Neurofilaments(NFs) are the most abundant intermediate filaments that make up the inner volume of axon, with possible phosphorylation on their side arms, and their slow axonal transport by molecular motors along microtubule tracks in a “stop-and-go” manner with rapid, intermittent and bidirectional motion. The kinetics of NFs and morphology of axon are dramatically different between myelinate internode and unmyelinated node of Ranvier. The NFs in the node transport as 7.6 times faster as in the internode, and the distribution of NFs population in the internode is 7.6 folds as much as in the node of Ranvier. We hypothesize that the phosphorylation of NFs could reduce the on-track rate and slow down their transport velocity in the internode. By modifying the ‘6-state’ model with (a) an extra phosphorylation kinetics to each six state and (b) construction a new ‘8-state’ model in which NFs at off-track can be phosphorylated and have smaller on-track rate, our model and simulation demonstrate that the phosphorylation-induced decrease of on-track rate could slow down the NFs average velocity and increase the axonal caliber. The degree of phosphorylation may indicate the extent of velocity reduction. The Continuity equation used in our paper predicts that the ratio of NFs population is inverse proportional to the ratios of average velocity of NFs between node of Ranvier and internode. We speculate that the myelination of axon could increase the level of phosphorylation of NF side arms, and decrease the possibility of NFs to get on-track of microtubules, therefore slow down their transport velocity. In summary, our work provides a potential mechanism for understanding the phosphorylation kinetics of NFs in regulating their transport and morphology of axon in myelinated axons, and the different kinetics of NFs between node and internode. Neurofilaments (NFs) are the most abundant intermediate filaments in axons of mature neurons, their population inside the axon may shape the morphology of axon –6. NFs aIt was observed that NF transport in myelinated axon decreased which induced a fatter axonal caliber in experimental observations , 19–24. In this work, the kinetics of phosphorylation of NFs are added into the ‘6-state’ model of NFs transportation, and a new ‘8-state’ model is developed based on the kinetics of phosphorylation and dephosphorylating of NFs. We construct a model of single axon with both internode and node of Ranvier, and propose a possible mechanism for understanding the slowing down of NFs at myelinated internode meanwhile velocity acceleration at unmyelinated node of Ranvier, and morphological difference between node and internode. Our results demonstrate that NF kinetics of getting on-track based on the phosphorylation state regulates the average velocity of NFs and thus shape the morphology of axon.The organization of the paper is shown in the following. In the result section 2.1, the result from modification of the ‘6-state’ by adding phosphorylation kinetics is shown; in section 2.2, the result of new ‘8-state’ model is shown to demonstrate the regulation of phosphorylation in NF kinetics and morphology of axon, and the result of NF kinetic difference and morphological difference of axon are shown by reconstruction of on-track rate in node and internodes in both PDE solution and Monte Carlo simulation. In section 3, conclusion and discussion are presented. In section 4 of model and methods, in section 4.1, the original ‘6-state’ model  is descrWe use mathematical analysis to predict the velocity of NFs at node and internode. When NFs transport reach dynamically equilibrium state, and it leads to: Pph: Pde = γde: γph.Therefore, the ratio of the probability of NFs at phosphorylated and dephosphorylated states is The final average velocity of NFs is contributed by two parts: one is the velocity of NFs at the phosphorylated state, and the other is the velocity of NFs at the dephosphorylated state. Therefore, an equation can be written as the summation of the following two terms as in In the following, we can calculate the average velocity in both node and internode sections along the axon. The probability ratio between dephosphorylation and phosphorylation is set as:Then the average velocity at both node and internode can be calculated as:γde: γph could be different for different nerves or species, therefore, the result can also be different according to This is the theoretic prediction. Our theoretical calculation successfully predicts the simulation result as shown in shown in , it can To simulate the effects of phosphorylation on the transport dynamics of NFs and axonal morphology, we construct a single axon with 50 μm long and the section of node of Ranvier is from 20 μm and 30 μm. The length of node of Ranvier ranges around 1 or 2 μm, here we assume that the node length is 10μm in order to illustrate the simulation results clearly; and our model and simulation are independent of the length of node, but depend on the kinetics of NFs phosphorylation and other parameters related with transport, and therefore can be applied to any type of axon or species.