Clémentine Puech
email: c.puech@univ-st-etienne.fr

Xavier Delavenne

Zhiguo He

Valérie Forest

Patrick Mismetti

Nathalie Perek

Direct oral anticoagulants are associated with limited damage of endothelial cells of the blood-brain barrier mediated by the thrombin/PAR-1 pathway

Keywords: Endothelial cells monolayer, Blood-brain barrier, Direct oral anticoagulants, Protease activated receptor-1, Thrombin, VE-cadherin, ZO-1

Anticoagulant therapy presents iatrogenic effects such as intracerebral hemorrhage (ICH). The latest anticoagulants on the market, direct oral anticoagulants (DOACs) such as apixaban, dabigatran and rivaroxaban, are reported to cause less ICH than other anticoagulants. Next to the ICH area, the thrombin is accumulated and the blood-brain barrier (BBB) is opened. The effects of thrombin on the BBB are largely mediated by the protease activated receptor (PAR) family, especially the PAR-1 isoform. Our hypothesis is that DOACs may limit the effects of thrombin on endothelial cells (of the BBB) alteration by a mechanism probably involving PAR-1 activation. To test this hypothesis in vitro, we used HBEC-5i human brain endothelial cells as a human BBB model. The effects of thrombin under warfarin, heparin, rivaroxaban, apixaban, and dabigatran treatment on endothelial cells were then investigated by measuring of permeability and junction proteins: ZO-1 and VE-cadherin expressions and PAR-1 cleavage.

Depending on the anticoagulant used, we observed three profiles of response of the endothelial cells after thrombin exposure: i) dabigatran treatment allowed maintaining the tightness of the endothelial monolayer; ii) other DOACs limited thrombin-induced alteration of the endothelial monolayer; and iii) pretreatment with warfarin and heparin did not protect from thrombininduced BBB breakdown. Pretreatment with DOACs clearly limited the impact of thrombin on PAR-1 cleavage in our model, contrary to other anticoagulants, associated with ZO-1 and VEcadherin expressions. In conclusion, DOACs seem to limit the alteration of the monolayer of endothelial cells of the BBB mediated by the thrombin/PAR-1 pathway.

Introduction

In the last decade, direct oral anticoagulants (DOACs) have emerged as alternatives to other anticoagulants, such as vitamin K antagonists (VKAs) and low molecular weight heparin (LMWH), in the prevention of thromboembolic events. DOACs are reported to cause less intracerebral hemorrhage (ICH) effect than VKAs [START_REF] Ezekowitz | Dabigatran with or without concomitant aspirin compared with warfarin alone in patients with nonvalvular atrial fibrillation (PETRO Study)[END_REF][START_REF] Miller | Meta-analysis of efficacy and safety of new oral anticoagulants (dabigatran, rivaroxaban, apixaban) versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Ruff | Comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials[END_REF]. There are two types of DOACs: direct thrombin inhibitor (dabigatran)and factor Xa inhibitors (rivaroxaban and apixaban).

ICH is a known iatrogenic effect occurring under anticoagulation therapy. In the literature, phase III clinical studies have shown a decrease in intracranial bleeding under DOACs compared to VKAs (warfarin) in patients suffering from atrial fibrillation [START_REF] Connolly | Dabigatran versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Granger | Apixaban versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Ogawa | Safety and efficacy of the oral direct factor xa inhibitor apixaban in Japanese patients with non-valvular atrial fibrillation. -The ARISTOTLE-J study[END_REF]. It has also been reported in an in vivo murine model that rivaroxaban and dabigatran prevented hemorrhage in comparison to warfarin [START_REF] Ploen | Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke[END_REF][START_REF] Sun | Anticoagulation with dabigatran does not increase secondary intracerebral haemorrhage after thrombolysis in experimental cerebral ischaemia[END_REF]. In ICH, thrombin, a known multifunctional serine protease and a fundamental component in the coagulation cascade to produce fibrin clots, has been largely involved. The activity of thrombin is the target of anticoagulant therapy.

During ICH, the blood-brain barrier (BBB) disruption and more precisely the alteration of the endothelial monolayer is an important contributor to brain hematoma expansion. The BBB is a physical and metabolic barrier which separates the central nervous system (CNS) from the blood circulation [START_REF] Arai | Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in Alzheimer disease brain[END_REF][START_REF] Ishida | Upregulation of protease-activated receptor-1 in astrocytes in Parkinson disease: astrocyte-mediated neuroprotection through increased levels of glutathione peroxidase[END_REF][START_REF] Yin | Brain endothelial cells synthesize neurotoxic thrombin in Alzheimer's disease[END_REF]. The BBB is composed of endothelial cells, pericytes, astrocytes, and neurons, the whole forming the neurovascular unit. Endothelial cells are the main components of the BBB because they restrict and control the passage of molecules inside and outside the brain. During ICH, the level of thrombin has been shown to increase and provoke disruption of endothelial cells of the BBB [START_REF] Brailoiu | Mechanisms of modulation of brain microvascular endothelial cells function by thrombin[END_REF][START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF].

Integrity barrier properties are ensured by the close opposition between brain microvascular cells. These properties are mainly provided by tight junction proteins, with the most important in the BBB being zonula occludens1 (ZO-1).As with other vascular barriers, thrombin significantly compromises BBB integrity, mediates ZO-1 decrease associated with damage to an adherent junction protein specific of vascular cells, i.e., vascular endothelial cadherin (VE-cadherin) [START_REF] Festoff | HMGB1 and thrombin mediate the bloodbrain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer's disease[END_REF][START_REF] Rabiet | Thrombin-Induced Increase in Endothelial Permeability Is Associated With Changes in Cell-to-Cell Junction Organization[END_REF].

The effects of thrombin are largely mediated by the protease activated receptor (PAR) family composed of several isoforms, PAR-1 to 4. It has been reported that thrombin exposure causes barrier disruption via activation of PAR-1 [START_REF] Cheng | Thrombin-induced cerebral hemorrhage: role of protease-activated receptor-1[END_REF][START_REF] Guan | Effect of thrombin on blood brain barrier permeability and its mechanism[END_REF][START_REF] Xi | The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective?[END_REF].

If clinical studies have shown that DOACs induced fewer ICHs than VKAs [START_REF] Miller | Meta-analysis of efficacy and safety of new oral anticoagulants (dabigatran, rivaroxaban, apixaban) versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Ogawa | Safety and efficacy of the oral direct factor xa inhibitor apixaban in Japanese patients with non-valvular atrial fibrillation. -The ARISTOTLE-J study[END_REF][START_REF] Ruff | Comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials[END_REF], the cellular mechanisms have not yet been clearly established for human BBB. [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF] demonstrated that a 24-h pretreatment with dabigatran limited the effects of thrombin on an in vitro rodent BBB model [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF]. It has also been described that thrombin exposure activates PAR-1 receptor and provokes BBB disruption. As dabigatran is a direct thrombin inhibitor, we could suppose that dabigatran limits PAR-1 activation and prevents BBB disruption [START_REF] Chen | Characterization of thrombinbound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro[END_REF].

In this study, our hypothesis was that DOACs may limit the effects of thrombin on brain endothelial cells by a mechanism probably involving PAR-1 activation. To investigate the effects of anticoagulant agents on thrombin-induced damage to human BBB, HBEC-5i human brain endothelial cells were used as a BBB model. Several DOACs, i.e., dabigatran (direct thrombin inhibitor), rivaroxaban and apixaban (direct Xa factor inhibitor), were compared to warfarin and heparin, two indirect inhibitors of thrombin.

Firstly, pretreatment with different anticoagulants were performed prior to the addition of thrombin. Trans endothelial electrical resistance (TEER) and apparent permeability (Papp)

were investigated. Secondly, we evaluated the activation of the receptor of thrombin, PAR-1, and the expression of VE-cadherin and ZO-1 junction proteins.

