPS (3-morpholino-1-propanesulfonic acid) SDS running buffer (Invitrogen)

SDS-PAGE gel deposition blue Laemmli

SDS 4%, glycerol 20% v/v, 2-mercaptoethanol 10% v/v, bromophenol blue 0.004% and Tris HCl 0.125 M, pH 6.8

Coomassie blue staining solution

Coomassie blue G 250 0,9 % w/v ; ammonium sulfate 10 % w/v ; orthophosphoric acid 1,6% w/v; ethanol 20 % v/v S200 buffer for dialysis (500 ml) NaCl (100 mM), HEPES (50 mM), pH 7.5

Buffer solutions

Commonly used buffers are described in table 3.

Solutions for crystallisation tests

Buffer solutions used in cristallisation tests are described in annex 2.

VII. Methods

Production and purification of MGLs: MGLs from Brevibacterium.aurantiacum quadruple mutant T92S+M97I+F187Y+M218T, double mutant M97I+M218T and MGL from Arabidopsis thaliana

While in sterile conditions.

a.

Transformation of competent E coli cells with the vectors containing MGL genes

The plasmid (4 µg) is centrifuged for 1 min 6000 g at 4°C, and diluted into 40 µl of sterile water (100 ng/ µl). 1 µl of plasmid solution was added to 50 µl of T7 express competent E. coli cells, then the mixture is placed on ice for 30 minutes. The cells are heat shocked at exactly 42 °C for 10 seconds to be made competent for transformation, followed by cooling in ice for 10 minutes. The cells containing the plasmids are subsequently regenerated by adding the SOC medium (950 μl) at 37 ° C for 1 h, then spread on a nutritive agar on a Petri dish (LB + kanamycin) overnight at 37 ° C.

c. Purification i) Extraction

After suspension of cells pellets (obtained from 500 ml of culture) in 20 ml of NaCl at 0.9% w/v, they are centrifuged again in the same conditions. The supernatant is discarded and the bacterial pellets are frozen in liquid nitrogen and then stored at -80 °C. The protein is retained on the resin thanks to specific interactions between the Ni 2+ ions, fixed beforehand to the resin, and the His-Tag sequence fused on the N-terminal side. These interactions are due to the properties of histidines whose imidazole nucleus has a pair of free electrons, carried by a nitrogen atom, which can form complexes with divalent metal ions such as Ni 2+ , Cu 2+ or Co 2+ .

The lysis supernatant is injected, using a 5 mL loop, at 0.5 ml / min on a Ni-Sepharose column coupled to an Äkta purifier system (GE Healthcare Life Sciences, Vélizy-Villacoublay, France).

The column was previously equilibrated with buffer A in the presence of 10-30 mM imidazole.

The protein has an affinity for nickel groups due to the 6His-tag at the N-terminal side provided by the pET-28a (+) -TEV plasmid. The protein is then eluted using a gradient of imidazole with buffer B (500 mM imidazole) which competes with the side chains of the histidines on the Histag of the MGL protein. Optical densities at 280 nm (proteins) and 422 nm (MGL-bound PLP cofactor) were measured continuously.

iii) Protein concentration and storage

The collected fractions were analyzed by electrophoresis under denaturing conditions (SDS-PAGE 12% polyacrylamide) revealed with Coomassie blue and then pooled and concentrated by ultrafiltration on an 30 kDa Amicon ® Ultra-15 centrifugal unit. The protein was finally stored at -20 °C and the concentration was determined spectrophotometrically at 280 nm.

iv) S200 increase size exclusion chromatography

In order to remove remaining contaminants and separate protein aggregates, a gel filtration step is carried out. The concentrated protein is injected, using a 500 µL loop, on a Superdex 200 column increase 10/300 GL (GE Healthcare) (fractionation range from 5 000 to 800 000 Da) filled with a resin consisting of porous beads, coupled with an Äkta system and previously equilibrated with Buffer A (table 3). This step allows separating proteins according to their hydrodynamic diameter using a molecular sieve. The largest proteins that cannot enter into the gel beads, leave the column first. The others are delayed by their interactions with the beads;

the smallest proteins, which can easily enter the beads, are the last to leave the column (Annex 2).

To be efficient, this technique requires a small volume of starting material. If the volume is too large, the proteins tend to spread along the column, which reduces the quality of their separation.

The fractions containing pure MGL protein, identified by electrophoresis under denaturing conditions with SDS-PAGE, are pooled and concentrated by ultrafiltration on a 30 kDa Amicon ® Ultra-15 centrifugal unit at 5000 g at 4 ° C to perform crystallization tests, then stored at -20 ° C.

A spectrophotometric UV-visible assay allows estimating the molar concentration of the protein and its PLP co-factor content, for this, the absorption of the protein at 280 nm and the absorption of the co-factor at 422 nm are measured.

Biochemical characterization of MGL variants from Brevibacterium aurantiacum

and MGL from Arabidopsis thaliana a.

SDS-PAGE electrophoresis under denaturing conditions

In order to check the purity of the protein after the purification steps, the samples are analyzed by SDS-PAGE electrophoresis under denaturing conditions, which is based on the principle of proteins separation according to their size on a 12 % polyacrylamide gel (NuPAGE from Invitrogen, sold by Thermo Fischer Scientific, Illkirch, France). The gel is revealed by colloidal coloring, allowing both better reproducibility, minimal background coloring and a better sensitivity due to the presence of ammonium sulfate, which increases the strength of hydrophobic interactions and therefore promotes interactions between G250 Coomassie blue and the basic and aromatic amino acids of the proteins fractionated on the gel b.

UV/ visible spectroscopy UV/visible spectra were performed on a Shimazu UV1800 spectrophotometer in order to characterize all the variants produced. It is the first measurement carried out on each chromatography fraction containing proteins after the purification step. This technique allows us to detect the PLP cofactor that in its form complexed with the enzyme absorbs at 422 nm, while the free PLP in solution has a pic around 390 nm, and also to compare the MGL variants with Pp-MGL from, which exhibits a better PLP retention, and identify those that retain PLP co-factor.

c. Dynamic light scattering (DLS)

This technic allows us to measure the size, from <one nm with MW <1000 Da, and the dispersity (homodispersity or polydispersity) of molecules present in purified MGL solution (or suspension), and subjected to random thermal movements by measuring their speed. In addition, it gives us information about the presence of aggregates. On the other hand, it requires the knowledge of the solvant viscosity and the centrifugation of our solution (Annex 3).

The hydrodynamic radius Rh is an estimate that gives us information about the MW of the MGL molecule. For a supposedly spherical molecule, its calculation is done according to the following known relation:

Rg/Rh = [3/5] 1/2 = 0,775
Rg is the radius of gyration.

For globular proteins, we have: [START_REF] Bernadó | A Self-Consistent Description of the Conformational Behavior of Chemically Denatured Proteins from NMR and Small Angle Scattering[END_REF] M: molecular weight in kDa d.

Rg (Å) ≈ 6.25 * 𝑀 1/3

Dialysis for assessment of PLP retention by MGL variants

We used this technic to separate the protein of interest from salts, imidazole, detergents or other small molecules that were present in the initial preparation or were introduced during a purification step, using their ability to cross the pores of a semi-permeable membrane often made of cellulose whose pores are smaller than the macromolecules (proteins of interest). Here we used dialysis cassettes (Slide-A-Lyzer, Thermo Fisher Scientific) that are immersed in PLPfree buffer and which contain the liquid to be dialyzed.

Proteins are retained in the dialysis cassette, whereas the diffusible molecules cross the membrane according to the concentration gradient. There will therefore be a net displacement of these latter molecules from the most concentrated side to the least concentrated side (principle is shown on Annex 4).

Structural characterization of MGL variants from Brevibacterium aurantiacum and MGL from Arabidopsis thaliana

Several techniques make it possible to gradually decrease the solubility of the protein in order to bring it to a crystalline state. In this project, we used vapor diffusion in two ways for crystallization: manually, or performing automated crystallization using commercial screens.

For the first step of automated crystallization, the sitting drop method has been performed, using various commercial screens: Crystal screen and Crystal screen 2 (Hampton Research, Aliso For manual optimization of the crystallization conditions, another application of the vapour diffusion technique is used with the hanging drop method. A volume of 1 mL of solution containing the precipitating agent (pH buffers, various polyethylene glycols (PEGs), salts, or other precipitants and additives) is deposited in the pits. A volume of 3 µL of protein solution and 3 µL of precipitating solution is deposited on a thin siliconized coverslip. The coverslip is then placed over the container and is hermetically sealed with vacuum grease. Therefore, the drop solution is diluted twice so that vapour diffusion will establish from the drop which is least concentrated in precipitant towards the pit, in order to balance the concentrations. Under favourable conditions, the slow concentration of the protein will lead to crystals formation (Figure 9). This technique was used in 24 wells VDX plates (Hampton Research, CA) for the reproduction of crystals and the gradual improvement of the crystals. When sufficiently large crystals (> 0.01 mm) are obtained, they are mounted on a cryoloop frozen in liquid nitrogen and exposed to X-rays on SOLEIL synchrotron. Collected diffraction data allows the resolution of the 3D structure.

RESULTS

UV-Visible spectra of MGLmut4

Production

The MGL quadruple mutant was transformed in pET-28a (+)-TEV plasmid (figure 8), which contains a histidine tag in the N-terminal position and a cleavage site by the TEV protease for possible removal of this tag, and produced in T7 Express competent E .coli cells. As we can see on the figure 10, the quadruple mutant MGLmut4 protein is well induced, showing an intense band (figure 10, lanes 5-6) whose apparent molecular weight is in accordance with the one calculated from the sequence: 43.3 kDa. It is the major band, which means that MGL is the major protein produced by E. coli. We can also observe some protein contaminants that we will try to eliminate by the ulterior purification steps.

