key: cord-103496-8tq78p2z
authors: Wang, Ting; Yegambaram, Manivannan; Gross, Christine; Sun, Xutong; Lu, Qing; Wang, Hui; Wu, Xiaomin; Kangath, Archana; Tang, Haiyang; Aggarwal, Saurabh; Black, Stephen M.
title: RAC1 nitration at Y32 IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury
date: 2020-11-13
journal: nan
DOI: 10.1016/j.redox.2020.101794
sha: 
doc_id: 103496
cord_uid: 8tq78p2z

Acute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y)34. Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y32, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y32, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y32. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.

The current pandemic of COVID-19 has caused over a hundred and sixty thousand deaths in USA, mainly due to a severe form of lung inflammation and edema, called acute lung injury (ALI) or its severe form acute respiratory distress syndrome (ARDS) [1] . ALI and ARDS represent the same disease spectrum characterized by hypoxemic respiratory insufficiency from pulmonary edema [2] . The acute lung inflammation and increased vascular hyperpermeability associated with ALI can be caused by systemic inflammation such as sepsis, or lung inflammation such as severe pneumonia induced by COVID-19 or other bacteria or viruses [3] . Besides supportive care with low tidal volume mechanical ventilation, general anti-inflammatory steroids, and extracorporeal membrane oxygenation (ECMO) in the intensive care unit (ICU), there are no specific drug therapies for ALI/ARDS, which still has an unacceptably high mortality of ~30% [4] .

Thus, a devastating demand for effective therapies for ALI/ARDS exists. respective primary antibody in blocking buffer, and the secondary antibody in blocking buffer after three time of wash in TBST. Reactive bands were visualized using chemiluminescence (Pierce) using either a Kodak 440CF image station or a LI-COR Biosciences Odyssey Imaging System. Band intensity was quantified using either Kodak 1D image processing software or Image Studio software version 5.

HLMVEC were homogenized in Pierce IP lysis buffer (Thermo-Fisher). After centrifugation, the supernatant was collected and mixed with A/G agarose beads (EMD/Calbiochem) with the pull-down antibody for 2h at 4ºC. The beads were collected after centrifugation at 5,000 rpm for 5 min and washed 3 times with IP wash buffer (Thermo-Fisher). The beads were then boiled with 2x Laemmli buffer for 5 min. Proteins were separated on 12% gels and transferred to PVDF membrane and analyzed using Western blot methods described above. In another experiment, immunoprecipitated proteins in gel stained with Coomassie blue dye were digested with chymotrypsin and used for mass spectrometry.

Rac1 activity was measured using the Rac1 Pull-Down Activation Assay Biochem Kit (Cytoskeleton Inc.). Briefly, HLMVEC and mouse lung tissue were lysed using the cell lysis buffer and the lysate with PAK-PBD beads was incubated at 4°C on a rotator for 1 h.

The PAK-PBD beads were centrifuged at 5,000 g and washed with wash buffer. Equal J o u r n a l P r e -p r o o f volume of 2x Laemmli sample buffer was added to each tube and boiled for 5 min.

Samples were analyzed by SDS-PAGE and Western blot analysis as described above.

All spectra were taken on an AB Sciex 5800 MALDI-TOF-TOF mass spectrometer in positive reflector mode (10 kV) with a matrix of CHCA. Masses were calibrated to Sciex nitration, we exposed NipR2 to authentic peroxynitrite (100μM) for 30 min. Samples were desalted using a C-18 ZipTip, mixed with CHCA matrix and were spotted directly onto a MALDI plate for further MS and MS/MS analysis. The ability of NipR2 or NipR2F peptides to bind to Rac1 was also analyzed by mixing 100ng/ml of peptide with 100ng/ml of human recombinant Rac1 protein for 45 min at RT and using MALDI MS Linear mode acquisition with sinapinic acid as a matrix. 

A nitro-Y 32 Rac1 specific antibody (Rac1-Y 32 -NO 2 ) was raised against a synthetic peptide antigen (LLISYTTNAFPGEY NO2 IPTVFD), where Y-NO 2 represents 3-nitrotyrosine as previously described [23] . The peptide was used to immunize rabbits. Tyrosine nitration-reactive rabbit antiserum was first purified by affinity chromatography. Further purification was carried out using immunodepletion using non-nitrated peptide (LLISYTTNAFPGEYIPTVFD) resin chromatography, after which the resulting eluate was tested for antibody specificity by immunoblotting and immunoprecipitation followed by mass spectrometry. The RhoA-Y 34 -NO 2 antibody was generated as previously described [24] .