γon in node and internode. At the initial section of the axon, we input NFs continuous into the axon (input one NF every 10s). We set the NFs at the initial of axon are at the state of running anterograde, if the displacement of the NFs is beyond 0–50 μm, we will remove it from our system. After reaching the steady state, the distribution of NFs population along the axon and their kinetic states are observed and recorded.We add the kinetics of phosphorylation and dephosphorylation into each state of ‘6-states’. Based on To improve the accuracy of simulation, the experiment was repeated 300 times. And we count the number of NFs in each position of the 300 axons, and calculate their mean value and the standard deviation .In our simulation, we recorded each NF location and state information. According to continuity equation, In order to test our simulation results, we can calculate the average velocity of NFs in the Monte Carlo simulation by the following formula:According to the results of 1 and in the node section N2 by counting the number of NFs in each position along the axon in We can obtain the average number of NFs in internode section NRemarkably, it can be seen that The consistence of theoretical analysis and computational simulation has several indications.γon has to be made? Given the relation and distribution of phosphorylation and dephosphorylation unchanged. Based on The first indication is that, if the velocity ratio between node and internode has to be 7.6, then how much modulation of We assume n = 149.5. This calculation indicates that if a velocity ratio between node and internode needs to be 7.6, the velocity of fast transport has to be boosted by 149.5 folds, i.e. If n, q4, where In addition, the ratios of average velocity between internode and node can be written as functions of If it is set r values in the node and internode. Therefore, if the two factors can be measured in experiments, then the average velocity between internode and node can also be predicted. We plot the velocity ratio between node and internode as function of n in According to γph: γde for a fixed value of x. As is shown in the red curve (r = 8.0) in For different r values, the colored curve show different dependence of y on the n and r, the velocity ratio between node and internode is increasing with r = γon in the node to make the velocity faster than the internode, and the supposed boost of velocity is 7.6 fold; while the phosphorylation kinetics interfere the ‘6-state’ model of NFs, by dividing each state into phosphorylated and dephosphorylated states, with different transition rates in the node and internode, the originally constructed velocity ratio between node and internode of 7.6 drops to be only 3.96. In another word, it demonstrates that the phosphorylation kinetics has decreased the influence of on-track rate γon in determining the average velocity ratio of NFs between node and internode.The second indication is, we chose to increase As can be seen in γph: γde = ∞ in the internode, then the average velocity will become γph ≪ γde in the node, then the average velocity will become γph: γde = 1 in the internode, then the average velocity will also be the same between node and internode The last point is that, as the ratio r changes, the velocity ratio between the node and internode will be regulated. We take an extreme example to illustrate the effect. If r = *  = 0; P*  = P* ; x0 and xl denotes the proximal boundary axon and the distal boundary of axon, P* denotes each probability of eight states. Initial condition of the PDE of NFs distribution is set by the equilibrium distribution according to The partial differential equations in In order to observe the distribution of NFs along the axon in the equilibrium state, NFs are continuously input into the axons from the proximal section (input one unit of NF population every 1s). The results of the numerical simulation are shown in the As can be seen in Pa, Pa0, Pr, Pr0 increase, while the probability of Pap, Prp decrease. At the node of Ranvier, the NFs at off-track states are dephosphorylated, thus NFs at Pap1, Prp1 are much lower; while NFs in the internode are highly phosphorylated in the axon, thus the NFs at states of Pap1, Prp1 are much higher in the internode. Therefore, the introduction of transition kinetics with different rates of phosphorylation and dephosphorylation in node and internode, and its modulation on the “on-track” rate As can be seen from γde and γph are shown in Fig 9 