Results

Impact on the tightness

Cells were treated with or without thrombin (1-5-10 U/mL) for 1 h. TEER, Papp with Na-Fl and dextran-FITC were evaluated (Figure 1). The highest thrombin concentration (10U/mL) decreased TEER by 26% (Figure 1A) and increased Papp 5-fold to Na-Fl or dextran-FITCcompared to control (Figure 1B-C). Our results clearly demonstrated that thrombin produced an alteration of the endothelial cell monolayer, the TEER and Papp values were modified in a concentration-dependent manner. Pretreatment effects with anticoagulant alone on endothelial cells of the BBB showed no effects on TEER and permeability values (Figure 1CD).

We then evaluated the pretreatment of anticoagulant during 24h with the highest thrombin concentration of 10U/mL on the endothelial monolayer (Figure 2). Depending on the anticoagulant used, we observed three responses of the endothelial cells after thrombin exposure.

Dabigatran pretreatment allowed to maintain the BBB monolayer exposed to thrombin: TEER= 40.3±1.7 Ω.cm² (Figure 2A) and Papp= 7.4±0.8 x10 -6 cm.s -1 for Na-Fl and 4.7±0.2x10 -6 cm.s -1 for dextran-FITC (Figure 2BC).

Treatment with other DOACs limited the alteration of endothelial cells induced by the thrombin. With apixaban and rivaroxaban, we obtained TEER values of 36.2±0.7 Ω.cm² and 37.2±0.7 Ω.cm² respectively (Figure 2A), Papp value for Na-Fl of 16.9±1.17x10 -6 cm.s -1 and 20.5±0.31x10 -6 cm.s -1 for Na-Fl respectively (Figure 2B). We obtained Papp value of 12.4±0.8x10 -6 cm.s -1 and 11.8±0.2x10 -6 cm.s -1 for dextran-FITC respectively (Figure 2C).

On the contrary, thrombin under treatment with warfarin and heparin provoked an alteration of endothelial cells, as represented by a low TEER (32.3±0.9 and 32.5±1.2 Ω.cm² respectively) (Figure 2A) and an increase in Papp (27.9±0.70x10 -6 and 28.8±2.82x10 -6 cm.s -1 for Na-Fl (Figure 2B) and 15.9±0.6x10 -6 and 16.9±0.4x10 -6 cm.s -1 for dextran-FITC respectively (Figure 2C).

Our hypothesis was that the breakdown of the BBB is mediated by the modulation of the activation of the receptor PAR-1 by thrombin.

2.2Thrombin/PAR-1 pathway

First, we used an activator (SFLLRNPNDKYEPF) and an inhibitor (vorapaxar) of PAR-1 to observe the effects on TEER and Papp.

Vorapaxar alone had no impact on TEER and permeability (Figure 3). Preincubation of vorapaxar blocked the effects of thrombin on the endothelial cells. Effectively, in this condition, we found the same result as the control for TEER (Figure 3A), Papp for Na-Fl (Figure 3B) and Dextran-FITC (Figure 3C). As expected, the use of the activator of PAR-1 induced an alteration of the endothelial monolayer, in the same manner as for the thrombin 10IU/mL exposure (Figure 3ABC).

Then we evaluated the release of the soluble fraction of PAR-1, since to be activated PAR-1 must be cleaved (Figure 4). The soluble fraction of PAR-1 increased after thrombin exposure in a dose-dependent manner (Figure 4A). The lowest concentration of thrombin allowed a cleavage of PAR-1 to 196±21pg soluble fraction of PAR-1/µg protein; and this value was increased approximately 2.5 times with the highest concentration of thrombin to obtain a high cleavage value of 476±6.4 pg soluble fraction of PAR-1/µg protein.

Anticoagulants alone did not cleave PAR-1, as observed for the control condition (Figure 4B). Depending on the anticoagulant used, we observed three responses of PAR-1 cleavage after thrombin exposure (Figure 4C). Under dabigatran, we obtained a low cleavage: 51.6±7.8 pg soluble fraction of PAR-1/µg protein. Under rivaroxaban and apixaban, we observed a limited cleavage (half of thrombin alone values). Under heparin, the cleavage fraction was as important as that observed with thrombin exposure alone. Pretreatment with DOACs clearly limited the impact of thrombin on PAR-1 cleavage in our model.

Effect of thrombin under anticoagulant on brain endothelial cell junctions

We verified that decrease in BBB tightness was associated with an alteration of cell junctions, the most represented being ZO-1 and VE-cadherin. Thrombin exposure (10 U/mL) provoked a 60% decrease in ZO-1 and a 40% decrease in VE-cadherin expressions with whole cell ELISA assay.

The DOACs rivaroxaban and dabigatran prevented the decrease of both ZO-1 and VEcadherin expressions with whole cell ELISA assay (Figure 5AB). For the other anticoagulants, warfarin, heparin and apixaban, we observed a 60% decrease in ZO-1 (Figure 5A) and a 40% decrease in VE-cadherin expression (Figure 5B), similar to that observed in the thrombin condition. Immunofluorescence ZO-1 assays confirmed whole cell assays, the BBB showed more important disruptionlinearity at cell-cell junction zones (white arrows, Figure 5C) under warfarin, compared to DOACs. Immunofluorescence assays allowed to observe disrupted ZO-1 and VEcadherin linearity at cell-cell junctions (Figure 5CD). Immunofluorescent staining showed a decreased expression in VE-cadherin with thrombin. It seemed that there was more important disruption linearity at cell-cell junction zones (white arrows, Figure 5D) under warfarin, compared to DOACs.

Discussion

Iatrogenic effects of anticoagulants are associated with BBB breakdown. Thrombin is a key factor in this rupture of the BBB [START_REF] Krenzlin | The Importance of Thrombin in Cerebral Injury and Disease[END_REF]. In the literature, it was described that DOACs cause less intracranial bleeding compared to other classes of anticoagulants [START_REF] Connolly | Dabigatran versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Miller | Meta-analysis of efficacy and safety of new oral anticoagulants (dabigatran, rivaroxaban, apixaban) versus warfarin in patients with atrial fibrillation[END_REF][START_REF] Ruff | Comparison of the efficacy and safety of new oral anticoagulants with warfarin in patients with atrial fibrillation: a meta-analysis of randomised trials[END_REF]. The involvement of the BBB in the limited number of hemorrhages under DOACs was observed in vivo in rodent models with a lower blue Evans diffusion [START_REF] Ploen | Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke[END_REF]. Few studies on the biological mechanisms are available to date. It seems that the activation of PAR-1 and other PARs such as PAR-3 and PAR-4 are implicated in the alteration or the maintaining of the barrier integrity [START_REF] Festoff | HMGB1 and thrombin mediate the bloodbrain barrier dysfunction acting as biomarkers of neuroinflammation and progression to neurodegeneration in Alzheimer's disease[END_REF][START_REF] Luo | Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection[END_REF][START_REF] Rohatgi | Protease-activated receptors in neuronal development, neurodegeneration, and neuroprotection: thrombin as signaling molecule in the brain[END_REF]. Our objective was to study the effects of thrombin/PAR-1 on the endothelial cells of the BBB model under different anticoagulants and to evaluate the activation of PAR-1.

The concentration of thrombin was based on the literature; we wanted a short but sufficient time to induce effects and to observe a modulation(Alabanza and Bynoe, 2012; [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF]. In human endothelial cells, maximal permeability is obtained after a minimum of 60 min of treatment with thrombin (Alabanza and Bynoe, 2012). First, we checked the effects of thrombin on BBB permeability with different parameters such as TEER and Papp.

Our results were in agreement with the literature, we observed a decrease in TEER and an increase in permeability to small molecules in a thrombin concentration-dependent manner (Alabanza and Bynoe, 2012;[START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF].

The concentration of dabigatran was based on Hawkins' works, it is a clinically relevant concentration [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF]. For warfarin, apixaban and rivaroxaban, we chose the same concentration as dabigatran. Heparin can be used in many models as an adjuvant to reinforce BBB properties; we chose a concentration without any impact on BBB tightness. Pretreatment with anticoagulants before exposure of endothelial cells to thrombin was evaluated, and there was no effect on the tightness in the endothelial monolayer.