J o u r n a l P r e -p r o o f

His-tagged Rac1 recombinant protein was expressed in E. coli as previously described [19] . Briefly, isopropyl-beta-D-thiogalactopyranoside (IPTG, 1mM) was added and the cells were incubated for 18-20h at 25°C. Bacteria were then harvested by centrifugation and the pellet was immediately lysed in 40mM Tris-HCl, 5% glycerol, 1mg/mL lysozyme, 100mM NaCl, protease inhibitor cocktail, ribonuclease A (Sigma), and deoxyribonuclease I (Sigma). The pellet was gently rocked for 30 minutes, sonicated and subjected to ultracentrifugation. The supernatant was loaded onto a Hisprep FF 16/10 column (Sigma) using binding buffer (40mM Tris-HCl, 100mM NaCl, 5% glycerol, 30mM imidazole) at 0.1ml/min flow. The column was washed with 40mM Tris-HCl, 300mM NaCl, 5% glycerol, 30mM imidazole using a flow rate of 1.5ml/min. Elution of the histidine-tagged protein was accomplished using elution buffer (40mM Tris-HCl, 300mM NaCl, 5% glycerol, 400mM imidazole) at 1.0ml/min flow. Collected fractions were loaded for size-exclusion gel filtration on a HiLoad 26/600 Superdex 75 column (Sigma) using gel filtration buffer (60mM Tris-HCl, 100mM NaCl, 5% glycerol) at 0.2ml/min flow. Fractions were collected and analyzed by Coomassie blue staining and Western blot. All purification steps were performed at 4°C, and purified protein was stored at -80°C. Recombinant human RhoA protein was also purified using a bacterial expression system as previously described [19] .

J o u r n a l P r e -p r o o f Pulmonary arterial endothelial cells (PAEC) were cultured as described [25] . Ovine PAEC, isolated as previously described [25] , were transduced with a His-tagged Rac1 adenovirus (MOI = 5) and incubated for 48 h at 37°C. Cells were lysed and the Histagged Rac1 protein was purified using HisPur Ni-NTA Columns (Thermo Scientific) and stored at -80°C. Fractions were analyzed by Coomassie blue staining and Western blot analysis.

Peptides containing 10 amino acids (FPGEYIPTVF) from aa28-37 of Rac1 fused with the cell permeable TAT sequence were synthesized by Peptide 2.0 Inc (Chantilly, VA).

This peptide was designated NipR2 (nitration inhibitory peptide for the Rho GTPases 2) and designed to interact with the flap region of the Rac1 protein. A similar phenylalanine-substituted peptide (NipR2F) was also synthesized harboring a Y32F mutation.

The electrical resistance of the endothelial cell monolayer was measured with the electrical cell impedance sensor (ECIS) technique [26] . HLMVEC were cultured on gold plated electrodes (8E10+) until 95% confluence. The change in electric resistance across the monolayer was measured continuously. The data was normalized to the initial values of basal resistance. In the transwell permeability assay, HLMVEC were seeded on a semi-permeable membrane in the upper chamber of the transwell (1μm pore size, BD Biosciences, San Jose, CA). After appropriate treatments, 40,000 MW FITC-dextran J o u r n a l P r e -p r o o f (1μg/μl) (Sigma-Aldrich) was added to the upper chamber. The amount of FITC-dextran infiltrating into the lower chamber was determined using a Fluoroskan Ascent Fluorometer.