and  are shown in Fig 11 in the section of model and methods. At the initial section, NFs are continuously injected into the axon (input one NF every 10 seconds). To improve the accuracy of simulation, the experiment was repeated 300 times. The number of NFs in each bin of the axon (2μm) is counted for all the 300 axons, each NF’s state are recorded and their mean values and the standard deviation are calculated. The simulation result is shown in We use Monte Carlo method to simulate the ‘8-state’ model, the value of According to the results of N1 = 10.82 and node of Ranvier N2 = 1.44 and we have From the simulation result of ph: γde (and the ration will be 1/r at node of Ranvier) varies, and the on-track rate If the rate ratio between phosphorylation and dephosphorylation in the internode r = γOn the other hand, as the ratio of on-track rate Based on the “stop-and-go” hypothesis, firstly we tried to add phosphorylation kinetics in each state, and analyzed the effect of phosphorylation on the average velocity of NFs transport in the node and internode. The basic assumption is the on-track rate is modulated by the phosphorylation kinetics, and NF transport velocity and the distribution of NFs population are thus changed by the on-track rate, according to the continuity equation. Secondly, by assuming that only at off-track states, NFs have phosphorylation kinetics and the phosphorylation status will eventually regulate the on-track rate, in which we introduce the kinetic transition between phosphorylation and dephosphorylation of NFs and build the ‘8-state’ model. Our model demonstrated that the regulation of phosphorylation on NF transport and axon morphology, and provided a potential mechanism for NFs slowing down during myelination.The modulation of the on-track rates of NFs is based on the assumption that NFs in node of Ranvier are less phosphorylated thus the on-track rate is higher, while in the internode, due to highly phosphorylation, NFs on-track rate is lower. Another basis of our modulation is that the NFs flux is a constant at equilibrium state, which results in the inverse proportional relationship between population of NFs and the average velocity distribution along node and internode. Through the Monte Carlo simulation and numerical solution of PDE of ‘8-state’ model, our results demonstrate the dephosphorylation of NFs can accelerate NFs kinetics in the node of Ranvier and phosphorylation of NFs can decrease NFs kinetics in the internode. In the end, our simulation results show that ratio of the number of NFs is inverse proportional to the average of velocity of NFs at both node and internode. Our model provides a potential mechanism to understand the regulation of NFs kinetics at myelinated axon by NFs phosphorylation kinetics; besides, the dynamic equilibrium for the kinetics between phosphorylation and dephosphorylation determines the fraction of NFs at each state, which regulates the on-track rate of NF transport, and this mechanism can be applied to any axon for any species.10, γ01, γra, γar are put into our model according to the “stop-and-go” model [However, our model still needs to be improved in the following aspects. First, we assume that the NFs are independent of each other, and do not consider the interaction between them, which is certainly unrealistic in the real axonal transport. Besides, NF-NF association results in the formation of NF "bundles", the “bundles” may have important effect on the axonal transport. Second, the transition rate constants γo” model  and by to” model . Noting Our model assumed different rates of phosphorylation and dephosphorylation between nodes and internodes, which have not been experimentally observed, and may have different values in different species or types of nerves. However, Data presented in  clearly There are different results, regarding on the experimental observation of NFs transport due to the NFs phosphorylation. Some experiments show that the phosphorylation slow down the NFs transport , 40, 41,In this section, we add NF transition kinetics of phosphorylation and dephosphorylation into each state in the ‘6-state’ model. By assuming that phosphorylated NFs have lower on-track rate and dephosphorylated NFs have higher on-track rate, we show that NFs phosphorylation regulates the average velocity of the NFs and their final distribution along axon by both analytical solution and simulation results.Pa, Pr, Pa0Pr0, Pap, Prp). The model gave an analytical expression for average velocity expression; and the model can produce simulation results in a good approximation of experimental data for the group transport wave of Gaussian function. The model mathematical expression can be written by coupled Partial Differential Equations (PDE) in Pa, Pr, Pa0, Pr0, Pap, Prp represent the distribution