We used different types of anticoagulant: DOACs, warfarin, an antivitamin K which corresponded to an older generation of oral anticoagulants, and heparin sodium an injectable anticoagulant. To our knowledge, no publication has compared all these anticoagulants at the cellular level to date.

In our studies, we observed different responses depending on the anticoagulant used.

dabigatran, a direct inhibitor of thrombin, blocked the effects of thrombin on the BBB as described in the literature [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF].

The other DOACs apixaban and rivaroxaban are inhibitors of the factor Xa in the coagulation cascade, limiting the effects of thrombin on the BBB with maintaining of the integrity of the monolayer. The other anticoagulants warfarin and heparin at this concentration, did not limit the effects of thrombin on the BBB integrity. In the literature, an in vivo model with rodent showed a decrease of blue Evans permeability in rivaroxaban treated animals compared to warfarin treatment after a mechanic induction of a stroke [START_REF] Ploen | Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke[END_REF]. This observation seems in agreement with ours.

Our hypothesis was that the cleavage is modulated by different anticoagulants, so we evaluated the cleavage of PAR-1. As expected, in our model, thrombin cleaved the receptor PAR-1 [START_REF] Chen | Characterization of thrombinbound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro[END_REF][START_REF] Luo | Protease-activated receptors in the brain: receptor expression, activation, and functions in neurodegeneration and neuroprotection[END_REF]. As for the alteration of the endothelial monolayer, we observed different responses depending on the anticoagulant used. In the literature it has been demonstrated that the expression of the protein PAR-1 can be modulated in the presence of warfarin, this mechanism has not been observed with DOACs [START_REF] Morihara | Reduction of intracerebral hemorrhage by rivaroxaban after tPA thrombolysis is associated with downregulation of PAR-1 and PAR-2[END_REF].

As described in the literature, the thrombin-bound dabigatran blocks the cleavage of PAR-1 preventing its activation that leads to BBB breakdown [START_REF] Chen | Characterization of thrombinbound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro[END_REF]. We obtained the same results with dabigatran in our model.

The other DOACs limited the cleavage of PAR-1 by thrombin. The other anticoagulants (warfarin, heparin) did not limit the cleavage of PAR-1. Our results are original and have not been reported in the literature on the BBB. These results are in agreement with the modulation of the tightness of the endothelial cells in a BBB model.

We have studied the ZO-1tight junction protein which plays a key role in the tightness of the BBB. It has been described that this junction is impacted in response to treatment with thrombin on endothelial cells(Alabanza and Bynoe, 2012; [START_REF] Brailoiu | Mechanisms of modulation of brain microvascular endothelial cells function by thrombin[END_REF][START_REF] Machida | Contribution of thrombin-reactive brain pericytes to blood-brain barrier dysfunction in an in vivo mouse model of obesity-associated diabetes and an in vitro rat model[END_REF]. VE-cadherin is an adherent junction protein characteristic of vascular endothelial cells. It has been shown that this junction protein is the first to be altered in response to thrombin treatment on endothelial cells; its alteration can induce ZO-1 alteration in a second time [START_REF] Kustermann | A real-time impedance-based screening assay for drug-induced vascular leakage[END_REF][START_REF] Rabiet | Thrombin-Induced Increase in Endothelial Permeability Is Associated With Changes in Cell-to-Cell Junction Organization[END_REF].It seems that under the action of thrombin, mediated by PAR-1 activation, the VE-cadherin and ZO-1 junction proteins are phosphorylated and removed from the membrane for ZO-1, which would decrease their availability to bind to other junction proteins [START_REF] Sabath | Gα12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly[END_REF][START_REF] Timmerman | Phosphorylation at endothelial cell-cell junctions: Implications for VE-cadherin function[END_REF]. The activation of the receptor PAR-1 can also result in the endocytosis of junctions [START_REF] Keep | Brain endothelial cell junctions after cerebral hemorrhage: Changes, mechanisms and therapeutic targets[END_REF]. In our model, thrombin induced a modification in expression of ZO-1 and VE-cadherin, and this modulation was limited by pretreatment with dabigatran and rivaroxaban before thrombin exposure; whereas the other anticoagulants did not limit this effect. The results obtained with the cell ELISA assay can be explained by the non possibility of the antibody to bind to the protein due to phosphorylation or internalization of junctions. This result is in agreement with the observation in immunostaining assays.

Conclusion

DOACs seem to act on PAR-1 cleavageand is directly related to the permeability of the cell barrier. DOACs limit the rupture of the BBB mediated by the thrombin/PAR-1pathway.

All these different responses between DOACs and other classes of anticoagulants reinforce our hypothesis of a limitation, under DOACs, especially rivaroxaban and dabigatran, of the BBB breakdown mediated by the thrombin/PAR-1 pathway.

In this study, we have only observed the effect of anticoagulants and thrombin on the endothelial cell. To better understand the result of clinical studies that have shown that DOACs induced fewer ICHs than VKAs, a more complex model with other cells of the BBB is necessary.

Material and methods

Chemicals and reagents

Bovine Secondary antibodies for cell ELISA were purchased from Santa Cruz Biotechnology (Dallas, USA). The F2R ELISA kit for PAR-1 measurement was purchased from Aviva Systems Biology (San Diego, USA). Rivaroxaban, dabigatran, apixaban and vorapaxar were purchased from Alsachim (Illkirch, France). Heparin sodium was purchased from Panpharma (Luitré, France).

Coumadin (warfarin) was purchased from Brystol-Myers Squibb (NewYork, USA). Bovine thrombin was purchased from Siemens Healthcare (Erlangen, Germany). Normal goat supplement (NGS) was purchased from Millipore (Burlington, USA). Secondary antibody (Alexa Fluor 555 goat anti-rabbit (A-21429)) and 4', 6-diamidino-2-phenylindole (DAPI) for microscopy were purchased from Thermo Fischer Scientific (Massachusetts, USA). Vectashield

Mounting Medium (H-1000) was purchased fromVector Laboratories (Burlingame, USA).

Cell culture inserts for 24-well plates (high density pore, 0.4 μm pore diameter size, translucent or translucidepolyterephtalate ethylene (PET) membrane) were purchased from Becton Dickinson (Franklin Lakes, USA). The EVOMvoltohmmeter system was purchased from World Precision Instruments (Hertfondsire, UK). 25 cm 2 culture flasks and companion plates were purchased from Dominique Dutscher (Strasbourg, France).

HBEC-5i BBB culture

The model was composed of HBEC-5i endothelial cells (from ATCC-Manassas, VA, USA) cultivated with conditioned medium from human astrocytes(HA from ScienCell Research Laboratories, Carlsbad, CA, USA) as previously described (Puech et al., 2018b).Cells were cultured in DMEM-F12 HAM, supplemented with 10% FBS and 1% antibiotic antimycotic solution. Cells were maintained in a humidified incubator at 5% CO2 and 37°C.

Treatments

Cells were incubated with or without dabigatran, rivaroxaban, apixaban, warfarin (at 0.5 µM physiological dose based on Hawkins [START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF]), and heparin (at 10 IU/mL) for 24 h. Cells were then treated with or without thrombin at 1, 5, or 10 IU/mL for1 h to mimic a haemorrhagic event (Alabanza and Bynoe, 2012;[START_REF] Hawkins | Dabigatran abrogates brain endothelial cell permeability in response to thrombin[END_REF].We used vorapaxar and SFLLRNPNDKYEPF as inhibitor and activator of the thrombin/PAR-1 pathways respectively.