The Committee on Animal Research at Georgia Regents University and University of Arizona approved all animal protocols and procedures. In the pre-injury model, male C57BI/6 mice (10 weeks) received vehicle (saline) or MnTMPyP (5mg/kg body weight) via an intraperitoneal injection 30 min before intratracheal installation of E. coli 0127:B8 LPS, 6.75x10 4 Endotoxin Units/g body weight, Sigma-Aldrich). Mice were examined 12 h after LPS challenge. At the end of the treatment, animals were anesthetized, and the lungs were flushed with 3 ml of ice-cold PBS (to remove blood) gently, and excised. A portion of the lung was quickly snap-frozen in liquid nitrogen and stored at -80ºC. In the post-injury model, male C57BI/6 mice (7-8 weeks) received vehicle (0.9% saline) or E.

coli 0127:B8 LPS (1.25mg/kg body wt, Sigma-Aldrich) prepared in 0.9% saline via an intratracheal injection 4 days before administration of NipR2 or NipR2F peptide (intraperitoneal injection, 1mg/kg). On Day 7 (post LPS) mice were euthanized, lungs were flushed with 3 ml of ice-cold PBS (to remove blood) gently, and excised. A portion of the lung was quickly snap-frozen in liquid nitrogen and stored at -80ºC. The remaining lung tissue was fixed in PBS buffered formalin for histology analysis.

J o u r n a l P r e -p r o o f BALF was obtained by instilling and withdrawing 1 mL of ice-cold HANKS buffer (Sigma) via a tracheal cannula. BAL fluid was then centrifuged 500 g for 10 min to collect pelleted cells. BAL cells were then resuspended in red cell lysis buffer (RCLB) for 15 s, centrifuged 500 g for 10 min to remove RCLB, and resuspended in 200 µl of PBS. Total cell count was then determined using a haemocytometer. Cell suspension (50 µl) was also separated by Cytospin centrifuge (Thermo-fisher) 600 g for 10 min onto glass slides. After air dry, the slides were stained with Dif-Quik staining (VWR) for differential cell count.

Lungs were inflated with 10% formalin under 15cm H 2 O pressure and immersed in the same solution before tissue processing into paraffin-embedded blocks and 4μm sections were then cut stained with H&E. Histopathological assessment was conducted by two researchers who were masked to treatment group. H&E stained sections were scored for the presence of neutrophil in the alveolar space, neutrophils in the interstitial space, the existence of hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening as described previously [27] .

Mice were remaining anesthetized with isoflurane (5% with oxygen), tracheostomized with a metal 1.2 mm (internal diameter) cannula and connected to a Flexi Vent (Scireq Inc) ventilator. Ventilation was initiated at a tidal volume of 10 ml/kg and a rate of 150/min. A TLC maneuver was performed, followed by 15 sec later, by a sinusoidal 1Hz J o u r n a l P r e -p r o o f oscillation. Subsequently, an 8 sec forced oscillatory signal (0.5-19.6 Hz) was applied, the mechanical input impedance of the respiratory system was calculated, and a model containing a constant phase tissue compartment was fit to input impedance in order to evaluate tissue elastance. Dynamic pressure-volume maneuvers were performed by stepwise increasing the airway pressure to 30cm H 2 O and then reversing the process.

Transcutaneous oxygen saturation were monitored via a small animal pulse oximeter (MouseOx Plus, STARR Life Sciences Corporation, Oakmont, PA, USA) by placing the non-invasive sensor on the neck, as previously described [28] .

Statistical analysis was performed using GraphPad Prism version 4.01 (GraphPad). The mean ± SD or SEM was calculated for all samples, and the significance was determined either by the unpaired t-test (for 2 groups) and ANOVA (for > 3 groups). For the ANOVA analyses, Newman-Kuels post-hoc testing was employed. A value of P <0.05 was considered significant.

In cultured HLMVEC, LPS challenge (1 EU/ml) induced significant decrease in Rac1

GTPase activity and this was attenuated by the peroxynitrite scavenger, MnTMPyP ( Figure 1A) . Utilizing immunoprecipitation analyses we demonstrated that Rac1 is subject to protein nitration and again this is attenuated by MnTMPyP ( Figure 1B&C ).