Purification

The cell lysate supernatant was purified using 2 steps of chromatography of 6His-MGLmut4

(histidine-tagged at the N-terminus, 6 histidine residues): affinity chromatography on immobilized Ni 2+ complex, and size exclusion chromatography (SEC) for further purification by removing unwanted small molecules, such as salts or aggregates. We observe 3 peaks with different intensities that may correspond to the purified MGLmut4 (Figure 11). Overall, we observe 3 pink peaks that could reflect PLP bound to the protein with an absorbance at 422 nm on UV-Visible spectrum. The major peak is composed by fractions F11 and F12 for which we observe a high absorbance measured at 280 nm on the UV-Visible spectrum (figure 12).

The spectra of the 3 peaks show an absorbance at 260 nm much lower than that at 280 nm for the fractions F8, F9, F11 and F12. It means that there are therefore very few nucleic acid contaminants in these fractions. About PLP retention, the UV-Visible spectrum indicates that we have little PLP in these fractions showing a very low peaks at 422 nm (figure 12).

RESULTS

16 SDS-PAGE shows a high intensity band at an apparent mass of 43.3 kDa (figure 13, lanes 2-4-5-6) which corresponds to the molecular weight of the MGL protein. The major band of MGL is observed in the F12 fraction, which is coherent with the chromatogram result. We can also observe a small amount of protein contaminants represented by bands that appear blurry and not very intense (figure 13, lanes 2-4-5-6). It means that most of contaminants proteins have been removed after the two purification steps. These protein contaminants suggest that they are aggregates of different proteins including that of MGL. F15 and F16 are probably non-proteic contaminant molecules (not visible on SDS-PAGE).

Crystallization tests

A high-resolution structure of Ba-MGL or of one of its variants would be very useful to determine a rational strategy for enzyme engineering. The crystallization of the wild-type enzyme in the presence of PLP resulted in nice crystals, but they diffracted X-rays only to low resolution (3.6 Å). A possible problem is the weak occupancy of the cofactor PLP in the crystals, due to the too low affinity of the enzyme for its cofactor, and progressive leakage of it during the time (several days) needed for protein crystallization. This probably results in a mixture of Ba-MGL-PLP complex and free Ba-MGL (apo Ba-MGL), unfavourable for crystallization. As the quadruple mutant seems to fix very few PLP, we hoped that it could give crystals of the apo-enzyme. We performed crystallization tests (crystallization conditions are described in Annexes 5 and 6). For that, the purified Histag-MGLmut4 from Brevibacterium aurantiacum, which fixes very little PLP, was concentrated at 11 mg/ml and diafiltered 2 times with a buffer without PLP to facilitate the crystallization of the apo-enzyme.

Crystallization tests showed small needle crystals. This first encouraging result was not followed up due to lack of experimentation time related to Covid-19 pandemy.

II. Production and purification of Ba-MG double mutant M97I+M218T

The double mutant MGLmut2 was produced and purified under the same conditions as those of the quadruple mutant MGLmut4. On the SDS-PAGE gel, we see many proteins but the major band remains that of MGLmut2 after IPTG induction with a molecular mass of approximately 43.3 KDa (figure 14, lane 3).

The chromatogram showing the result of the second step of chromatography purification (SEC) for MGLmut2 shows two peaks between fractions from F11 to F25 (Annex 7). The major peak consists of fractions F13-F14-F15-F16 and the other one is composed of F21-F22-F23.

The UV-visible spectrum of these peaks shows a clear absorbance measured at 280 nm on the UV-Visible spectrum (figure 15). The chromatogram also shows two low peaks at 422 nm, which correspond to the PLP in its complexed form. Therefore, these fractions do not mainly contain functional MGL since they contain little complexed PLP as confirmed on UV-Visible spectrum (figure 15). We note that for these two peaks (F13 to F16 and F21 to F23) the absorbance at 280 nm is higher than the absorbance at 260 nm: there is a satisfying protein / nucleic acid ratio (considering that the absorbance at 260 nm of 1 mg of DNA is 25 times higher than the absorbance at 280 nm of 1 mg of protein, the percent of nucleic acids contaminants in our protein sample should be very low).

SDS-PAGE profile of the different protein fractions F14-F21 from S200 increase size exclusion chromatography shows an apparent intense band with the expected molecular weight of the MGL (43.3 KDa) (figure 14). This confirms the chromatogram result that showed two peaks at F14 and F21 fractions, with a small amount of protein contaminants: most protein contaminants have been removed after the steps of purification.

III. Production and purification of Ba-MGL variants M94I and M94L

In a second step, we looked to methioninases of other species (beyond Pp-MGL). We searched sequence differences in the PLP-binding region (Annex1). The MGL of Citrobacter freundii has a better affinity for PLP than that of Clostridium novyi, the Kd of C. novyi being 23 times higher than that of C. freundii [START_REF] Kulikova | Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi[END_REF]. Among the six residues that participate in binding of PLP, the only sequence difference is M92 from C. novyi, which is replaced by I (isoleucine) in C. freundii. This methionine is conserved in many MGLs including Ba-MGL (M94). Interestingly, Hs-CGL, the human cystathionine gamma lyase, a PLP dependent enzyme, has a sequence similar (~ 40%) to that of Ba-MGL, and is much more stable in human plasma than Ba-MGL and even Pp-MGL. Thus, it was attractive to test Ba-MGL M94I and M94L mutants.

MGL from Brevibacterium aurantiacum variant M94I

The Ba-MGL mutant M94I was produced in T7 Express Competent E .coli cells, purified by

Nickel affinity chromatography as a first step (Figure 16) followed by S200 increase size exclusion chromatography (Figure 17), and analysed by SDS-PAGE. The result showed that this mutant is well expressed in the presence of IPTG with the right molecular weight (43.3 kDa) (Figure 18, lanes 4-5). It is eluted in the fraction F12 with a sufficient purified amount after the two steps of purification (Figure 18, lane 7-8-9).

MGL from Brevibacterium aurantiacum variant M94L

The Ba-MGL mutant M94L was produced and purified by two steps of purification: Firstly, we used Nickel affinity chromatography (Annex 8). The analysis by SDS-PAGE showed that this mutant is well induced in the presence of IPTG, and eluted in the fractions F33 and F42

with a small amount of contaminants after the first step of purification (Figure 19, lanes 8-9).

It was followed by S200 increase size exclusion chromatography and under the same conditions as those of the other tested mutants.

Analysis of UV-visible spectra of Ba-MGL M94I, Ba-MGL M94L and the wild type show that Ba-MGL M94I retains less PLP than that of the wild type. For the other mutant Ba-MGL M94L, the absorbance at 422 nm is much lower than that of the wild type and even negligible.

IV. Production and purification of MGL from Arabidopsis thaliana

As already mentioned in the objectives section, along with Ba-MGL, the MGL of Arabidopsis thaliana (At-MGL) is the only known MGL that exhibits a high activity toward methionine but spares cysteine. In addition, there is no published crystal structure of either of these enzymes, which possess a glycine residue instead of the catalytic cysteine present in other enzymes of this family. The plant enzyme was produced in T7 Express Competent E .coli cells and its purification was undertaken by Nickel affinity chromatography as a first step, results are shown on Annexes 9 and 10.

Expression and purification were checked on SDS-PAGE gel. The protein is poorly expressed in the presence of IPTG, thus the induction is not satisfactory. This is represented by two sparse bands (figure 20, lanes 2-3) with an apparent molecular weight close to the one calculated from the sequence. It is higher than that for the Ba-MGL (43.3 kDa), and in accordance with the calculated 50.51 kDa molecular weight. This is logical because the sequence of the At-MGL is longer than that of Ba-MGL (Annex 1). The analysis of the fractions eluted after nickel affinity chromatography shows a band that hardly appears at a molecular weight of 50.5 kDa (figure 20, lane 6). It means that the protein is eluted, but with a small amount that could be explained by: a poor induction, a washing problem that resulted in the loss of the protein, or it may be due to the remaining of the protein in the cell pellet after the lysis.

In order to improve the induction of At-MGL, we thought of changing the culture medium from LB medium to TB medium which is described as producing higher cell yields than those of LB medium, as already described in the material and methods section. We also decreased the imidazole concentration in the washing buffer to 10 mM in order to eliminate the non-specific fixation of certain E.coli proteins naturally rich in histidine. Moreover, in addition to the cell lysis supernatant, we also analysed the cell lysis pellet to see if it contains the protein. For that, it is washed with washing buffer, centrifuged and its volume adjusted as that of the supernatant.

SDS-PAGE corresponding to the induction in the two LB and TB media in addition to the various purification fractions obtained by Nickel affinity chromatography is presented in figure 21. weight is in accordance with the one calculated from the sequence: 50.5 Kda. Changing the culture medium to TB does not improve the poor induction of the protein. We also observe other protein bands that may correspond to At-MGL oligomers, or to different proteins. On the other hand, we note that the cell lysis pellets do not contains the protein of interest, whatever culture medium is used.

Fractions from Nickel affinity chromatography of the two culture media were concentrated and pooled to be injected for S200 increase size exclusion chromatography. Chromatogram analysis showed a major peak composed by fractions F11-F12-F13 (Annex 11); It contains protein that absorb at 280 nm on UV-Visible spectrum (figure 22), as well as a low absorbance peak at 422 nm which indicates that these fractions contain a little complexed PLP, as was the case for Ba-MGL .

F11-F12-F13 fractions of purified At-MGL, that retains a little PLP cofactor, are concentrated at 5.5 mg/ml. Tests of crystallization with the robot are performed on these fractions in similar conditions as the quadruple mutant. Results did not allow to the formation of crystals.

RESULTS

21

DLS experiments carried out on the pooled and concentrated fractions F11-F12-F13 (5.5 mg/ml, figure 23). It is essentially composed of At-MGL oligomers with a diameter close to 11 nm. We also see larger aggregates with a diameter close to 100-200 nm. Remembering that in DLS experiments the scattering intensity of a particle is proportional to the sixth power of its diameter, the result on Figure 19 indicates that the aggregates are far less abundant than the MGL oligomers. The monomer of At-MGL has a calculated MW of 50.515 kDa. Thus, a tetramer of At-MGL should give, if perfectly spherical, a radius of approximately 9.5 nm.