Barrier properties

The apparent permeability (Papp) was assessed using two hydrophilic fluorescent molecules: Na-Fl (MW = 376 Da) or dextran-FITC (MW= 4 kDa). HBSS transporter buffer supplemented with 10 mM HEPES containing 10µg/mL of Na-Fl or dextran-FITC was loaded onto the apical side of the insert and incubated at 37°C for 1 h. Fluorescence was measured with a fluorescence spectrophotometer (FluoroskanAscent TM , Thermo Fischer scientific, France) with 485 nm excitation and 530 nm emission wavelengths. The permeability for all molecules was computed using the following formula:

𝑃 𝑎𝑝𝑝 = 𝑉 𝑟 𝐶 0 × 1 𝑆 × 𝐶 1 𝑡 (Formula 1)
Where Papp is the apparent permeability, Vr is the volume of medium in the receiver chamber, C0 is the concentration of fluorescent compound in the donor chamber at T0, S is the surface area of the monolayer, C1 is the concentration of fluorescent molecule in the receiver chamber after 1 h of incubation and t is the incubation time. The results are express in cm.s -1 (Puech et al., 2018a).

Trans endothelial electrical resistance (TEER) was recorded using an EVOM ® resistance meter with STX-2 electrode to characterize the formation of a tight endothelial cell monolayer. The measurement of a blank filter, i.e. without cells, was subtracted from those of a filter with cells. Then the resulting value was multipliate by the membrane area to obtain the TEER measurement in Ω.cm².

Thrombin, PAR-1 pathway

PAR-1 is activated by proteolytic cleavage of its extracellular N-terminus domain, resulting in the formation of a novel N-terminus (with sequence: SFLLRNPN) that serves as a tethered ligand folding back into the ligand-binding pocket of the receptor PAR-1.

PAR-1 activation is evaluated with the cleavage of PAR-1 based on standard sandwich ELISAs.

Measurements were realized according to the manufacturer's recommendation. The result was normalized by micro BCA total protein assay according to the manufacturer's instructions.

5.6 Protein expression 5.6.1 Whole cell ELISA Cells were seeded onto a 96-well plate at a density of 10,000 cells/well. At confluence, cells were treated with anticoagulants for 24 h and/or thrombin at 10 IU/mL for 1 h.

Then cells were fixed with 4% paraformaldehyde and permeabilized with methanol with 3% H2O2. ZO-1 and VE-cadherin expressions were evaluated in our model with primary polyclonal antibodies diluted 1/200 and incubated at room temperature. Secondary IgG antibody diluted at 1/500 was incubated at room temperature. Then, streptavidin was added for20 min at room temperature. TMB substrate was added in each well. At the end the reaction was stopped by addition of hydrochloric acid 1N and was measured with a spectrophotometer (Multiskan TM RC, Thermo Fischer Scientific, France) at 450 nm. Our results were normalized with the total protein content as assessed by Coomassie blue total protein assay, according to the manufacturer's instructions.

Immunofluorescence studies

BBB on inserts were fixed in glacial methanol for 6 min and rehydrated. Following a blocking step, the inserts were cut and placed in 24-well plates. Primary antibodies were diluted at 1/200 and incubated for 1 h. Secondary antibodies were diluted 1/500 and incubated with an insert membrane for 45 min. Nuclei were finally counterstained with DAPI in PBS at room temperature for 30 min. The staining of all biomarkers had been imaged using an epifluorescence inverted microscope IX81 (Olympus, Tokyo, Japan) equipped with CellSens imaging software (Olympus, Munster, Germany). FIJI software was used to treat the image.

Statistical Analysis

Graphpad software was used for data analysis and graphic outputs. Data were expressed as the mean ± standard deviation. Statistical analyses were performed using Graphpad software (Graphpad Software Incorporation, San Diego, USA). A Mann-Whiney test was used. Treated samples were compared to untreated control. The differences were considered statistically significant when p-value<0.05.

Figures legends

  serum albumin (BSA), sodium fluorescein (Na-Fl), BCA assay kit QuantiPro, DMEM-F12 HAM (Dulbecco's Modified Eagle Medium: nutrient Mixture F-12), Hank's balanced salt solution (HBSS), Dulbecco's phosphate buffered saline without sodium and magnesium (DPBS), fetal bovine serum (FBS), trypsin, antibiotic and antimycotic solution,fluorescein isothiocyanate dextran (dextran-FITC 4kDa), HEPES buffer, 3,3′,5,5′tetramethylbenzidine (TMB), hydrogen peroxide solution (H2O2), Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SFLLRNPNDKYEPF) peptide were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Rabbit anti-ZO-1 (ab59720) and rabbit anti-VE cadherin (ab33168) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Figure 1 :

 1 Figure 1: Effect of thrombin and anticoagulants on the endothelial cells of the blood brain barrier (BBB) model. A: Mean Trans endothelial electrical resistance (TEER) values for BBB model with three concentrations of thrombin after an incubation of 1h. Mean ± SD n= 3 N=5. B: Apparent permeability (Papp) values for sodium fluorescein (Na-Fl) (full circle) and dextran-FiTC (empty circle) in the same configuration. C: Mean TEER values for BBB model with several anticoagulants after an incubation of 24 h n=3. D: Papp values for Na-Fl with the same anticoagulant. * p-value<0.05; ** p-value<0.01; ***p-value<0.005 compared to untreated control as determined by a Mann-Whitney test.

Figure 2 :

 2 Figure 2: Effect of anticoagulant pretreatment on the endothelial cells of blood brain barrier (BBB) damage caused by thrombin. A: Mean trans endothelial electrical resistance (TEER) values for the effect of pretreatment with anticoagulant for24 h before an exposure of thrombin at 10 IU/mL for 1 h Mean ± SD n=3 N=2. Apparent permeability (Papp) values for sodium fluorescein (Na-Fl) (B) and dextran-FITC (C) on the same configuration. Mean ± SD n=3. * p-value<0.05; ** p-value < 0.01; ***p-value<0.001 compared to untreated control as determined by a Mann-Whitney test.

Figure 3 :

 3 Figure 3: Effect of activator and inhibitor of the protease activated receptor-1 (PAR-1) pathways on the blood brain barrier (BBB). A: Mean Trans endothelial electrical resistance (TEER) values for vorapaxar (inhibitor of PAR-1) for24 h alone or with thrombin for1h and SFLLRNDKYEPF (activator of PAR-1) for1 h. Mean ± SD n=3, N=2. Apparent permeability (Papp) values for sodium fluorescein (Na-Fl) (B) and dextran-FITC (C) on the same configuration. Mean ± SD n=3. * p-value<0.05; ** p-value < 0.01; ***p-value<0.001 compared to untreated control as determined by a Mann-Whitney test.

Figure 4 :

 4 Figure 4: Effect of thrombin with or without anticoagulant on the cleavage of the protease activated receptor-1 (PAR-1). A: Evaluation of the expression of soluble fraction of PAR-1 in the supernatant of HBEC-5i after exposure to different concentrations of thrombin for1h. B: Evaluation of the expression of the soluble fraction of PAR-1 in the supernatant of HBEC-5i after exposure to anticoagulants for24 h. C: Evaluation of the expression of the soluble fraction of PAR-1 in the supernatant of HBEC-5i after a pretreatment with anticoagulant before

Figure 3 Figure 5

 35 Figure 3

Funding:

This work was supported by Jean Monnet University (Saint Etienne, France).

Acknowledgements

We thank Fina Liu for her correction of the manuscript text 8 References Alabanza, L.M., Bynoe, M.S., 2012. Thrombin induces an inflammatory phenotype in a human brain


Kryštof Březina

Elise Duboue-Dijon

Vladimír Palivec

Jiří Jiráček

Tomáš Křížek

Cristina M Viola

Timothy R Ganderton

Andrzej M Brzozowski
email: marek.brzozowski@york.ac.uk

Pavel Jungwirth
email: pavel.jungwirth@uochb.cas.cz

Elise Duboué-Dijon

Can Arginine Inhibit Insulin Aggregation? A Combined Protein Crystallography, Capillary Electrophoresis, and Molecular Simulation Study

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human insulin is a hormone behind control of blood glucose levels, lipid metabolism, and life span, being also delivered exogenously to patients with diabetes. [START_REF] Cohen | The Twentieth Century Struggle to Decipher Insulin Signalling[END_REF] Mature human insulin is a 51-amino acid protein consisting of two disulfide-linked chains: A1-A21 and B1-B30, which triggers cellular responses by binding as a monomer to the (αβ)2 hetero-dimer tyrosine-kinase type insulin receptor (IR). In vivo, insulin is synthesized first as a single chain (pre)proinsulin. Its mature two-chain form is formed by excision of the C-peptide from proinsulin at two-arginine rich sites, and it is maintained in the storage granules of pancreatic β-cells, which release the hormone to the bloodstream in response to elevated blood glucose levels.