These findings suggest that LPS-mediated Rac1 nitration is associated with reduced Rac1 GTPase activity. To further confirm the nitration on Rac1 peptide and identify the J o u r n a l P r e -p r o o f nitration site, we first utilized recombinant human Rac1 protein purified using an expression purification system based in E.coli [19] . Purity of Rac1 was determined to be >90% using both SDS PAGE with Coomassie staining (Figure 2A ) and Western blot analysis ( Figure 2B ). The purified recombinant human Rac1 protein was exposed to authentic peroxynitrite (100 µM, 30 min) and then digested using chymotrypsin endoproteinase. The resulting peptide fragments were subjected to MALDI-TOF MS and MS/MS analysis. A nitrated peptide (1044.4Da) with sequence TTNAFPGEY 32 was identified with a single nitration site present on the tyrosine residue. A MS peak with +45Da in molecular weight difference (equal to nitro group addition) was observed with the nitrated Rac1 peptide fragment, within the region of aa24-32 ( Figure 2C ). Further MS-MS analysis confirmed the peptide sequence and the position of the nitro-group as Y 32 ( Figure 2C ). We next investigated the nitration of Rac1 in cultured pulmonary arterial endothelial cells (PAEC). In order to achieve a quantifiable intracellular level of Rac1, PAEC were transduced with an adenovirus construct containing a His-tagged human wildtype Rac1. We confirmed that human Rac1 protein could be purified utilizing a HisPur Ni-NTA Column ( Figure 2D ). Rac1 purification was confirmed by SDS PAGE with Coomassie staining ( Figure 2D ) and the identification of a single band by Western blot analysis ( Figure 2E ). Transduced PAEC were then exposed to the peroxynitrite donor, SIN-1 (200 µM for 1 h) and Western blot analysis utilized to confirm that peroxynitrite did not alter total Rac1 levels in the cells ( Figure 2F ). However, utilizing a nitrotyrosine antibody which specifically binds nitrated tyrosine we were able to see a strong band in Western blot at 21.5 kDa indicating a nitrated Rac1 protein in transduced PAEC exposed to SIN-1 ( Figure 2G ). The nitrated Rac1 protein was then purified using HisPur J o u r n a l P r e -p r o o f Ni-NTA Column and the peptide fragments generated by overnight chymotrypsin digestion were subjected to MALDI-TOF MS and MS/MS analysis. After exposure to SIN-1, the same nitration site Y 32 was detected in the Rac1 protein isolated from PAEC ( Figure 2H ). These results indicate that peroxynitrite is capable of inducing Rac1 nitration at a specific site, Y 32 both in vitro and in vivo. Next, using the known Rac1 protein structure and a previously established computational modeling method [19] , we simulated the structural impact of Y 32 nitration on Rac1. This modeling predicts that the Y 32 containing region (the "flap" next to the Switch I region of the catalytic domain) leads to a "close" state in the flap region ( Figure 2I ), compared to the "open" state induced by nitration of Y 34 in the flap region of RhoA [19] ( Figure 2J ). These computational modeling data, coupled with our Rac1-GTPase activity measurements, ( Figure 1A ) clearly demonstrate that LPS-mediated peroxynitrite stress induces Rac1 nitration at Y 32 and that this is inhibitory for Rac1 GTPase activity.

In order to increase the detection sensitivity for Y 32 nitrated Rac1, we next generated a polyclonal antibody (Rac1-Y 32 -NO 2 ) that specifically recognizes Y 32 nitrated Rac1 following the protocol from the previous study which used an antibody (RhoA-Y 34 -NO 2 ) that recognized Y 34 nitrated RhoA [24] . We confirmed that the Rac1-Y 32 -NO 2 antibody selectively detects Y 32 nitrated Rac1 in recombinant protein exposed to authentic peroxynitrite in-vitro ( Figure 3A ) and in-vivo HLMVEC exposed to SIN-1 ( Figure 3B ).

Further, we validated that Rac1-Y 32 -NO 2 and RhoA-Y 34 -NO 2 antibodies are preferential for nitrated Rac1 and nitrated RhoA respectively ( Figure 3C ). Using Rac1-Y 32 -NO 2 J o u r n a l P r e -p r o o f antibody we were next able to confirm that there was significant increase in Rac1 nitration in the mouse lung 12 h after I.T. LPS challenge, and that again Rac1 nitration was significantly suppressed by MnTMPyP ( Figure 3D ). These changes in Rac1 nitration correlated with change in Rac1 GTPase activity ( Figure 3E ). These data demonstrate that nitration of Rac1 at Y 32 , is intimately involved in the loss of Rac1 activity in the mouse lung during sepsis-mediated ALI.