of running-anterograde, running-retrograde, pausing-anterograde, pausing-retrograde, off-track-anterograde, and off-track-retrograde, respectively. In our simulation, the anterograde and retrograde velocities va = 0.52 μm/s, vr = −0.36μm/s are adopted from research [We model NFs transport based on a previous model: ‘6-state’ model , where erp+γarPapWhere Paresearch , 40, 41.P = Pa + Pr, Pa0 + Pr0 + Pap + Prp.The spatial distribution of NFs population is: If NFs enter the axon at the proximal section and leave the axon at distal section at a constant rate, after long time, the steady distribution of NFs in the kinetic states can be obtained readily by solving the above equations. The theoretical solution of probability at each of the six states are :pa=ρ,pr10 = 0.14 s−1, γ01 = 0.064 s−1, γoff = 0.00445 s−1, γra = 1.4 × 10−5s−1, γar = 4.2 × 10−6s−1. The rate of on-track will be modified in the latter section.For the paper, the rate parameters are set as : γ10 = 0According to the experimental result, the average movement velocity of NFs in the node of Ranvier is 7.6 times as that in the internode sections . Why thePph and Pde represent the distribution of phosphorylated and dephosphorylated NFs respectively; the rate of γde and γph are assumed to be dependent on the location where NFs transport through, which is related to myelination function along the axon. According to the experimental observations [ph = 0.8, γde = 0.1. while the node, the rates are set opposite: γph = 0.1, γde = 0.8.The kinetics of phosphorylation and dephosphorylation can be written in the following equation:rvations , 30, 32,on. On the contrary, NFs dephosphorylation can reduce side arm length, and facilitate NFs to jump on-track for running, which results in higher on-track rate. In the following part, the on-track rate reconstruction is illustrated based on the continuity equation [We assume that phosphorylation of NFs makes NFs difficult to get on-track of microtubule by either tangling with neighboring NFs or affecting the interaction between the NFs and molecular motor kinesin or dynein, therefore, therefore, it decrease the rate of on-track γequation .From paper , the ave1, q2 and q3. The on-track rate of γon can be re-written as a function of average velocity as follows:The magnitude of Based on on = 2.75 × 10−4s−1, Therefore, for each six states, when NFs are at the state of phosphorylation, the on-track rate will be lower  is much lower than the rate of dephosphorylation (γde = 0.8 s−1), indicating that NFs are less phosphorylated; while in the internode section (x ≤ 20 μm and x > = 30 μm), the phosphorylation rate (γph = 0.8 s−1) is much higher than the rate of dephosphorylation (γde = 0.1 s−1), indicating that NFs are more phosphorylated. In the constructed configuration, the model can demonstrate the effect of phosphorylation on the transport of NFs.The rate parameter about phosphorylation of NFs of γIn this section, we build a new model- “8-state” model by considering two extra states of phosphorylated and dephosphorylated in the “6- state” model, and derive a mathematical expression for the average velocity of NFs.Based on the experimental observations of axonal morphology and the NFs population difference between the internode and node of Ranvier , we assuConsidering that NFs have transition kinetics of phosphorylation and dephosphorylation, we modify the ‘6-state’ model by subdividing two off-track pausing states at both anterograde and retrograde directions, resulting in an ‘ 8-state’ model. and The basic assumptions of our model is that we assume only NFs at off-track states have to be divided into two sub-states, : one is NFs of phosphorylation with Correspondingly, the kinetic equations for NFs transport can be written as follows:The spatial distribution of NF population is the summation of all the motion states of individual NF, which can be written as2:If NFs enter the axon at the proximal end and exit the axon at a constant rate, the equilibrium distribution of NFs can be reached, and can be obtained readily by solving the above The reproduction of NFs distribution in node and internode can readily adopt the strategy of Continuity Equation , since wThe on-track rate γph = 0.8 s−1, γde = 0.1 s−1.For the internode, the average velocity is assumed to be γph = 0.1s−1, γde = 0.8 s−1. The distribution of on-track rates along axon can be seen in While at node of Ranvier, the average velocity is By the analytical calculation in pa = 2.17 × 10−1, pr = 6.05 × 10−2, pa0 = 4.74 × 10−1, pr0 = 1.42 × 10−1, For NFs at node of Ranvier, the distribution of NFs at equilibrium in each state is calculated, and the result is: Therefore, the average velocity of NFs at node and internode is the same as we constructed in the beginning: S1 Data(RAR)Click here for additional data file.