In the pancreatic secretory granules, insulin is stored at mildly acidic pH in the presence of high concentrations of zinc and other ions and ligands. [START_REF] Foster | Elemental Composition of Secretory Granules in Pancreatic Islets of Langerhans[END_REF] It has been suggested that proinsulin assembles in the Zn 2+ -containing granules into soluble hexamers, which aggregate, precipitate or even crystallize, upon excision of the C-peptide. [START_REF] Dodson | The Role of Assembly in Insulin's Biosynthesis[END_REF][START_REF] Emdin | Role of Zinc in Insulin Biosynthesis. Some Possible Zinc-Insulin Interactions in the Pancreatic B-Cell[END_REF][START_REF] Kemmler | Studies on the Conversion of Proinsulin to Insulin. 3. Studies in Vitro with a Crude Secretion Granule Fraction Isolated from Rat Islets of Langerhans[END_REF][START_REF] Carroll | A Mutant Human Proinsulin Is Secreted from Islets of Langerhans in Increased Amounts via an Unregulated Pathway[END_REF] Thus, an aggregated crystalline, or amorphous, hormone material is likely the stored form of the hormone in the granules. [START_REF] Dodson | The Role of Assembly in Insulin's Biosynthesis[END_REF][START_REF] Greider | Isolation and Properties of Secretory Granules from Rat Islets of Langerhans. II. Ultrastructure of the Beta Granule[END_REF][START_REF] Dodson | Structural Relationships in the Two-Zinc Insulin Hexamer[END_REF] The formation of aggregating hexamers is supposed to play a role in protecting the insulin from degradation, while the existence of the initial proinsulin form ensures proper folding of the hormone and prevents its fibrillation. [START_REF] Dunn | Zinc-Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer -A Review[END_REF][START_REF] Weiss | Proinsulin and the Genetics of Diabetes Mellitus[END_REF][START_REF] Landreh | Insulin, Islet Amyloid Polypeptide and C-Peptide Interactions Evaluated by Mass Spectrometric Analysis[END_REF][START_REF] Huang | Proinsulin Is Refractory to Protein Fibrillation: Topological Protection of a Precursor Protein from Cross-Beta Assembly[END_REF] Although the sequence of events for insulin oligomerization and aggregation is not fully established yet, the path connecting monomeric insulin with its hexameric form goes likely through a dimeric intermediate. [START_REF] Dunn | Zinc-Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer -A Review[END_REF][START_REF] Lord | Insulin Self-Association. Spectrum Changes and Thermodynamics[END_REF] Over 200 crystal/NMR insulin structures are deposited the Protein Data Bank (PDB), which include mostly dimers and hexamers of this hormone. Remarkably, the native monomeric form of the hormone has eluded crystallization efforts except for a case where concentrated acetic acid was employed, [START_REF] Zhang | Crystallization and Preliminary Crystallographic Investigation of a Low-PH Native Insulin Monomer with Flexible Behaviour[END_REF] while mutants of insulin yielded several monomeric crystalline forms. [START_REF] Brange | A Model of Insulin Fibrils Derived from the X-Ray Crystal Structure of a Monomeric Insulin (Despentapeptide Insulin)[END_REF][START_REF] Jiráček | Implications for the Active Form of Human Insulin Based on the Structural Convergence of Highly Active Hormone Analogues[END_REF] The in vitro insulin hexamer is an allosteric protein, which exhibits ligand-mediated interconversion between different conformational states referred to as T6, T3R f 3, and R6. These states differ by the conformation of the B1-B8 residues of the B-chain, where T correspond to its fully extended form, and R f /R to a helical conformation which contributes to the long B1-19 helix. [START_REF] Dunn | Zinc-Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer -A Review[END_REF] These insulin allosteric forms exhibit different physical properties such as dissolution rate that is particularly relevant for diabetes treatment. [START_REF] Kaarsholm | Comparison of Solution Structural Flexibility and Zinc Binding Domains for Insulin, Proinsulin, and Miniproinsulin[END_REF][START_REF] Brader | Insulin Hexamers: New Conformations and Applications[END_REF] While the addition of Zn 2+ to insulin in the absence of other ligands initiates formation of the hexameric T6 form, smaller phenolic-like cyclic compounds such as meta-cresol that is used in clinical formulations of insulin can stabilize the B1-B19 helix in a fully helical R6 conformation. [START_REF] Dunn | Zinc-Ligand Interactions Modulate Assembly and Stability of the Insulin Hexamer -A Review[END_REF][START_REF] Rahuel-Clermont | Mechanisms of Stabilization of the Insulin Hexamer through Allosteric Ligand Interactions[END_REF][START_REF] Berchtold | Binding of Phenol to R6 Insulin Hexamers[END_REF] We have been interested in the impact of granule-endogenous ligands on insulin conformation and it quaternary states. Our previous work focused on such a role of neurotransmitters such as serotonin and dopamine, which are involved in the exocytosis of the storage granules and may affect insulin aggregation. We provided the first crystallographic evidence that arginine binds to T3R f 3 insulin hexamers forming a ternary complex with serotonin and insulin. There, arginine penetrated the insulin T3R f 3 hexamer filling the voids around Zn 2+ and HisB10. [START_REF] Palivec | Computational and Structural Evidence for Neurotransmitter-Mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules[END_REF] Arginine is a biogenic molecule that may be abundant in the granules upon processing of proinsulin at its two arginine-rich sites. [START_REF] Davidson | Pro)Insulin Processing[END_REF] Its presence in insulin solution is also of a clinical relevance as small arginine-rich protamines are employed in some injection formulations of this hormone. [START_REF] Norrman | Structural Characterization of Insulin NPH Formulations[END_REF][START_REF] Hagedorn | Protamine Insulinate[END_REF] Arginine is also known as a potent stimulator of insulin secretion from pancreas. [START_REF] Leiss | Insulin Secretion Stimulated by l -Arginine and Its Metabolite l -Ornithine Depends on Gα I2[END_REF][START_REF] Thams | L-Arginine Stimulation of Glucose-Induced Insulin Secretion through Membrane Depolarization and Independent of Nitric Oxide[END_REF] Here, we investigate further in vivo allostery of insulin oligomers by studying the sole impact of arginine on the formation of insulin hexamers and its binding to different oligomeric forms of this hormone by a combination of molecular dynamics (MD) simulations, capillary electrophoresis, and X-ray crystallography.

Methods

Experimental details

Crystallographic experiments

Crystallizations of insulin at 5-10 mg/mL in 10 mM HCl were performed in in-house crystallization screens that consisted of known crystal growth condition of this hormone. All crystallizations were performed in the presence and absence of Zn The structure was solved by Molecular Replacement [START_REF] Storoni | Likelihood-Enhanced Fast Rotation Functions[END_REF][START_REF] Vagin | Molecular Replacement with MOLREP[END_REF] using the structure of B1-B6 truncated insulin monomer (PDB ID 1mso) as the model. Model building and refinement were performed by COOT [START_REF] Emsley | Coot: Model-Building Tools for Molecular Graphics[END_REF] and the CCP4 suite of programs. [START_REF]N. 4. The CCP4 Suite: Programs for Protein Crystallography[END_REF] Details of X-ray data collection, and refinement statistics are in Table S3 in the Supporting Information. Figures were made using

Capillary electrophoresis

Chemicals

Human insulin, recombinant, expressed in yeast (Sigma, USA), L-arginine (>98 %, Sigma, USA), sodium hydroxide (p.a., Penta, Czech Republic), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (≥ 99.5 %, Carl Roth, Germany), and zinc nitrate hexahydrate (p. a., Lachema, Czech Republic) were used as purchased. Deionized water obtained by a water purification system (Premier MFG'D, USA) was used to prepare all aqueous solutions.