We next aimed to design a peptide which specifically targets the flap region of the Rac1 protein to prevent its nitration at Y 32 . Docking simulations ( Figure 4A ) were used to design a nitration shielding approach that would bind to the flaps region of Rac1 similar to that used earlier for RhoA [19] . This resulted in the design of a peptide with sequence HRKKRRQRRRQFPGEYIPTVF designated nitration inhibitory peptide for Rho GTPases 2 (NipR2). The molecular mass of the NipR2 peptide of 2755.5 Da was confirmed by MALDI MS ( Figure 4B ) and peptide sequence was confirmed by MS/MS ( Figure 4C ). The NipR2 peptide was then exposed to authentic peroxynitrite (100 µM, Figure 4E ). As expected, when the NipR2F peptide was exposed to peroxynitrite (100 µM, 30 min) there was no mass difference observed in MS and MS/MS spectrum because of absence of the target tyrosine residue required for nitration ( Figures 4D and E) . Using MALDI MS Linear mode acquisition, with sinapinic acid as a matrix, we also confirmed that both NipR2 and NipR2F peptides we able to bind efficiently to intact human recombinant Rac1 ( Figure 4F ). 

To evaluate the therapeutic potential of NipR2, we utilized a mouse model of ALI Figure 6A ). As expected, NipR2, but not NipR2F, significantly attenuated LPS-mediated Rac1 nitration ( Figure 6B ) and restored Rac1 GTPase activity ( Figure 6C ). Mice were also used to perform additional physiological, biochemical and morphological studies to evaluate the efficacy of the NipR2 peptide in attenuating symptoms of ALI. Cell infiltration into the BALF was significantly increased in LPS treated animals ( Figure 6D ). NipR2, but not NipR2F, reduced this increase ( Figure 6D ). We also assessed histopathological changes in the lungs. LPS induces severe alveolar damage that includes the presence of large numbers of neutrophils and red blood cells in the alveolar and interstitial space, formation of hyaline membranes, septal thickening and debris accumulation in the J o u r n a l P r e -p r o o f alveoli ( Figure 6E ). Again, NipR2, but not NipR2F, reduced these pathological changes ( Figure 6E &F) .

We next assessed the effect of NipR2 on lung function in LPS challenged mice. NipR2 restored oxygen saturation from ~72% to ~95% ( Figure 7A ). Using FlexiVent technology, we also performed respiratory pressure-volume loop (PV loop) measurement and analysis. LPS induced a characteristic downward shift of the PV loop, compared to control mice ( Figure 7B ). Consistent with the oxygen saturation findings, NipR2, but not NipR2F, restored the PV loop, indicative of an improvement in respiratory mechanical function. Specifically, NipR2 increased the respiratory maximal volume at highest pressure ( Figure 7C ). NipR2 also prevented the LPS-mediated attenuation of total respiratory compliance ( Figure 7D ) and the increase in respiratory elastance ( Figure 7E ). Together these data demonstrate that NipR2 significantly improved lung function in this murine model of acute lung injury.

ALI is a complex syndrome with an unacceptable mortality. ALI pathogenesis involves disruption of the epithelial and endothelial barriers leading to an increased permeability and decreased edema fluid clearance [29] . Lung barrier permeability is tightly regulated by adherens junctions, tight junctions, and gap junctions. Junctional integrity is vital for cell-cell adhesion, actin cytoskeleton remodeling, intercellular signaling, and transcriptional regulation [30] . Rac1 regulates lung endothelial integrity via cytoskeleton J o u r n a l P r e -p r o o f rearrangements that determine cell shape and junctional integrity of the lumen [31] .

Interestingly, within the Rho-family small GTPases, Rac1 and RhoA are antagomirs [32] .

Rac1 is involved in the maintenance and stabilization of microvascular endothelial barrier functions (maintains junctional formation and cell relaxation), whereas RhoA primarily acts antagonistically to impair barrier properties [33] . Post-translational modification can directly influence the structure, function and stability of the Rho GTPases [34] . This particular study confirms, for the first time, that Rac1 can be nitrated at Y 32 , which leads to a persistent inhibition of GTPase activity. Crystal structure analysis of Rac1 indicates that Y 32 NMR and X-ray crystal structures for Rho family GTPases propose that the Cys18 thiol in the GXXXXGK(S/T)C motif is accessible for solvent and suggest reactive oxygen species and reactive nitrogen species possibly target the Cys18 thiol [35] [36] [37] . Further, we have shown that S-nitrosylation of RhoA attenuates the activity of RhoA [38] . The effect of S-nitrosylation on Rac1 is unresolved but we speculate that as nitration activates while S-nitrosylation inhibits RhoA that S-nitrosylation may stimulate Rac1 activity. However, further studies will be required to test this possibility. Besides nitration a number of other post translational modifications have been reported that have the potential to alter its activity/function. These include ubiquitination [39] [40] [41] , phosphorylation [42, 43] , and adenylylation (AMPylation) [44] , all of which have all been shown to play important roles in the regulation of Rac1. Interestingly, it has been reported that Y 32 in Rac1 is a target for AMPylation [44] . Therefore, we speculate that nitration might inhibit phosphorylation and/or AMPylation of Y 32 in Rac1 which could permanently alter its activity. Thus, it is likely that the competition among these various potential modifications likely has both physiological and pathological implications. Again, further studies will be required to investigate this.