Taking in account the uncertainty of such a measurement, this is in the range of that of the observed peak. The theoretical Rh calculated from the molecular weight supposed to be a sphere should be approximately 12 nm, which would correspond for a perfect sphere to an octamer.

This difference is rather due to the non-sphericity of the tetramer. In addition, the sample was not centrifuged before DLS measurements, and was conserved at 20°C for a long time, which explains why results are not very precise.

RESULTS

22

V. MGL characterization by dialysis

The enzyme, in the blood, undergoes a depletion of its cofactor. In order to mimic these environmental conditions with a simple in vitro system, the method that was developed is the dialysis to assess the ability of MGL to retain PLP cofactor.

Ba-MGL and Pp-MGL wild types (purified Ba-MGL 17p, Ba-MGL 38p, Pp-MGL 04p

provided by the GTP society, and Pp-MGL from the Shionogi pharmaceutical laboratory) were concentrated at 0,5 mg/ml and 1mg/ml respectively, then dialyzed against a buffer without PLP (described in table 3, Material and Methods). To remove the free PLP which was present in the enzyme buffer solution and which leaked to the dialysis buffer, we renewed the buffer after 2h of dialysis. Analysis of UV-visible spectra at different times shows that concentration of the dialyzed Ba-MGL 17p decreases with time, due to the swelling of dialysis compartment (Figure 24 a). Between t=2h and t=68h, the decrease is due to the withdrawn of 100 µl of sample for the spectrum at t=2h.

In order to compare better the spectra at different times, we have normalized the 280 nm absorbance at the same value for all spectra (Figure 24b). The PLP signal decreases with time, because during dialysis PLP leaks from the sample to the dialysis buffer. Histogram shows that 37 % of PLP remains complexed to the enzyme after 68h of dialysis (figure 25). This result is close to that obtained in 2019 internship (47%) (Figure 6).

For the three other enzyme samples, technical problems occurred: i) values indicated by the spectrophotometer were shifted, ii) the samples were probably too often frozen/thawed, leading to excessive leakage of PLP, iii) PLP was too much exposed to light. Therefore, these results are not shown.

DISCUSSION & PERSPECTIVES

DISCUSSION

MGL is known for nearly half a century as an attractive drug strategy for anti-cancer therapy [START_REF] Sato | Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers[END_REF]. Pp-MGL has been tested by the partner team of Dr.Machover and many other laboratories: it induced growth arrest and death of cancer cells in vitro and in vivo [START_REF] Yoshioka | Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase[END_REF]. However, it has three serious limitations: immunogenicity, cysteine degradation and rapid inactivation in plasma. Another MGL (Ba-MGL) has been identified at INRA Grignon. It has two crucial properties interesting for anticancer therapy: i) it is abundantly present in food and therefore, its likely to comply with human immune tolerance and ii) it specifically degrades methionine, notably sparing cysteine, as already explained.

The studies described in this work aimed to the development of a novel drug (MGL from a non-pathogenic bacterium Brevibacterium aurantiacum) for cancer therapy based on engineered methionine gamma lyase with hight affinity for PLP cofactor. Previously, the first step of engineering used single point mutations. The obtained variants did not retain PLP better than the wild type, contrary to what was expected. In a second step, we tried new mutants with multiple mutations.

Our results establish that double mutant M97I+M218T and quadruple mutant T92S +M97I+

F187Y+M218T are correctly produced and well expressed in E.coli cells. As regards to PLP retention, UV-visible spectra of these two mutants showed that little PLP is complexed. The cofactor binding is not closer to that of Pp-MGL. It is even weaker than that of the wild type.

Therefore, we did not perform dialysis experiments on these variants. In addition, crystallization trials of the quadruple mutant in the absence of PLP produced small needle crystals of the apo-enzyme. In a third step, mutations of a residue that differs between MGLs of C.novyi and C.freundii was performed. Variants were produced, expressed and purified with the idea to dialyze. However, the PLP signal of M94I variant was lower than that of the wild type; that of M94L was even negligible.

In parallel, we studied MGL enzyme from the model plant Arabidopsis thaliana. Most MGLs have a conserved catalytic cysteine (Cys 116 in Pp-MGL). This cysteine is not conserved in other PLP dependent enzymes, and is substituted by glycine or proline in cystathionine γ-lyase, cystathionine β-lyase, and cystathionine β-synthase, and thus was previously suggested to be involved in the recognition and γ-elimination of methionine [START_REF] Sato | Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers[END_REF]. Ba-MGL also has a glycine at the position equivalent to that of Cys 116 in Pp-MGL, and it is the only known bacterial methioninase that does not degrade cysteine. These remarkable properties are also encountered in At-MGL. In order to understand the basics of MGL specificity, At-MGL was produced and purified to homogeneity. Its PLP signal was significant. Crystallization tests were initiated but did not provide crystals yet.

PERSPECTIVES

It will be necessary to perform enzymes activity tests in order to ensure that mutations and added HisTags do not affect activity and specificity of the enzyme. As explained above, cysteine degradation by MGL have several undesirable effects.

In order to progress with Ba-MGL engineering, we looked for new mutation strategies that do not target the PLP binding residues. Based on the sequence alignment of different bacterial MGLs, At-MGL and Hs-MGL, there is a loop extension (W48-L55) in Ba-MGL which is not present in other MGL enzymes (Figure 26). This extension of the loop in Ba-MGL may alter the stability of the enzyme and prevent its crystallization. Thus, it will be attractive to design a mutant without this loop extension in order to solve the enzyme structure, and to improve its stability.

The sequence alignment shows conserved residues that flank this loop: G42 in bacterial MGLs, plus T33 and Y63 in all aligned MGLs including Hs-MGL. This suggests various possibilities to design Ba-MGL mutants that could be more stable.

Finally, a new crystallization strategy is proposed. We hypothesize that previously obtained wild type Ba-MGL crystals diffracted X-rays to limited resolution because of the heterogeneity of the enzyme preparation used: As PLP is weakly bound, there might be a mixture of methioninase alone and bound to PLP. In this context, new mutant in which the lysine binding the PLP is replaced by an alanine was ordered to crystallize Ba-MGL or At-MGL apo-enzymes.

A high-resolution crystal structure would be very helpful for enzyme engineering.

ANNEXES ix

Annex 6-Example of a series of manual tests Pits 1 ml, drop 6 µl (3 µl of the protein + 3 µl of the well) Solutions : MES 0.1 M pH 6.5 ; PEG 4000 at 40 % w/v in MES buffer 0.1M pH 6.5 ; NaCl 2 M in MES buffer 0.1 M pH 6.5.

N°

Figure 2 :

 2 Figure 2: MGL catalysed reaction, here the α, γ-elimination and γ-replacement of L-methionine.

Figure 3 :

 3 Figure 3: The overall crystal structure of MGL from Pseudomonas putida (Cartoon representation; Protein Data Bank access code 1PG8). Each subunit of the homotetramer is indicated in green, yellow, magenta and cyan. PLP is indicated in blue (ball-and-stick representation).

Figure 5 :

 5 Figure 5: Residues close to PLP that were targeted by site directed mutagenesis. Left: Superposition of crystal structure of Pp-MGL (5dx5MGL-PLP, C sporogenes) in grey and a 3D model of Ba-MGL (provided by the I-Tasser program) in yellow, showing the two residues (F187 and the corresponding Y189) that are close to PLP (green). Right: sequence alignment of residues close to PLP that differ between Ba-MGL and Pp-MGL.

Figure 6 :

 6 Figure 6: Percentage of initial PLP present after 65 hours of dialysis against buffer without PLP (evaluated by absorbance at 422 nm) for different MGLs (unpublished results obtained in previous 2019 internship).

Figure 8 :

 8 Figure 8: Schematic representation of the construction commissioned from GenScript

  produced in T7 Express Competent E. coli strain, transformed by pET-28a (+) -TEV vector from GenScript (Netherlands) in LB or TB medium supplemented with kanamycin 100 mg/ml in order to promote the production of MGL protein. Cells are seeded at 0.1 OD600nm cell density and incubated overnight at 37°C at 200 rpm. Then the cultures are incubated at 37 °C and monitored for active growth by continually measuring optical density at 600 nm until obtaining an OD600nm of 0.6. Then the protein expression is induced with IPTG at 0.1 mM. Cultures are then incubated overnight at 18°C at 200 rpm in order to slowdown bacteria generation time, allowing more time for the expression of chaperone proteins which help the folding of the expressed protein. Cells are harvested by centrifugation at 6000 g at 4 °C for 10 min.

  -Trap column: Immobilized Metal Affinity Chromatography (IMAC) Cells pellets are re-suspended into Buffer A (Table3) in the presence of an anti-protease cocktail (Roche tablet). The cells were lysed with a ONE Shot cell disruptor (Constant Systems Ltd, UK) operated at a pressure of 1.96 Kbar. Cellular debris were removed by centrifugation at 12 000 g for 15 min at 4 °C.

  Viejo, CA), PEGII suite from Nextal (Qiagen, France), Morpheus screen (Molecular dimensions limited, Cambridge, UK), allowing to test many crystallization conditions. The sitting drop (1 µl) is deposited on a support located close to the well (150 µl). The advantages of the automated crystallization using commercial screens are protein saving, time saving and reproducibility. The commercial screens are made up of conditions that have already enabled protein crystals to be obtained and offer the advantage of exploring a wide variety of precipitating agents (salts, polymers, organic solvents), pH, and additives. These tests were carried out in 96 wells boxes (CrystalQuick plates, NatX-ray, Saint Martin d'Hères, France) in which each of the 96 wells contains 150 µl of a given crystallization condition (precipitant) in equilibrium with the 1 µl drop containing the protein solution.