Electrophoretic conditions

Capillary electrophoresis experiments were carried out on an Agilent 1600 CE instrument (Agilent Technologies, Germany) using unmodified fused-silica capillaries (Polymicro Technologies, USA), of 50 μm I.D., 375 μm O.D., 35.0 cm total length, and 26.5 cm effective length. Prior to the first measurement of the day, the capillary was activated by rinsing with 1 M NaOH for 10 min, followed by water for 10 min using a pressure of 93 kPa. Between individual runs, the capillary was flushed for 2 min with background electrolyte. The samples were introduced with a pressure of 5 kPa for 5 s. The diode array UV detector was operated at 200 nm wavelength. The temperature of the capillary cassette was maintained at 25 °C. The separation voltage was set to 25 kV, inducing an electric current of approximately 6 μA.

Background electrolyte and sample solutions

Background electrolyte solutions were prepared as follows. First, a 10 mM HEPES solution was prepared and its pH was adjusted to 7.5 with 1 M NaOH. Zinc nitrate and/or arginine were added to the buffer as 0.1 M aqueous solutions. Insulin was dissolved in water to obtain a 1 mg mL -1 stock solution. From this solution, each sample was prepared by mixing appropriate amounts of the insulin stock solution, of an aqueous solution of thiourea (1 mg mL -1 ) as electroosmotic flow marker, and of the background electrolyte. The insulin concentration in all the samples was of 8.6 μmol L -1 , and the ionic strength was in the range 5.2 -5.7 mM, depending on the arginine and zinc concentrations.

Computational details

Arginine force field

The Amber force field for arginine was generated using the antechamber 33 module. Partial atomic charges were determined using the Gaussian09 software [START_REF] Frisch; Trucks | Gaussian 09[END_REF] at the HF/6-31+G* level of theory using the RESP [START_REF] Burger | Quantum Mechanics/Molecular Mechanics Restrained Electrostatic Potential Fitting[END_REF] method, after geometry optimization of the molecule at the same level of theory. Full description of the employed arginine force field is provided in the Supplementary Information.

MD simulations

We performed force field MD simulations of the insulin dimer under different physicochemical conditions, which are summarized in Table 1. The insulin dimer was simulated in the presence of arginine at both low (System II) and high ionic strength (1.5 mol L -1 , system III). To assess the stability of the experimentally observed binding site of arginine, we also performed simulations at minimal ionic strength conditions of the crystallographic complex obtained in this work, which consists of an insulin dimer with one arginine in each of its monomers (System I). Since a crystallographic complex of the insulin hexamer exhibiting insulin-bound arginine molecules was previously obtained in the presence of arginine and serotonin, [START_REF] Palivec | Computational and Structural Evidence for Neurotransmitter-Mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules[END_REF] we also explored the stability of the arginine binding site within the insulin hexamer. To this end, we performed simulations at minimal ionic strength conditions (i.e., only compensation of the protein charge) starting from the crystallographic structure (PDB 5MT9), after removal of the crystallographic serotonin molecules (system V).

System

No. To mimic a neutral pH, histidine residues were singly protonated in the δ-nitrogen sites in all cases. In systems II and III, the structure of the insulin dimer was taken from the first two monomers of the T6 hexamer structure (PDB ID 1OS3) and 24 arginine molecules were randomly added using the antechamber utility of Amber 33 . The system was then explicitly solvated, before addition of the counterions (Na + and Cl -). System IV was prepared by extracting a representative structure (protein with an arginine molecule bound to the Glu B13 binding site) from the trajectory of system II. This structure was subsequently solvated, and ions added as previously. In systems I and V, the protein structure together with the ligands were taken from the X-ray structures and the missing C-terminal threonine B30 residues were added using the antechamber utility.

Insulin

All simulations were performed with the Gromacs 2016.4 software. [START_REF] Abraham | GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers[END_REF] The SPC/E model was used for water molecules. [START_REF] Berendsen | The Missing Term in Effective Pair Potentials[END_REF] As the standard force fields are known to overestimate the interaction between ions and charged protein groups, [START_REF] Duboué-Dijon | Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations[END_REF] we described the ions using the Electronic Continuum Correction (ECC). [START_REF] Leontyev | Polarizable Mean-Field Model of Water for Biological Simulations with AMBER and CHARMM Force Fields[END_REF][START_REF] Leontyev | Polarizable Molecular Interactions in Condensed Phase and Their Equivalent Nonpolarizable Models[END_REF][START_REF] Leontyev | Accounting for Electronic Polarization in Non-Polarizable Force Fields[END_REF] This is a mean field approach that implicitly accounts for the fast electronic polarization, absent in classical force fields, by a dielectric continuum. This approach is equivalent to scaling the ionic charges by the factor of (εel)⁻ 1/2 , where εel is the fast electronic part of the dielectric constant of water (1.78). Previously developed ECC force fields were used for the Na + (Ref. [START_REF] Dang | Ion Solvation in Polarizable Water: Molecular Dynamics Simulations[END_REF] ) and Cl -(Ref. [START_REF] Pluhařová | Hydration of the Chloride Ion in Concentrated Aqueous Solutions Using Neutron Scattering and Molecular Dynamics[END_REF] ) ions (see SI, Table S2). Consistently, the protein was described using an ECC, scaled charge version, of the AMBER99SB-ILDN force field. [START_REF] Duboué-Dijon | Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations[END_REF][START_REF] Lindorff-Larsen | Improved Side-Chain Torsion Potentials for the Amber FF99SB Protein Force Field[END_REF] For comparison, we provide in the Supplementary Information the results obtained with the standard (full charge) force field, which lead to the same conclusions.

The systems were first equilibrated using the steepest descend method with a force tolerance threshold of 100 kJ mol⁻ 1 nm⁻ 1 . The equilibration was continued by rising the temperature up to 300 K within a 100 ps long run in the NVT ensemble using a Berendsen thermostat [START_REF] Berendsen | Molecular Dynamics with Coupling to an External Bath[END_REF] a time constant of 0.1 ps. This was followed by equilibration in the NpT ensemble for 100 ps using a Parinello-Rahman barostat [START_REF] Parrinello | Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method[END_REF] (1 bar) with a 2 ps time constant. During this equilibration sequence, the backbone of the protein was constrained with harmonic restraints using a force constant of 100 kJ mol⁻ 1 nm⁻ 2 to retain its initial position for systems II and III. A similar approach was employed in the case of systems I and V, but the arginine and zinc ions (in system V) were additionally restrained using a harmonic force constant of 500 kJ‧mol⁻ 1 ‧nm⁻ 2 to preserve their binding mode during the equilibration procedure. The production runs were then carried out in the NpT ensemble as previously described and without any constraints on the protein (except for system V, were the zinc-histidine distance was restrained even during the production run). We used a 2 fs integration time step and the LINCS algorithm [START_REF] Hess | A Linear Constraint Solver for Molecular Simulations[END_REF] to constrain hydrogen-containing bonds. Long-range electrostatic interactions were accounted for by the Particle Mesh Ewald method [START_REF] Ewald | Die Berechnung Optischer Und Elektrostatischer Gitterpotentiale[END_REF][START_REF] Essmann | A Smooth Particle Mesh Ewald Method[END_REF] with 9 Å cutoff.