During sepsis-like conditions, LPS exposure stimulates the production of peroxynitrite which leads to protein nitration of tyrosine residues, a nonreversible event which often affects protein structure and function. Previously we had reported that peroxynitrite J o u r n a l P r e -p r o o f stress induced by LPS challenge causes RhoA nitration at Y 34 which facilitates its persistent activation [19] . Rac1 and RhoA, as a balanced control mechanism of lung endothelial integrity, is disrupted by peroxynitrite stress. Even though RhoA and Rac1 share structural homology, RhoA is involved in endothelial barrier disruption during the development of ALI, while Rac1 is involved in endothelial barrier recovery during the resolution phase of ALI. The fact that each protein acts in a different phase of ALI could provide a therapeutic window to target Rac1, instead of RhoA, in the patients in ICU, most of whom have passed the earlier phase of endothelial disruption and are most likely in the slow recovery phase of this devastating disease. This possibility is supported by our animal studies in which we have utilized our NipR2 peptide to target Rac1 nitration in a "reversal"-, instead of "prevention"-, strategy to effectively "treat" experimental ALI. Our study reveals a new mechanism by which endothelial integrity loss is maintained through nitration-mediated Rac1 inhibition during sepsis-associated ALI. Importantly, we developed two new research tools in this study, a polyclonal antibody which specifically detects Y 32 -nitrated Rac1, and NipR2, a synthetic peptide which prevents Rac1 nitration at Y 32 . Since Rac1 is wildly expressed in multiple tissues with multidisciplinary function, our nitration specific Rac1 antibody has significant potential as a research tool in a number of pathologic states. Further, we validated the nitration shielding potency NipR2 in both cultured cells and a murine model of sepsis induced ALI. Thus, NipR2, or its next generation, may have clinical utility given the fact that ALI still does not have a selective drug therapy. However, this possibility should be tempered by the fact that previous ALI drug clinical trials have all failed. We propose that this is due to the fact that ALI is a syndrome that results from different insults (e.g., J o u r n a l P r e -p r o o f COVID-19 induced ALI is clearly different from trauma induced ALI), and the involvement of multiple systems failure such as endothelial barrier disruption, macrophage activation, and leukocyte infiltration. In this study, we report that NipR2 selectively targets Rac1 nitration and effectively blocks murine lung injury, but the therapeutic efficacy in ALI patients is hard to predict. We anticipate that a successful clinical efficacy of NipR2 or similar product might require: 1) precision medicine approach to identify patients in the sub-group with satisfactory responsiveness of Rac1 nitration blockade, as not all triggers of ALI (e.g., trauma) will lead to endothelial oxidative stress and peroxynitrite generation; 2) combination therapy with other effective reagents, including suppressor of the cytokine storm and/or neutrophil eliminators; 3)

selective tissue delivery vehicle, to increase therapeutic index. Further, we propose that conditional genetically modified in-vivo models will be needed to fully elucidate the biological functions of the Rho GTPases in different physiological environments and pathological conditions, and this information will likely be required to develop precise pharmacological inhibitors.