Figure 9 :

 9 Figure 9: Principle of crystallization by diffusion of vapour phase with suspended drop. A large volume of precipitating agent solution (1 ml) is placed in the well. The crystallization drop is made up of a 1/1 ratio of protein solution and precipitating agent solution (3 µL each). Due to the difference in the concentration of precipitating agent between the drop and the well, a diffusion of water(vapour) from the drop to the reservoir will occur. Thus, the concentration of protein and precipitating agent in the drop will gradually increase until reaching a state of equilibrium

Figure 10 :

 10 Figure 10: SDS-Polyacrylamide Gel Electrophoresis showing induction efficiency for MGLmut4. MW: molecular weight markers (Mark12).

Figure 11 :Figure 12 :

 1112 Figure 11: SEC chromatography of 5 ml of At-MGL from cultures with LB medium.

Figure 13 :

 13 Figure 13: SDS-PAGE of MGLmut4 after 2 steps of purification: selected fractions obtained after size exclusion chromatography on S200 increase column.

Figure 14 :

 14 Figure 14: SDS-Polyacrylamide Gel Electrophoresis showing induction efficiency and S200 increase size chromatography results for MGLmut2. 1: molecular weight; 2: Cell pellet lysate before IPTG induction; 3: Cell pellet lysate after IPTG induction; 4: F14 fraction of the S200 increase size exclusion chromatography; 5: F21 on S200 increase column.

Figure 15 :

 15 Figure 15: UV-Visible spectra of F16-F23 from size exclusion chromatography on S200 increase column

Figure 16 :

 16 Figure 16: Immobilized Metal affinity Chromatography of 5 ml of Ba-MGL mutant M94I.

Figure 17 :

 17 Figure 17: Chromatogram of S00 increase size exclusion chromatography of 1 ml purified Ba-MGL mutant M94I from Nickel affinity chromatography

Figure 18 :

 18 Figure 18: SDS-Polyacrylamide Gel Electrophoresis showing induction efficiency and purification results of MGL Brevibacterium aurantiacum mutant M94I (Ba-MGL M94I). 1: molecular weight markers; 2-3: Cell lysate supernatant before IPTG induction; 4-5: After IPTG induction; 6 HisTrap F7; 7 HisTrap F36; 8 S200 F9; 9 S200 F12.

Figure 19 :

 19 Figure 19: SDS-Polyacrylamide Gel Electrophoresis showing induction efficiency and purification results after HisTrap purification of MGL Brevibacterium aurantiacum mutant M94L (Ba-MGL M94L). 1: molecular weight markers; 2-3: Cell lysate supernatant beforeIPTG induction;[4][5] 6 HisTrap F12; 7 HisTrap F18; 8 HisTrap F33; 9 HisTrap F42; 10 HisTrap F80.

Figure 20 :

 20 Figure 20: SDS-Polyacrylamide Gel Electrophoresis showing induction efficiency and purification of At-MGL. 1: molecular weight markers; 2: cell lysate before IPTG induction; 3: cell lysate after IPTG induction; 4,5,6: fractions of the Nickel Affinity Chromatography.

Figure 21 :

 21 Figure 21: SDS-Polyacrylamide Gel Electrophoresis for At-MGL, showing (a) induction efficiency and (At-MGL) (b) purification results. 1: molecular weight markers; 2: Cell lysate supernatant before IPTG induction, obtained in LB; 3: After IPTG induction; 4: LB pellet; 5: Before IPTG induction; 6: After IPTG induction; 7: TB pellet. b) 1: molecular weight markers; 2 HisTrap F4; 3 HisTrap F7; 4 HisTrap F15; 5 HisTrap F31; 6 HisTrap F44; 7 HisTrap F3; 8 HisTrap F13; 9 HisTrap F28; 10 HisTrap F41

Figure 23 :

 23 Figure 23: DLS of fractions F11-F12-F13 of the S200 increase size exclusion chromatography (5 different measurements of the same sample superimposed).

Figure 24 :

 24 Figure 24 : Dialysis results for Ba-MGL17p. a) raw spectra observed at three different times during dialysis. b) spectra observed during dialysis normalized to the same absorbance level at 280 nm.

Figure 25 :

 25 Figure 25: Histogram of Ba-MGL17p (wild-type enzyme without HisTag, external supplier) before and after dialysis showing the percentage of initial PLP.

Figure 26 :

 26 Figure 26: The extended loop of Ba-MGL. Upper part sequence alignment. Lower part Superposition of the crystal structure of Pp-MGL (5x2w) in purple and a 3D model of Ba-MGL (I-Tasser) in green, showing extension loop of Ba-MGL (W48-L55) in red and conserved residues G42 and Y63. Other subunits of Pp-MGL are indicated in cyan. PLP from Pp-MGL structure is indicated in orange.

  P A I D L S T T N P V N D V A T G G D S Y E W L A T G H T L K D G D SA V Y Q R L W Q P G V A R F E T A L A G L E 82 Pp-MGL -L V P P V Y Q T A T F T F P T V E Y G A A C F A --------G E Q A G H F Y S R I S N P T L N L L E A R M A S L E 78 Cf-MGL -L S T P I F Q T S T F V F D S A E Q G A A R F A --------L E E S G Y I Y T R L G N P T T D A L E K K L A V L E 77 Cn-MGL -L A T P I F Q T S T F I F D S A E Q G G N R F A --------G K E D G Y I Y S R I S N P T V A E I E R K I A L L E 80 At-MGL -V N M S I E A S A T F T V M E P D T ----M R R M F T G E L G P D N D F Y V Y S R H F N P T V L N L S R Q M A A L E 114 Hs-CGL A V V P P I S L S T T F K Q --------G A P --------G Q H S G F E Y S R S G N P T R N C L E K A V A A V N D V A T G G D S Y E W L A T G H T L K D G D S A V Y T F T F P T V E Y G A A C F A --------G E Q A G H F Y T F V F D S A E Q G A A R F A --------L E E S G Y I Y T F I F D S A E Q G G N R F A --------G K E D G Y I Y T F T V M E P D T ----M R R M F T G E L G P D N D F Y V Y T F K Q --------G A P --------G Q H S G F E Y N 48 55 43

Table 1 :

 1 Specificity of MGL from Brevibacterium aurantiacum for Methionine.[START_REF] Machover | Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination[END_REF]

Table 2 :

 2 Cytotoxicity

of MGL-BL929 in human cells

[START_REF] Machover | Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination[END_REF]

Figure 4: A) Enzymatic activity of native MGL-BL929 (Ba-MGL) incubated for 8 hours was assayed at 37°C under four different conditions. Conditions were (I) phosphate-buffered saline; (II) PBS supplemented with human serum albumin at 40 g/l; (III) pure human plasma; and (IV) pure rat plasma. Measurement of enzyme activity was performed without additional PLP

Production and purification of Brevibacterium. Aurantiacum MGL quadruple mutant T92S+M97I+F187Y+M218T

							RESULTS
		RESULTS
	Abs I.
	Absorbance at 280 nm greater than
	absorbance at 260 nm: small amount
	of nucleic acid contaminants
			Absorbance at 422 nm: little PLP is
			complexed to the protein
							λ nm
	F7	F8	F11	F12	F14	F15	F16
							15

  Annex 11-Chromatogram of S200 increase size exclusion chromatography of 1 ml purified and concentrated At-MGL obtained from selected Nickel affinity eluted fractions.

	NaCl 2 M 300 µl 300 300 300 300 300 Solutions: Trisodium Citrate 0.1 M pH 5,6 ; PEG 4000 at 40 % in citrate buffer pH 5,6 ; PEG 4000 40% MES buffer 6.5 M µl % µl 1 0.6 450 18 250 2 0.6 500 20 200 3 0.6 525 21 175 4 0.6 550 22 150 5 0.6 625 25 75 6 0.6 675 27 25 (NH4)2SO4 1 M in citrate buffer pH 5,6. N° PEG 4000 40 % (NH4)2SO4 Trisodium buffer µl % µl M µl 7 400 16 200 0.2 400 8 450 18 200 0.2 350 9 500 20 200 0.2 300 10 550 22 200 0.2 250 11 575 23 200 0.2 225 12 625 25 200 0.2 ANNEXES
	xii

Pp-MGL as an anticancer drug was abandoned.

Ba-MGL Pp-MGL Ba-MGL Ba-MGL Pp-MGL Pp-MGL

ANNEXES

Here example of the PEGII,1 suite screen from Nextal.

With the robot : Pits 150 µl, drop 1 µl (0.5 µl protein and 0.5 µl pit)

N°

Engineering methionine gamma lyase from the cheese bacterium Brevibacterium aurantiacum for cancer therapy

The cancer incidence is increasing worldwide, where the disease is the second cause of mortality. The methionine depletion induced by the methionine γ-lyase enzyme (MGL, methioninase) is a promising strategy against cancer, whose cells are more dependent on methionine than normal cells. MGL is a pyridoxal phosphate (PLP) dependent enzyme that degrades methionine. It is present in bacteria, in plants, but absent in mammals. This work studied the methioninase from Brevibacterium aurantiacum (Ba-MGL). This enzyme from a cheese bacterium should comply with human immune tolerance. In addition, it has the advantage to be specific of methionine, sparing cysteine whose depletion could induce undesirable side effects. However, it is quickly inactivated in human plasma, due to PLP leakage. The aim was to improve enzymatic engineering the affinity of Ba-MGL for PLP, in order to extend its activity in human blood so that it could be used in anticancer therapy. Quadruple (M97I+M218T+T92S+F187Y) and double (M97I+M218T) mutants, designed to mimic the MGL from Pseudomonas putida, were produced in sufficient quantities and purified. According to UV-visible spectra, these mutants did not retain enough PLP, as was initially expected. Thus, they were not assayed for dialysis. A second series of variants (Ba-MGL M94L and M94I), based on sequence comparison with various bacterial MGLs and human cystathionine γ-lyase, were produced and purified for dialysis assays. In parallel, we studied the MGL from the model plant Arabidopsis thaliana (At-MGL), which, as Ba-MGL, does not degrade cysteine. Crystallization tests were performed on the quadruple mutant and At-MGL. We obtained first needle-shaped crystals. Overall, five variants were successfully expressed and purified. Our results subsequently obtained show the limits of designing enzyme variants merely from sequence and 3D structure comparisons. In cooperation with a bioinformatics laboratory, new designed mutations are underway to improve stability of Ba-MGL. Crystallization trials of variants from Ba-MGL and At-MGL are also underway, in order to solve the structure of MGL, which would help new engineering strategies.