Free energy calculations

Calculations of the binding free energy of arginine to the insulin dimer binding sites were performed using the umbrella sampling 50 method. A starting configuration of the protein together with the ligand in the studied binding site was extracted from a previous MD simulation and solvated into a rectangular box prolonged in the z-direction (System IV). Starting configurations for the different umbrella sampling windows were generated by pulling the arginine into the bulk in the direction of the z vector using steered molecular dynamics [START_REF] Izrailev | Steered Molecular Dynamics[END_REF] , with moving harmonic restraints on the arginine with a force constant of 150 kJ mol⁻ 1 nm⁻ 2 , and static harmonic restraints on the protein with a force constant of 150 kJ mol⁻ 1 nm⁻ 2 . The free energy profile along the distance between the center of mass (COM) of arginine and COM of the B13 glutamate carboxylic groups was obtained using 60 roughly equidistant windows distributed between 2 and 50 Å. The windows between 2 and 10 Å were sampled for 100 ns. The rest of the windows was sampled for 20 ns. The simulation windows were combined and unbiased using the weighted histogram analysis method [START_REF] Kumar | Multidimensional Free-Energy Calculations Using the Weighted Histogram Analysis Method[END_REF] (WHAM) to yield a free energy profile along the distance coordinate, which was corrected by adding the standard entropic term RTln(4πr 2 ). [START_REF] Pohorille | Free Energy Calculations : Theory and Applications in Chemistry and Biology[END_REF]

Results and Discussion

X-ray Crystallography

Crystallization conditions consisting of 0.6 M Na2SO4, 0.3 M Tris pH 8.2, 0.6 mM Zn Acetate, and 75-100 mM arginine yielded a typical insulin dimer crystal form (one insulin molecule/asymmetric unit), with each monomer of the dimer in a complex with one arginine residue. Although the electron density maps show some semi-continuous features on the surface of the dimer, only one Arg/monomer was modeled and refined (Figure 1). However, the initial electron density for this Arg ligand does not fully cover this amino acid, and refined 2wFo-DFc map is not entirely unambiguous, indicating its possible alternative conformation (Figure 2).

Nevertheless, the arginine molecule is shown here in only one orientation that fits the most optimum network of ligand:insulin hydrogen bonds (Figure 2). Control crystallizations at pH > 7.5 without the arginine gave typical T3R f 3 hexamer insulin crystals. Crystallizations with arginine at pH 5.5, similar to the pH in the storage granules, gave also insulin T3R f 3 hexamers without any bound arginine. Network of H-bonds (dashed lines) at the arginine binding site. The 2wFo-DFc electron density map in blue mesh is contoured at 1 level; chains/atoms color coding as in Figure 1. The side chain of HisB10 is modelled in two alternative conformation according to its corresponding electron density.

In the crystal dimer form at pH 8.2, arginine occupies a shallow insulin cavity between the T-state N-terminus of the B-chain and its B9-B19-helix, being involved in a network of hydrogen bonds (Figure 2, Figure 3). Here, the N atom of arginine is hydrogen bonded (2.37 Å) to N1 atom of HisB10, although a broader electron density of this side chain indicates its alternative conformations, and possible involvement in hydrogen bond (3.05 Å) with O of serine B9. The half-occupancies of the HisB10 side chain reflect likely the mobility of the ligand, which is also outlined by patchy, and partial in some places, electron density. The other arginine interaction that may impact assembly of insulin hexamer is between its free-NH3 + group and carboxyl moiety of GluB13. It appears, that the crystal-observed arginine:insulin binding results more from ligandshape -hormone-surface fitting and hydrogen bond network that engage guanidine group of the arginine, rather than electrostatic surface attractions (Figure 3). The arginine also seems to have some 'natural affinity' for insulin surface, as there is plenty of tubular, but low level, electron density network on this insulin dimer, which may indicate highly dynamic and weak multi-ligand:insulin associations. Such phenomenon has also been observed in insulin:spermine-like rich environment, which may contain many arginine molecules as well. [START_REF] Norrman | Structural Characterization of Insulin NPH Formulations[END_REF] HisB10 and its protonation state thus emerges as a potential key factor in arginine binding, as the likely protonation of HisB10 N1 atom at pH 5.5 did not yield any crystalline arginine:insulin complexes.

Consequent investigations of the insulinarginine interactions

MD simulations at low ionic strength. The atomistic details of arginine binding to the insulin dimer were studied by low-ionic strength molecular dynamics simulations, starting from the Arg:insulin dimer crystal structure determined here by X-ray crystallography. Within 2 ns, the arginine ligands left their proposed crystallographic binding site and moved into the bulk solution (see Supplementary Information). This suggests that the crystal-observed binding site is not stable at the low ionic strength conditions of the simulation.

Possible arginine-insulin dimer interactions were investigated further by running MD simulations of the insulin dimer in the presence of arginine molecules in solution (See Methods, system II). Spatial distribution maps of arginine around the insulin dimer obtained from simulations performed at low ionic strength (system II) and at high ionic strength (system III) are depicted on panels A and B, respectively, in Figure 4. The binding sites discussed in the text are circled. The spatial distribution maps of arginine around the insulin dimer (Figure 4A) indicate that at low ionic strength, arginine molecules do not show any affinity to the crystallographic binding site. Rather, a high arginine density is found next to the pair of GluB13 side chains coming from each monomer (red circle in Figure 4A). Arginine also interacts with several negatively charged (glutamates A4, A17 and B21) or aromatic (phenylalanines B25) residues, but these interactions are unlikely to affect the insulin hexamerization process, as these residues are exposed on the surface of the hexamer.

Figure 4. Spatial distribution maps of arginine around the insulin dimer obtained from simulations performed at low ionic strength (system II) and at high ionic strength (system III) are depicted on panels A and B, respectively. In panel A, the arginine distribution at the B13 glutamate region is circled in red. The glutamate residues are colored red and the Zn 2+ binding HisB10 are in blue. In panel B, the binding interaction in the GluB13 region is missing. However, the crystallographic binding sites (one for each monomer, circled in red) is captured here (compare with Figure 1). Chain color coding as in Figure 1.

It seems then that GluB13 emerges as a residue that may impact hexamer formation. The general importance of GluB13 for hexamer stability is well known, as it occupies the central cavity, forming there a Ca 2+ binding site consisting of six circularly arranged glutamate side chains contributed by each insulin monomer. [START_REF] Coffman | Insulin-Metal Ion Interactions: The Binding of Divalent Cations to Insulin Hexamers and Tetramers and the Assembly of Insulin Hexamers[END_REF][START_REF] Palmieri | Proton Fourier Transform NMR Studies of Insulin: Coordination of Calcium to the Glu(B13) Site Drives Hexamer Assembly and Induces a Conformation Change[END_REF] We computed the free energy of arginine GluB13

pairing as G = -17.8±1.7 kJ mol -1 (Figure 5). To assess whether such binding mode could affect the hexamerization process, we compared it to the G change associated with the hexamerization.

Previous ITC calorimetry measurements found the overall free energy change associated with the hexamerization process, i.e., bringing together three insulin dimers and binding of two Zn 2+ , to be around G= -109 kJ mol⁻ 1 . [START_REF] Carpenter | Thermodynamics of Formation of the Insulin Hexamer: Metal-Stabilized Proton-Coupled Assembly of Quaternary Structure[END_REF] For a putative hexamerization in the presence of arginine, three arginine molecules (one from each dimer) must be displaced, which would correspond to a 55 kJ mol -1 free energy cost, which is small enough to maintain the overall negative G of the

Capillary electrophoresis experiments.

The effect of arginine on the hexamerization process was investigated further by capillary electrophoresis, performed at low ~ 5.5 mM ionic strength. We have shown in a previous study [START_REF] Duboué-Dijon | Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations[END_REF] that the formation of insulin hexamers upon addition ofn  gives rise to a very characteristic increase -in absolute value -of insulin electrophoretic mobility. Analogous experiments were thus repeated here in the presence of arginine and the same trend in the insulin mobility increase was observed. The magnitude of the change in the electrophoretic mobility remains constant over a wide spectrum of arginine concentrations (Figure 6). Hence, these results agree with the MD simulations, and suggest that arginine does not inhibit insulin hexamerization at low ionic strength.