LPS is a known activator of endothelial ROS generation [45, 46] . Interestingly the generation of ROS might be "designed" a result of detoxification of oxidative species in oxygen-rich environment, and sometimes as a signaling cascade, since it can be generated and removed quickly. However, these protective or regulatory signaling pathways can be overwhelmed, leading to "oxidative stress". In the lungs, multiple cell types, including endothelial cells, macrophages, epithelial cells, and infiltrated inflammatory leukocytes, are good ROS generators. LPS can activate several J o u r n a l P r e -p r o o f endogenous machineries to generate ROS including NADPH oxidase (NOX), uncoupled NOS, dysfunctional mitochondria, and xanthine oxidase, all of which have been reported to be involved with the oxidative stress associated with ALI [47] [48] [49] [50] [51] . The link between increased oxidative stress and lung injury, as well as the success of antioxidant therapy in preclinical models makes ROS generation an attractive target in ALI. However, the complex roles of ROS in ALI development and recovery have restricted the efficacy of antioxidant therapy. ROS is involved in tissue injury [52] , barrier disruption [53] , proinflammatory cytokine production [54] . While it is also involved in endothelial barrier recovery [55] , and endothelial adherens junction re-assembly [56] .

Endothelial cells have abundant NO which is mainly produced by eNOS, which, upon LPS-challenge mediated uncoupling, also starts to generate ROS. The downstream effector of NOS uncoupling is likely peroxynitrite, formed from the interaction of NO with superoxide generated from different sources. We propose that targeting adverse events downstream of ROS, such as Rac1 nitration, will be more selective and effective to terminate ROS associated adverse outcome, and reduce ALI. Nitration unlike phosphorylation, which is selectively mediated by kinases and phosphatases, and modulated by signaling cascades regulating these enzymes, is not dependent on enzymes to facilitate the post-translational modification. Peroxynitrite mediated tyrosine nitration is a covalent modification that adds a nitro group (-NO 2 ) to one ortho carbon of tyrosine's phenolic ring to form 3-nitrotyrosine. Protein tyrosine nitration introduces a net negative charge to the nitrated tyrosine at physiological pH, thus altering structural properties. Previously, we had reported that like Rac1, its antagomir RhoA can also be nitrated at Y 34 , a similar location in the flexible flap region, leading to its persistent J o u r n a l P r e -p r o o f activation. We speculate that nitration will only occur at accessible tyrosine residues that are already sites for other post-translational modifications such as phosphorylation.

Indeed, nitration is very similar to phosphorylation in terms of charge, and thus could lead to a similar structural/functional outcome to that obtained by phosphorylation.

Alternatively, the presence of a 3-nitrotyrosine modification could potentially prevent the tyrosine residue being available for phosphorylation events. Since no enzyme has been identified as being responsible for the removal of nitration sites, mimicking the counter part of phosphatase for phosphorylation, the structure/function effect is persistent and thus deleterious as it is not balanced and countered. This might also explain the opposite functional outcome between Rac1 and RhoA nitration at a similar domain.

In conclusion, for the first time, we have used mass spectrometry as well as cell and molecular biology methodologies to identify Rac1 as being susceptible to nitration. This single nitration site located at Y 32 leads to Rac1 GTPase inhibition and enhances LPSmediated barrier disruption. We have also demonstrated that Rac1 nitration plays an important role in sepsis-mediated ALI and that blocking Rac1 nitration using a shielding peptide approach has therapeutic potential to reverse LPS-induced lung injury. Further studies will be required but this approach has the potential to produce a treatment for ALI that has so far proven to be intractable to all the pharmacologic approaches tried to date. 

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IRAK-1 contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing NOX-1 transcription and Rac1 activation and suppressing the expression of antioxidative enzymes

In vivo lipid-derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS

Oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization

Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells

Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: Role of asymmetric dimethylarginine

Lipopolysaccharide induces mitochondrial dysfunction in rat cardiac microvascular endothelial cells

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury

Signaling in the Pathogenesis of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS)

Autophagy maintains the integrity of endothelial barrier in LPS-induced lung injury

Superoxide potentiates NF-kappaB activation and modulates endotoxin-induced cytokine production in alveolar macrophages

Regulation of endothelial barrier function by reactive oxygen and nitrogen species

Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells

• Endotoxin exposure induces site specific nitration of Rac1 at Y 32 via peroxynitrite stress.• Rac1 nitration at Y 32 leads to persistent Rac GTPase inhibition and endothelial barrier disruption. • Novel Rac1 nitration shielding peptide, NipR2 blocks Rac1 nitration and rescues endotoxin induced lung inflammation. • NipR2 is potentially an effective therapy for sepsis induced lung injury by targeting Rac1 Y 32 nitration.

The authors have no conflict of interest to declare related to this research article .