Sylvain Grellet -Brgm

OGC HYDRO DOMAIN WORKING GROUP -and WaterML 2.0 suite of standards

•o••

  Joint OGC -World Meteorological Organization (OMM / WMO) group ○ Memorandum of Understanding between OGC -WMO • Target: water data standards => WaterML 2.0 suite : https://www.ogc.org/standard/waterml/ • Organizing Interoperability Experiments -(IEs) focused on the water sub-domains • Entry point https://external.ogc.org/twiki_public/HydrologyDWG/WebHome • Chairs o Tony Boston (Australia) o Silvano Pecora (Italy) -WMO Liaison o David Blodgett (USA) Iterative Development http://www.opengeospatial.org/projects/groups/hydrology Group dynamics • A long history of joint activities o -Earth Systems Science Domain Working Group (DWG) o -Hydrology DWG o -Groundwater Interoperability Experiment (IE) o -Water Information Services Concept Development Study o -Surface Water Interoperability IE o -Hydrology Forecasting IE o -Climate-Hydrology Information Sharing Pilot o -GroundWater IE2 o -Water ML 2.0 Standards Working Group (SWG) o -Hydrographic Features SWG o -Research Data Alliance Global Water Information IG (Hydro DWG sister group) o -Groundwater SWG o -Geoscience DWG o -Environmental Linked Features IE (ELFIE) o -Borehole IE o -Second ELFIE (SELFIE) o -Water Quality IE => on going o NR-Can, GSC, USGS, US EPA, CUASHI, SDSC, BRGM, UK CEH, DELTARES, GRDC, BaFG, Univ Tartu, Fraunhofer IOSB, DataCove, NIWA/LAWA (NZ), Federation University (Australia), BoM Australia, CSIRO, Open and documented o implemented in WMO Information System and in many organizations : UNESCO, USGS, US EPA, NrCan, NIWA, BRGM, etc… o And in opensource tools : CUASHI Hydro-Server, Kisters, 52°N etc… o Updated with a regular contribution from projects involving Hydro DWG partners 6 //portal.ogc.org/files/?artifact_id=57222 WaterML2: Part 1 -Timeseries Harmonization Contributed to the birth of OGC TimeSeries ML WaterML2: Part 1 adoption• WaterML2 announcement regarding US national strategy for civil earth observations (2013) o http://www.opengeospatial.org/pressroom/pressreleases/1831 • US Federal Geographic Data Committee (FGDC) endorses WaterML2 (2014) o http://www.fgdc.gov/standards/news/WaterML • WaterML2 recommended in EU legislation on data sharing (2013) => INSPIRE o http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2013:331:0001:0267:EN:PDF • USGS implements WaterML2 in Water Information System (2014) o http://help.waterdata.usgs.gov/news/april-10-2014 • BoM (Bureau of Meteorology) supports WaterML2 via Water Data Online (2017) o http://www.bom.gov.au/waterdata/ • WMO implements it natively in its Information System • And others …WaterML2: Part 2 -Ratings, Gaugings and Sections • Encodes rating conversions (e.g. stage to discharge), gauging observations and river cross • https://docs.ogc.org/is/15-018r2/15-018r2.html WaterML2: Part 3 -Surface Hydrology Features • Conceptual model describing surface water hydrologic features such as rivers, lakes, catchments• https://docs.ogc.org/is/14-111r6/14-111r6.Conceptual model describing surface water hydrologic features such as rivers, lakes, catchments• https://docs.ogc.org/is/14-111r6/14-111r6.htmlWaterML2: Part 4 -GroundWater Markup Language 2 (GWML2)• Conceptual and logical model describing surface water hydrologic features such as rivers, lakes, catchments• https://docs.ogc.org/is/16-032r2/16-032r2.html• Hydrogeological units, Conceptual and logical model describing surface water hydrologic features such as rivers, lakes, catchments• https://docs.ogc.org/is/16-032r2/16-032r2.html• WaterML-WQ -an O&M and WaterML 2.0 profile for water quality data• https://docs.ogc.org/bp/14-003/14-003.html • Scope o Observations & Measurements and WaterML2 -Part 1 specialisation for time series of chemical concentrations o Beginning of work on 'Water quality' vocabularies based on QUDT, ChEBI, NVS P01, S06 I-ADOPT ⇒ Currently being updated by the running Water Quality Interoperability Experiment WaterML2: Part 5 -Water Quality Best Practice • OGC Water Quality Interoperability Experiment : https://github.com/opengeospatial/WaterQualityIE o Co-Lead : BRGM, Internet of Water o Membres: USGS, US EPA, WMO, UNESCO, BfG, BRGM, OFB -Pôle INSIDE, University of Tartu, Australia & NZ participants o Scope o Sharing water quality data (surface or ground water) between organisations o Types of water data : in-situ sensors, one time measurements, samples (and sub-samples), lab analysis, biodiversity observations and associated vocabularies o 'OGC Baseline' o Semantics: "Observations, Measurements & Samples" o API : SensorThings API, OGC API -Features La Dordogne -Février La Dordogne -Pont de Lalinde -Août Merci s.grellet@brgm.fr

Thanks to

Tony Boston tony.boston@anu.edu.au Blodgett, David L dblodgett@usgs.gov Boisvert, Eric eric.boisvert@NRCan-RNCan.gc.ca


pour le cas français

Protection de l'emploi et formation des travailleurs en n de carrière : l'expérience française de la contribution Delalande

Résumé

Ce papier vise à estimer à partir de données françaises l'eet d'une protection de l'emploi spécique à l'âge sur le taux de formation des travailleurs en n de carrière. Nous nous appuyons sur l'expérience française de la taxe Delalande, qui de 1987 à 2008, était due par toute rme qui licenciait un travailleur senior. Nous exploitons un changement de barème de cette taxe en 1999 qui n'a concerné que les entreprises de plus de 50 salariés et nous adoptons une approche par double diérence combinée avec une méthode d'appariement sur le score de propension. Nous montrons que cette réforme a fortement augmenté le taux de formation des travailleurs âgés de 45-49 ans, soit juste avant l'âge seuil de la taxe Delalande.

Travailleurs en n de carrière, protection de l'emploi, formation continue JEL Classication : J14, J24, J26 1 Introduction Depuis 40 ans, la majorité des pays de l'OCDE ont connu une très forte chute des taux d'emploi des 55-64 ans. Ainsi, alors que l'âge moyen de cessation d'activité pour les hommes était en 1977 de 67 ans au sein de l'OCDE, il n'était plus que de 62,5 ans en 2007. Face à ce phénomène qui menace à terme l'équilibre nancier des systèmes de retraite, de nombreux pays tels que la France mais aussi la Belgique, la Finlande ou l'Autriche ont renforcé la protection de l'emploi pour les travailleurs âgés, soit sous forme d'une taxe sur le licenciement plus élevée ou de délais de préavis plus importants pour les rmes (OCDE, 2006). De nombreuses études révèlent que ces dispositifs ont bien réduit le taux de sépération parmi les seniors concernés 1 .

Cette observation soulève la question suivante : en incitant les employeurs à instaurer une relation de plus long terme avec leurs travailleurs âgés, dans quelle mesure cette protection de l'emploi spécique a permis d'augmenter leur taux de formation ? Cette question est d'autant plus pertinente que cette catégorie de travailleurs présente un important désavantage comparatif concernant l'accès à la formation continue [START_REF] Behaghel | Changement technologique et formation tout au long de la vie[END_REF], compte-tenu d'une part de leur faible horizon de carrière [START_REF] Montizaan | The eects of pension rights and retirement age on training participation : Evidence from a natural experiment[END_REF][START_REF] Khaskhoussi | La proximité de l'âge de la retraite. Quels eets sur l'accumulation du capital humain et sur la recherche d'emploi ?[END_REF] et d'autre part de leur faible capacité à s'adapter aux changements organisationnels ou technologiques 2 . Ainsi, en France la probabilité qu'un senior bénécie d'une formation est de 20% inférieure à celle pour un travailleur âgé entre 30 et 34 ans [START_REF] Lainé | Métier, accès à la formation continue et mobilité professionnelle[END_REF].

Dans la littérature, la relation entre protection de l'emploi et formation a déjà été explorée. Certains papiers mettent l'accent sur les eets sur l'investissement en formation des travailleurs, en soulignant qu'une forte protection de l'emploi encourage les oreurs de travail à accumuler du capital humain spécique [START_REF] Wasmer | General versus specic skills in labor markets with search frictions and ring costs[END_REF][START_REF] Belot | Welfare-improving employment protection[END_REF]. D'autres s'intéressent davantage aux incitations qu'ont les rmes à orir des formations à leurs salariés. Ainsi [START_REF] Acemoglu | Training and innovation in an imperfect labour market[END_REF] et [START_REF] Lechthaler | The interaction of ring costs and rm training[END_REF] montrent qu'une forte protection de l'emploi incite les employeurs à investir davantage dans le capital humain général de leurs travailleurs. De même, [START_REF] Chéron | Inecient Job Destructions and Training with Holdup[END_REF] mettent en évidence un eet de complémentarité entre protection de l'emploi et investissement en capital humain spécique. L'idée est qu'en cas de forte protection de l'emploi, l'entreprise anticipant un choc de productivité sur l'un de ses postes va investir dans de la formation pour rendre ce poste moins vulnérable aux chocs et ainsi éviter de payer le coût de séparation.