.

MD simulations at high ionic strength.

There is a seeming discrepancy between the high ionic strength crystallographic experiments, which suggest that arginine binding to the insulin dimer prevents the formation of the hormone hexamer on the one side, and the low ionic strength electrophoresis and MD simulations on the other side, which find the experimental binding site to be unstable and suggest that the insulin-arginine interaction is not strong enough to inhibit hexamerization. To further investigate the cause of these apparent differences, we performed MD simulations at high ionic strength (see Methods, system III), chosen to mimic the conditions employed in the crystallographic experiments. In contrast to the previous MD simulations performed at low ionic strength, the obtained spatial distribution map (Figure 4B) now shows significant binding of arginine to the site located in the B1-B8 loop (red circles in Figure 4B), with specific hydrogen bonding to the backbone carbonyl oxygen atom of HisB5, hydrogen )

c arginine (mmol L -1 )
bonding of the α-amino group of the arginine to the GluB13 residue, and additional π-π stacking interaction with the HisB10. This reflects the arginine binding mode identified in the crystallographic experiments (see Figure 2). In addition, the obtained trajectory shows high mobility of the arginine molecule in the binding site, that confirms poor electron density for this ligand in the crystal structure. These simulations revealed also other less specific, and short-lived, binding interactions such as hydrogen bonding of the arginine guanidinium group to the HisB10/HisB5 imidazole rings, and argininehistidine stacking interactions.

MD simulations of arginine binding to the insulin hexamer.

Since MD simulations as well as capillary electrophoresis and previous crystallographic experiments [START_REF] Palivec | Computational and Structural Evidence for Neurotransmitter-Mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules[END_REF] indicated that insulin hexamerization appears to be possible in the presence of arginine at least at low ionic strength, we also probed the arginine binding to the insulin hexamer using MD simulations. Firstly, we assessed the stability of the crystallographic binding site for arginine in a T3R f 3 hexamer:serotonin:arginine complex. [START_REF] Palivec | Computational and Structural Evidence for Neurotransmitter-Mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules[END_REF] When simulations of the insulin hexamer at low ionic strength were started with arginine in its experimental binding site (Methods, system V), the arginine molecule remained in the crystallographic binding sites for the entire course of the 20 ns simulations (see Supplementary Information). Arginine binds to a rather specific cavity at the surface of the hexamer, which is created by several amino acid side-chains (Figure 7). Its bottom is formed by the B13 glutamate pair, to which the arginine binds via its positively charged guanidinium group. The guanidinium position is further stabilized by a π-π stacking interaction with the B10 histidine and a hydrogen bonding to the B9 serine oxygen atom. On top of this, the carboxylate group of the arginine is coordinating the zinc ion cofactor of the hexamer. Therefore this arginine binding mode corresponds to the starting crystallographic structure, [START_REF] Palivec | Computational and Structural Evidence for Neurotransmitter-Mediated Modulation of the Oligomeric States of Human Insulin in Storage Granules[END_REF] since the structure remained stable, and did not evolve significantly during the course of the simulation.

Conclusions

The possible scenario of arginine-insulin binding that emerges from our crystallographic, MD simulation, and capillary electrophoresis studies can be summarized as follows: (i) At high -~molarionic strength there is good agreement between arginine binding observed in the MD simulations and the crystal structure. Here, arginine binds to the insulin dimer at the shallow site located in the B1-B8 loop, with specific hydrogen bonding interactions with HisB5, GluB13, and additional interactions with HisB10. (ii) At lowmilimolarionic strength arginine binds to the insulin dimer at a different site than at the high ionic strength. While binding to GluB13 side chains could, in principle, interfere with insulin-insulin interactions, simulations and electrophoretic measurements indicate that it is not strong enough to prevent insulin hexamerization. (iii) Arginine can bind rather stably to the hexamer on its own, even in the absence of serotonine, in a mode observed in the arginine-serotonin-hexamer complex. (iv) Although the exact conditions in the storage granules in the -cells are not known, the ionic strength may be as high as 0.25-1.0 M. [START_REF] Foster | Elemental Composition of Secretory Granules in Pancreatic Islets of Langerhans[END_REF] Hence our experiments and simulations performed at the high salt conditions may closely reflect the in vivo molecular situation. (v) Insulin granule concentration is estimated to be at ~15 mM [START_REF] Foster | Elemental Composition of Secretory Granules in Pancreatic Islets of Langerhans[END_REF][START_REF] Suckale | The Insulin Secretory Granule as a Signaling Hub[END_REF] and the pro-insulin cleavage generated arginine may reach 50 mM. This implies that the higher ionic strength may promote arginine:insulin binding at physiologically relevant conditions.

In summary, our findings expand the possible in vivo patterns of insulin oligomerization and storage, being also relevant for the understanding of insulin stability in arginine-rich injections.

They also indicate that the quantitative understanding of the in vivo effects of putative insulin allosteric ligands, such as arginine, on aggregation of the hormone need to be scrutinized in detail in granule-mimicking physico-chemical environment.

Acknowledgment

PJ and JJ acknowledge support from the European Regional Development Fund OP RDE (project ChemBioDrug no. CZ.02.1.01/0.0/0.0/16_019/0000729). EDD acknowledges support from the

CCP4mg. 32

 32

Figure 1 .

 1 Figure 1. Crystal structure of insulin dimer in complex with arginine. B-and A-chains are in white and coral respectively; C-, N-and O-atoms in arginines are in green blue and red, respectively.

Figure 2 .

 2 Figure 2.Network of H-bonds (dashed lines) at the arginine binding site. The 2wFo-DFc electron density map in blue mesh is contoured at 1 level; chains/atoms color coding as in Figure1. The side chain of HisB10 is modelled in two alternative conformation according to its corresponding electron density.

Figure 3 .

 3 Figure 3. The arginine binding site at the insulin monomer electrostatic surface. Arginine

Figure 5 .

 5 Figure 5. Free energy profile for the arginine binding to B13 glutamate pair.

Figure 6 .

 6 Figure 6. Electrophoretic mobilities of insulin (cins = 8.6 μmol L -1 ) in the absence (blue) and presence (red) of 20 μmol L -1 zinc nitrate measured at varying arginine concentrations.

Figure 7 .

 7 Figure 7. Snapshot from MD simulations showing arginine bound to the insulin hexamer. The binding is mediated through the existence of a cavity (zoom in) cornered by the Ser B9 (orange), His B10 (blue) and Glu B13 (red) residues and the Zn 2+ cofactor (gray) at the top.

  2+ , and at varying concentrations of arginine of 25, 50, 75 and 100 mM. No-arginine controls have also been carried out. All trials

	were done at 203K. Resulting crystals obtained at 0.6 M Na2SO4, 0.3 M Tris/HCl pH 8.2, 0.6 mM
	Zn 2+ acetate, were flash-cooled directly in liquid N2. X-ray data were collected at Diamond Light,
	Didcot, UK and processed by xia. 27

Table 1 .

 1 Characteristics of simulated systems.

		Form	Number of Arginine	Na + ions	Cl ⁻ ions	Box Vectors* (Å)	No. of water molecules	Sim. Time (ns)
						7.84
	I	Dimer	2	2	0	7.84	8 030	20
						7.84
						11.60
	II	Dimer	24	0	20	11.60	50 239	2000
						11.60
						11.60
	III	Dimer	24	797 817	11.60	50 239	2000
						11.60
						6.52
	IV	Dimer	1	3	0	6.32	15 929	---
						11.92
						7.49
	V	Hexamer	3	5	0	7.49	12 164	100
						7.49

*Column [x,y,z] notation

EMBO and Marie Curie Actions (fellowship ALTF 952-2015). AMB, TRG, CMV work was supported by the Medical Research Council (Grant MR/K000179/1 and MR/R009066/1 to AMB).

The project was also supported by the Academy of Sciences of the Czech Republic (Research Project RVO:6138963, support to the Institute of Organic Chemistry and Biochemistry).