La contribution de ce papier est essentiellement empirique. En nous appuyant sur l'expérience française, nous cherchons à estimer l'eet d'une forme de protection de l'emploi spécique à l'âge sur le taux de formation des travailleurs en n de carrière, c'est à dire âgés de 45 ans et plus 3 . Depuis 1987 et jusqu'à 2008 en France, toute rme qui licenciait un travailleur âgé devait payer une taxe exprimée en termes de mois de salaires du travailleur et croissante avec l'âge de celui-ci. Cette taxe, connue sous le nom de Contribution Delalande, a connu de nombreuses modications. Ainsi, en 1992, l'âge seuil d'éligibilité à ce dispositif pour un travailleur est passé de 55 ans à 50 ans, et toutes les rmes qui embauchaient des travailleurs de 50 ans ou plus en ont été éxonérées. De plus, en 1999, cette taxe a augmenté mais seulement pour les rmes de 50 salariés ou plus. Le tableau 1 permet d'illustrer les diérents changements de barème qu'a connus la taxe Delalande jusqu'à sa suppression en 2. L'enquête EGS50+ de la DARES menée en 2008 révèle que pour plus d'un tiers des employeurs interrogés, la principale limite d'un senior vient de sa faible adaptation au changement 3. Cette dénition large des travailleurs en n de carrière nous permet de considérer des travailleurs situés au-delà et d'autres en deça de l'âge seuil de la taxe. En eet, la littérature théorique traitant de l'eet d'une protection de l'emploi spécique à l'âge sur l'emploi [START_REF] Behaghel | La protection de l'emploi des travailleurs Âgés en France : une étude de la contribution Delalande[END_REF][START_REF] Chéron | Age-dependent employment protection[END_REF] montre que ce dispositif aecte diéremment l'emploi pour ces deux classes d'âge de travailleurs.

2008.

Tableau 1 Barème de la Contribution Delalande en fonction de la taille de l'entreprise et de l'âge du travailleur licencié (en mois de salaire bruts)

Age du travailleur 50 51 52 53 54 55 56-57 58 59

Janvier 1993-Decembre 1998 Firmes de toutes tailles 1 1 2 2 4 5 6 6 6

Depuis janvier 1999 Firmes de 50 salariés ou plus 2 3 5 6 8 10 12 10 8 Firmes de moins de 50 salariés 1 1 2 2 4 5 6 6 6 Source : [START_REF] Behaghel | The perverse eects of partial employment protection reform : The case of French older workers[END_REF], articles de loi.

Notre stratégie empirique consiste à exploiter le changement de barème de la Contribution Delalande à partir de janvier 1999, en protant du fait que la hausse de la taxe n'a concerné qu'une partie des rmes en fonction de leur taille 4 . Par conséquent, nous pouvons adopter une approche par double diérence, pour comparer l'évolution des taux de formation des travailleurs en n de carrière dans les petites entreprises de moins de 50 salariés entre 1998 et 1999 à celle observée dans les entreprises de 50 salariés ou plus (concernées par la hausse de la taxe) sur la même période. Néanmoins, cette méthode ne peut fournir des résultats robustes que sous l'hypothèse que les deux groupes d'individus (ceux travaillant dans les grandes entreprises et ceux travaillant dans les petites entreprises) sont comparables. Or, comme nous le verrons plus loin, les incitations à former pour les entreprises dièrent fortement selon leur taille, notamment du fait de l'existence de plans de formation dans les rmes de grande taille. Par conséquent, dans une deuxième étape, nous combinerons notre estimation par double diérence avec une stratégie d'appariement sur le score de propension, an de contrôler les diérences de caractéristiques observées entre les deux groupes d'individus.

L'article est organisé comme suit : après avoir présenté les données utilisées et fourni quelques statistiques descriptives dans la section 2, nous présenterons la méthode économétrique adoptée dans une section 3 puis nous exposerons et discuterons les principaux résultats dans la section 4. La section 5 conclut. Pour mesurer l'eet de la Contribution Delalande sur les incitations qu'ont les rmes à former leurs salariés, nous utilisons deux bases de données complémentaires. La première est l'enquête "Formation Continue" menée en mars 2000 par l'INSEE auprès de 28667 individus. Cette base de données nous fournit une information détaillée sur les épisodes de formation qu'a connus l'interrogé sur trois périodes principales : i) de sa sortie d'études jusqu'à février 1998, ii) de Mars 1998 à Décembre 1998 et iii) de janvier 1999 à mars 2000. L'enquête indique également le mode de nancement de ces formations. Nous ne considérons que les formations nancées par l'employeur, étant donné que la Contribution Delalande cible ces derniers. Au regard des données, ces épisodes concernent environ 80% des formations déclarées par les travailleurs en n de carrière en 1998 ou en 1999. De plus, si nous suivons le cadre théorique proposé par [START_REF] Chéron | Inecient Job Destructions and Training with Holdup[END_REF], nous cherchons à tester une complémentarité entre la protection de l'emploi et les incitations des rmes à former leurs travailleurs. Ainsi, nous examinons si le durcissement du barème de la contribution Delalande a pu se traduire par une augmentation du taux de formation des salariés dans le but de s'adapter à des changements survenus dans leur environnement de travail. De tels changements constituent en eet des chocs qui peuvent aboutir à des destructions d'emploi. Or, suite à la réforme, le licenciement d'un travailleur âgé de 50 ans et plus est devenu plus coûteux dans les rmes de 50 salariés et plus. Par conséquent, nous pouvons supposer qu'elles vont davantage former leurs travailleurs en n de carrière pour améliorer leur capacité d'adaptation à ces changements et ainsi économiser les coûts de séparation devenus plus élevés. Nous ne considérons donc pour notre étude que les formations dont le but est de s'adapter au poste de travail (soit plus de 80% des formations nancées par l'employeur et ciblées sur les travailleurs âgés) 5 .

Nous apparions ces données avec les deux vagues de l'enquête Emploi pour les années 1998 et 1999. Cette enquête concerne 135000 personnes et constitue un panel rotatif pour lequel un tiers de l'échantillon est renouvelé chaque année. Par conséquent, nous avons pour les deux vagues des informations détaillées sur deux tiers de l'échantillon de 1998. Ces informations concernent la situation socio-démographique de l'interrogé ainsi que les caractéristiques de son poste et de l'entreprise dans laquelle il est embauché, dans le cas où il est en emploi.

5. En guise de test de robustesse, nous avons refait les estimations en n'opérant pas de sélection sur le motif de la formation. Les résultats sont alors similaires à ceux obtenus lorsque l'adaptation à l'emploi est retenue comme seul objectif de la formation 2.2 Sélection de l'échantillon et statistiques descriptives Nous dénissons notre échantillon de la manière suivante. D'abord, nous excluons les agriculteurs ainsi que les individus occupant une profession libérale et ceux employés dans le secteur public. Pour atténuer l'eet de sélection, nous restreignons notre échantillon aux interrogés âgés entre 45 et 54 ans en mars 1998 6 . De plus, il est aujourd'hui bien reconnu que la distance par rapport à l'âge auquel l'individu peut liquider une pension à taux plein 7 est un facteur important pour comprendre les taux de formation des travailleurs âgés [START_REF] Montizaan | The eects of pension rights and retirement age on training participation : Evidence from a natural experiment[END_REF][START_REF] Khaskhoussi | La proximité de l'âge de la retraite. Quels eets sur l'accumulation du capital humain et sur la recherche d'emploi ?[END_REF]. En eet, la décision de formation reposant sur un arbitrage coût/bénéce espéré, plus le départ à la retraite est lointain, plus la période d'amortissement du coût de formation sera longue, et donc plus le bénéce net sera élevé, ce qui in ne rend l'entreprise plus disposée à former son salarié. De ce fait, il est important d'inclure la variable de distance à la retraite parmi les variables explicatives du taux de formation. Toutefois et à la diérence des périodes de chômage, la distance à la retraite à taux plein telle que nous la calculons ne permet pas de prendre en compte les interruptions de carrière liées aux congés maternité et congés parental d'éducation. Or, ceux-ci sont incontestablement majoritairement pris par les femmes et peuvent constituer un biais important dans la mesure de la distance à la retraite pour elles du fait de leur longue durée . En particulier, ne pas les prendre en compte est fortement susceptible de sous-estimer la distance réelle et donc de biaiser les estimations. Il nous semble donc plus raisonnable de mener l'analyse de la réforme du barème de la contribution Delalande sur les pratiques de formation en considérant un échantillon uniquement constitué d'hommes. Toutefois, nous étudierons dans une section ?? l'eet du changement de barème de la Contribution Delalande sur les taux de formation des femmes en n de carrière.

En supprimant les quelques valeurs manquantes ou aberrantes et en sélectionnant uniquement les individus en emploi en 1998 et 1999 8 , nous obtenons un panel cylindré avec 1000 observations pour chaque vague. Pour la suite de l'étude, nous considèrerons que la période de mars 1998 à décembre 1998 constitue la période préréforme et celle qui s'étend entre mars 1999 et décembre 1999 représente la période post-réforme 9 .

6. En 2000 en France, environ 50% des 55-59 étaient déjà hors de l'emploi 7. En nous appuyant sur les travaux d [START_REF] Hairault | Distance to retirement and older workers' employment : The case for delaying the retirement age[END_REF], qui soulignent l'importance de la distance par rapport à l'âge du taux plein dans l'étude des comportements des travailleurs âgés, nous approximons l'âge du taux plein en fonction de l'âge de sortie d'études et du nombre de trimestres requis pour atteindre le taux plein. Si une personne de moins de 60 ans a déjà cotisé susamment d'années pour prétendre à une pension à taux plein, alors on considère que l'âge du taux plein est égal à l'âge légal, soit 60 ans pour la période de notre étude.

8. Cette dernière sélection peut sembler un peu forte mais les données montrent que moins de 5% des individus employés en 1998 ne le sont plus en 1999. 9. Même si on observe la formation des travailleurs entre janvier 1999 et mars 2000 dans l'enquête, nous préférons nous restreindre aux épisodes de formation survenus entre mars et décembre Quelques statistiques descriptives sur notre échantillon sont fournies dans le tableau 3 en annexe. Le plus important est d'examiner les diérences entre les travailleurs employés dans les rmes de 50 salariés ou plus et ceux employés dans les rmes de moins de 50 salariés. Nous remarquons que la probabilité qu'une rme propose des plans de formation est bien plus élevée dans les grandes rmes que dans les petites 10 . De plus, l'ancienneté des travailleurs est plus importante dans les grandes rmes. Enn, il semble que la composition sectorielle dière fortement selon la taille de la rme. Tandis que le secteur de la construction ne représente que 6% des travailleurs dans les grandes entreprises, il comprend environ 20% des salariés des petites entreprises. A l'inverse, la proportion d'individus travaillant dans l'industrie est plus importante pour les grandes entreprises. Enn, concernant la distribution des salaires, les travailleurs employés dans les grandes rmes sont en moyenne mieux payés que ceux des petites rmes.

Par conséquent, ces deux groupes d'individus ne sont pas comparables. Nous devons donc estimer l'eet causal du changement de barème de la Contribution Delalande sur le taux de formation des travailleurs en n de carrière, en tenant compte des diérences de distribution de certaines variables observables entre les deux groupes. Dans ce but, nous combinons une approche par double diérence avec une méthode d'appariement sur le score de propension pour calculer l'estimateur de double diérence conditionnelle [START_REF] Heckman | Characterizing selection bias using experimental data[END_REF].

3 La méthode économétrique

L'approche par double diérence

Le but de cette étude est de mesurer l'impact d'un renforcement de la protection de l'emploi des travailleurs âgés sur les incitations des rmes à former leurs salariés. Nous exploitons une discontinuité dans la réforme de la taxe Delalande : en 1999, la loi conduit à une augmentation de la taxe pour les rmes employant 50 salariés ou plus, tandis que le niveau de la taxe pour les plus petites entreprises restait inchangé. En nous appuyant sur la littérature standard 11 , nous assimilons ce changement de barème à un traitement qui s'applique uniquement aux travailleurs dans les entreprises employant 50 salariés ou plus (que nous désignerons comme le groupe traité). Ce changement survient à partir de janvier 1999, ainsi nous pouvons dénir clairement une période pré-et post-réforme et nous pouvons adopter une approche par double diérence. L'idée consiste à estimer l'impact d'un traitement sur le groupe traité, en comparant la diérence entre l'évolution des taux de formation des travailleurs en n de carrière dans le groupe traité entre 1998 et 1999 à celle observée pour le groupe contrôle (comprenant les individus employés dans les petites entreprises) sur la même période. De manière plus formelle, soit P i,t une variable muette égale à 1 si le travailleur i a bénécié d'une formation nancée par son employeur à la date t, avec t ∈ {1998; 1999}. Le groupe traité et contrôle sont identiés par la variable muette T i , avec T i = 1 si le travailleur i est employé dans une grande rme12 . Nous incluons dans notre régression un ensemble de variables observables X i,t telles que le sexe, le statut marital, la situation professionnelle, le niveau d'éducation, la nationalité, l'ancienneté, la distance par rapport à l'âge de la retraite à taux plein et l'existence d'un plan de formation dans l'entreprise de l'interrogé. Il est important de rappeler que l'estimateur par double diérence permet de supprimer l'hétérogénéité inobservable spécique à chaque individu mais constante au cours du temps et aussi la tendance inobservable propre à chaque date mais commune à tous les individus. Cependant, il se base sur l'hypothèse identiante d'invariance temporelle : la variable dépendante observée chez le groupe contrôle aurait évolué de la même manière que celle observée chez le groupe traité, si ce dernier n'avait pas été traité. Formellement, cette hypothèse s'écrit :

E(P i,99 = 0|T = 1, X 1,99 ) -E(P i,98 = 0|T = 1, X 1,98 ) (1)
= E(P i,99 = 0|T = 0, X 0,99 ) -E(P i,98 = 0|T = 0, X 0,98 )

(2) Or, nous avons vu précédemment que les caractéristiques des travailleurs diffèrent fortement entre les deux groupes. Il n'est donc pas pertinent de supposer que l'évolution de leurs taux de formation auraient été identique en l'absence de traitement. Pour relâcher cette hypothèse, nous devons combiner cet estimateur avec une approche d'appariement sur le score de propension.

L'estimateur de double diérence conditionnelle

Pour tenir compte des diérences de caractéristiques entre les individus du groupe contrôle et ceux du groupe traité, nous calculons un estimateur de double diérence conditionnelle [START_REF] Heckman | Characterizing selection bias using experimental data[END_REF]. Cet estimateur combine une approche par double diérence avec une stratégie d'appariement, an d'estimer de manière semiparamétrique le contrefactuel, i.e. le taux de formation des individus du groupe traité, s'ils avaient travaillé dans les petites entreprises. L'appariement permet de rendre comparable la distribution des variables observables entre les deux groupes, et ainsi d'éliminer le biais de selection dans le groupe traité due à certaines caractéristiques observables.

Cet estimateur de double diérence conditionnelle se détermine en plusieurs étapes. D'abord il s'agit d'estimer le score de propension e(X) = P (T i = 1|X i ), c'est à dire la probabilité conditionnelle, calculée à partir d'un modèle logit ou probit, qu'un individu i soit dans le groupe traité (donc dans une rme de 50 salariés ou plus) sachant un ensemble de caractéristiques X i observées durant la période préréforme. Puis, dans une deuxième étape, nous estimons l'eet moyen du traitement sur le groupe traité en utilisant une approche par double diérence et en appliquant un poids W ij à chaque individu j du groupe contrôle, en fonction de sa proximité avec chaque individu traité i, selon leur score de propension. Soit α DiDM l'estimateur de double diérence conditionnelle que nous pouvons écrire de la manière suivante :

α DiDM = Σ i [(E(P i,99 = 1|T i = 1) -E(P i,98 = 1|T i = 1)) (3) -Σ j W ij (E(P j,99 = 1|T i = 0) -E(P j,98 = 1|T i = 0))]
Cette stratégie permet d'obtenir des résultats non biaisés sous l'hypothèse d'indépendance conditionnelle. Nous supposons donc que l'évolution du taux de formation d'un individu entre 1998 et 1999 est indépendante du fait qu'il appartienne au groupe traité, sachant son score de propension e(X). Plus formellement, cette hypothèse peut s'écrire :

E(P i99 = 1|T i = 0) -E(P i98 = 1|T i = 0) ⊥ T i |e(X i ) (4)
4 Résultats et discussion Avant de présenter les résultats de l'estimateur de double diérence conditionnelle et de les comparer à ceux obtenus avec une simple approche par double diérence, nous devons vérier la qualité de l'appariement, i.e., s'assurer que cette stratégie a bien permis de rendre comparable les individus employés dans les grandes rmes et ceux employés dans les petites rmes.

En premier lieu, nous devons vérier la qualité de l'ajustement lors de l'estimation du score de propension. Les résultats de ces estimations sont reportées dans le tableau 4 en annexe. Nous pouvons être conants en la signication de ce score estimé, car nos modèles probit permettent de prédire la véritable appartenance d'un individu soit au groupe traité soit au groupe contrôle dans environ 78% des cas. Il faut noter également que cette stratégie d'appariement n'est pertinente qu'au sein d'un support commun, i.e. un ensemble d'individus traités et non traités pour lesquels les distributions des scores de propension pour chaque groupe s'imbriquent susamment. En restreignant notre échantillon à ce support commun, nous éliminons seulement 2 observations pour les 45-49 ans et 28 pour les 50-54 ans, ce qui ne représente qu'une faible perte d'information. Enn, nous devons vérier si les distributions des caractéristiques au sein des deux groupes d'individus sont plus comparables après appariement. La gure 1 représente les distributions des scores de propension entre les deux groupes, avant et après appariement pour les 45-49 ans 13 . Nous constatons que les distributions des scores de chaque groupe s'imbriquent bien mieux après appariement. Par conséquent, cette première étape a bien permis de rendre plus comparables les deux groupes en termes de distributions des caractéristiques observables. Nous reportons ensuite dans le tableau 2 nos estimations de l'eet moyen du changement de barème de la taxe Delalande survenu en 1999 sur le taux de formation des individus du groupe traité. Nous comparons les résultats obtenus à l'aide de notre approche par double diérence conditionnelle à ceux obtenus par une simple double diérence. Bien que la deuxième approche semble surestimer l'eet moyen de la réforme, les deux méthodes conduisent à des résultats qualitativement similaires. Nous remarquons d'abord que la hausse de la Contribution Delalande à partir de janvier 1999 a augmenté de manière signicative le taux de formation des individus entre 45 et 49 ans employés dans les grandes entreprises. Avec la première approche, 13. La gure est très similaire pour les 50-54 ans cet eet est d'environ 10 points de pourcentage. De plus, nous observons que l'eet moyen de la réforme sur l'accès à la formation des travailleurs entre 50 et 54 ans est faible et non signicatif, avec une hausse de seulement 3 points de pourcentage si on se e à la première approche.

Tableau 2 Eet moyen de la réforme de 1999 sur le taux de formation des travailleurs dans les grandes entreprises 45-49 ans 50-54 ans

(1) (2)
(3) (4) Eet moyen sur la formation 0,099 Ces résultats peuvent paraître surprenants au premier abord. En eet, les travailleurs âgés entre 50 et 54 ans se situent au dessus de l'âge seuil de la taxe et de ce fait, ce sont leurs emplois qui sont davantage protégés suite à la réforme de 1999. Néanmoins, ces travailleurs pâtissent d'un faible horizon de carrière, compte-tenu de l'âge précoce de cessation d'activité14 . Or, nous pouvons supposer que la protection de l'emploi n'a d'eet sur les incitations à former des rmes que si elle augmente les rendements futurs anticipés de cet investissement. Comme l'horizon de carrière des travailleurs de 50-54 ans est trop faible, le retour sur l'investissement en capital humain de la rme est incertain, même si la protection de l'emploi est renforcée. Par conséquent, les employeurs sont réticents à former leurs travailleurs jugés trop proches de la sortie d'activité. Cet argument rejoint ainsi les précédents résultats de [START_REF] Montizaan | The eects of pension rights and retirement age on training participation : Evidence from a natural experiment[END_REF] et de Khaskhoussi et Langot (2008) et plaide pour un allongement de la durée d'activité an d'inciter davantage les entreprises à former leurs seniors.

Le résultat le plus innovant de notre papier concerne l'eet de la réforme de 1999 sur l'accès à la formation des 45-49 ans. En eet, leur emploi n'étant pas concerné par la hausse de la taxe Delalande, on peut se demander en quoi cette réforme a aecté les incitations des rmes à former ces travailleurs. Pour expliquer ce résultat, nous pouvons avancer l'argument théorique de Chéron et Rouland (2009) d'une complémentarité entre protection de l'emploi et formation. Dans notre cadre, les employeurs dans les grandes entreprises concernées par la réforme anticipent en 1998 qu'il sera plus coûteux de licencier un travailleur de 50 ans et plus à partir de 1999 en cas de choc de productivité sur son poste. Ces employeurs sont donc fortement incités à protéger leurs travailleurs de 45-49 ans contre des chocs futurs en les formant davantage, car leur coût de licenciement après quelques années aura fortement augmenté.

Conclusion

Dans ce papier, nous avons examiné l'eet d'un renforcement de la protection de l'emploi spécique à l'âge sur l'accès à la formation des travailleurs âgés entre 45 et 54 ans. Nous avons ainsi exploité une quasi expérience naturelle, à savoir une hausse de la Contribution Delalande à partir de 1999 qui n'a concerné que les rmes employant 50 salariés ou plus. Nous avons adopté une approche par double diérence conditionnelle, an de supprimer l'hétérogénéité inobservée constante soit entre individus soit au cours du temps et aussi de contrôler le biais de sélection sur observables.

Nos estimations révèlent un eet positif très signicatif de l'ordre de près de 10 point de pourcentage de la réforme de 1999 sur le taux de formation des 45-49 ans employés dans les grandes rmes. Nous expliquons ce résultat via un eet de complémentarité entre protection de l'emploi et formation : en anticipant que le coût de licenciement futur augmente fortement avec la réforme, les grandes entreprises sont incitées à protéger leurs travailleurs proches de l'âge seuil de la taxe en investissant davantage dans leur capital humain. Dans ce contexte, la hausse de la taxe Delalande a fortement augmenté les rendements anticipés de la formation pour les employeurs. Cependant, pour les travailleurs de 50-54 ans trop proches de la sortie d'activité, cette réforme n'a eu que peu d'impact sur leur taux d'accès à la formation, du fait de leur horizon trop faible.

Cette étude ouvre donc une piste intéressante de recherche mais comporte à ce stade de nombreuses limites. D'une part, nous ne fournissons que peu de détails sur le type ou la durée des formations reçues par les travailleurs. En particulier, nous ne cherchons pas à bien diérencier capital humain général et spécique, alors que cette diérence est la clé dans la littérature sur la formation [START_REF] Becker | Investment in human capital : a theoretical analysis[END_REF]. Néanmoins, il faut noter que notre échantillon concerne uniquement les travailleurs en n de carrière. Or, comme nous le montrons dans les données pour ce type de travailleurs, la majorité des investissements réalisés par la rme concerne leur capital humain spécique, avec pour seul but d'adapter les salariés à un changement survenu sur leur poste de travail. D'autre part, la vraie limite de cette étude vient du fait que nous ne mettons en évidence qu'un eet de court terme. En eet, nos données s'arrêtent en mars

4.

  Nous pourrions bien sûr soupçonner que les entreprises auraient anticipé ce changement avant qu'il ne rentre en vigueur. Dans ce cas, la variation observée des taux de formation entre 1998 et 1999 ne fournirait qu'une information partielle sur l'eet de la réforme sur les comportements des entreprises. Cependant, cette modication est entrée en vigueur via le décret n°98-1201 du 28 décembre 1998, publié au JORF n°301 du 29 décembre 1998, soit 3 jours avant son application (1er Janvier 1999). Ainsi, nous pouvons supposer que les rmes n'ont pas été en mesure d

  1999 ceci an d'homogénéiser nos périodes de pré-et post-traitement 10. Ce fait est cohérent avec les précédentes études menées par Bassanini et al. (2007) ou Montizaan et al. (2010). 11. cf notamment Imbens et Wooldridge (2009) pour une revue récente de la littérature sur ces méthodes d'évaluation.

Figure 1

 1 Figure 1 Distribution des scores de propension entre groupe traité et contrôle avant et après appariement

A moins qu'il n'ait été embauché à 50 ans ou plus. En eet, depuis 1992, les travailleurs embauchés dans les grandes rmes à 50 ans ou plus ne sont pas concernés par la taxe Delalande

En 2000, l'âge eectif de cessation d'activité en France se situait autour de 58 ans

On peut citer


Albane Saintenoy
email: albane.saintenoy@u-psud.fr

Eric Pohl

Christophe Grenier

Antoine Séjourné

Ivan Khristoforov

Pavel Konstantinov

Kencheeri Danilov

Kirill Bazhin

Alexander Fedorov

Overcoming data scarcity of active layer thicknesses using geophysics

Keywords: Ground-Penetrating Radar, Electrical Resistivity Tomography, Active Layer, River Influence

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Typical landscape features in Yakutia are small rivers that connect thermokarstic lakes within Alas valleys. Due to latent heat effects, rivers create a warmer soil below the riverbed compared to the surroundings. They influence the thermal equilibrium of the soil and the depth to permafrost in their vicinity. Here, we focus on a small river in a valley 100-km East of Yakutsk, close to Syrdakh village. The river is flowing from E to W, resulting in a S-facing right bank and a N-facing left bank, the latter largely covered with forest. The river width varies along its course from 2.5 m up to 15 m when the river forms larger water pools (Fig. 1). In September 2017, exceptional conditions occurred because of two consecutive very dry years. The river had mainly dried out and facilitated access to the riverbed allowed us to apply Ground-Penetrating Radar (GPR) and Electrical Resistivity Tomography (ERT) in order to measure detailed permafrost depth variations in 6 cross sections along the river (Fig. 1). Recently, [START_REF] Léger | Quantification of Arctic Soil and Permafrost Properties Using GPR and ERT Datasets[END_REF] successfully combined these two techniques to quantify the active layer depths in Arctic soil.

Geophysical data

GPR data were acquired using the Russian OKO system using two sets of antennas with nominal frequencies of 250 and 150 MHz. Measurements were acquired while walking at a constant speed, taking marks every 1 or 2 m. Traces were evenly interpolated every 5 cm in between each marks. The parameter settings of the GPR measurements were adjusted depending on the antenna size, using a time window from 100 ns down to 300 ns. One cross-section, CS9, is intensively equipped with 11 piezometers and 14 boreholes down to the permafrost as described in Pohl et al. (companion submission). These measurements were used to calibrate geophysical data (Fig. 2). In addition, four pits were dug after GPR data were acquired over a test profile (not shown here). Diffraction hyperbolas analysis and adjusting depth measured in situ of the unfrozen/frozen interface with reflection arrival time observed on radargrams, give an estimated electromagnetic wave velocity of 0.052 m/ns. All radargrams were migrated using Stolt's method (1978) using this velocity.

ERT data were acquired along CS7 and CS9 using a 16-channel instrument SibER-64 system (made in Novosibirsk) with 64 electrodes and a 0.5-m spacing between electrodes, using Schlumberger and gradient protocols. Data were inverted using three different softwares (Res2Dinv, ZondRes2D and BERT).

Results

Permafrost depths are estimated from GPR data and ERT data acquired in September 2017 (Fig. 3). All permafrost depths estimated from geophysical data are in reasonable ranges compared with those estimated from boreholes. The influence of the river on these depths is visible on all CS. GPR data suggest that for river channels narrower than 8 m, permafrost below the river is at least 0.5 m deeper than 10 m away. Insolation differences between right and left bank are also visible at some CS (e.g. CS3 and CS9), which are not completely shadowed by the forest (e.g. CS5). When the riverbed is wider than 8 m, electromagnetic loss is too strong for clearly resolving permafrost depth below the river with GPR data. ERT data acquired on CS7 show a high conductivity zone as deep as 6 m below the river (Fig. 3). Even though the limits of this zone are subject to some uncertainty and depend on the inversion software, ERT provides first order estimates where GPR cannot resolve the permafrost interface. GPR and ERT profiles (bottom: narrower riverbed, top: larger riverbed). River widths are provided on top of each graphics.

Figure 2 :

 2 Figure 2 : GPR profile (250 MHz) acquired on CS9 from S to N. Green bars localize boreholes with permafrost depths.

Figure 3 :

 3 Figure 3 : Summary of permafrost depths from boreholes, GPR and ERT profiles (bottom: narrower riverbed, top: larger riverbed). River widths are provided on top of each graphics.

Acknowledgments

We gratefully acknowledge funding from Univ. Paris-Saclay SPU Carbon Fate Emerging Project, Labex-IPSL and RFBR grants N°16-31-60082-mol-a-dk. We thank E. Léger for BERT inversions.