The process of inflammation occurs due to inflammatory mediators, including prostaglandins, cytokines, and tumor necrosis factor (TNF). All these mediators activate the process of tumorigenesis and dysplasia, leading to colitis-associated cancer. Several drugs used to decrease these mediators will help in the treatment of acute attacks and also help in prolonged remissions of the disease by using nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and biological factors. Reducing these inflammatory mediators also have a role in chemoprevention and prevent progression to colorectal carcinoma. The most researched drugs in this process of chemoprevention are NSAIDs as it has both cyclooxygenase-2 (COX-2) inhibitory and non-inhibitory effects. These drugs should be taken for a long time and in large doses to reach this effect, which puts the patient at risk for various side effects. Researchers will need to do more research in the future to find the lowest effective dose that can reach the chemopreventive effect. We used database Pubmed as the main source for data search and extracted articles exploring the relationship between NSAIDs and their role in chemoprevention of colorectal carcinoma in inflammatory bowel disease (IBD) patients. We chose 23 studies which included seven review articles. We found that inflammatory mediators have a key role in colitis-associated cancer. Inflammatory bowel disease (IBD) is a broad term that describes a group of conditions that affect the gastrointestinal system causing inflammation and several other changes to the mucosa in ulcerative colitis (UC) or all layers of intestines in Crohn's disease. All these changes can occur because of interactions between environmental and genetic factors and microbial, leading to immunological response and inflammation of the intestine .In 2015, an estimated 1.3% of adults (3 million) reported being diagnosed with IBD either UC or Crohn's. The estimate does not include children under 18 years, who may also have IBD, as most people with IBD usually diagnosed in their 20-30 according to Center of Disease Control and Prevention (CDCP) data and statistics ..9].The anticancer effect of NSAIDs was initially considered only to be due to COX inhibitory effect but later found to be due to both COX inhibitory and COX non-inhibitory effects. COX inhibitory pathway involves Prostaglandins. When the enzyme catalyzes the synthesis of PGE2 (prostaglandins), it stimulates the proliferation of the cancer cells. Hence, the inhibition of COX-2 leads to the inhibition of growth and induction of apoptosis in tumor cells. However, extra work is done with COX-independent effects, which was found to help significantly in the chemopreventive role.The COX non-inhibitory part involves multiple signaling pathways like wnt , β-catenin, nuclear factor of Kappa light chain enhancer of B cells (NF-κB), p53 (tumor gene), toll-like receptor 4, nitric oxide synthase NOS-2) and caspases (action for aspirin) sulindac , signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor(TNF-α), IL-1β (interleukins), IL-4, adenosine monophosphate protein kinase (AMPK), proliferating cell nuclear antigen (PCNA), cyclin D1, β-catenin, inducible NOS, COX-2, and p65 (for sulindac with dose 300 mg per day) ,11,17. A and caspTreatment with aspirin using a dose of 81-325 mg or more can reduce the 20-year risk of CRC by 24% and decrease cancer-associated mortality by 35%  . Also, tThe chemopreventive effect of NSAIDs  has been studied and found that these drugs, when prescribed to a patient with IBD, showed a decrease in colon cancer by 62%; these findings provoked considerable interest in permanently adding 5-ASA or its analogs to the protocol treatment of UC patients. These anti-tumor effects, however, could only be achieved with large doses as mentioned before, and when administered systemically may have a questionable safety profile, as the drug may lead to severe complications.Side Effects of Long-Term Usage of NSAIDs Long-term use of aspirin and NSAIDs in large doses can cause several side effects, which can prevent its use. COX-1 enzyme and its products like prostaglandin 1 (PG1) maintains the integrity of the mucosal layer of the gastrointestinal system. Inhibition of this protective effect by using these drugs can cause erosive gastritis, peptic ulcer, anemia due to chronic bleeding, and peptic ulcer perforation which may even cause death form acute hemorrhage. The bleeding is also aggravated by the antiplatelet effect of aspirin . Adding A possible association between the use of NSAIDs and the onset or flare-up of IBD has been suggested, using clinical evaluation and measurement of fecal calprotectin  demonstrate that using non-selective NSAIDs associated with 17% to 28% of disease relapse; however, lack of evidence to find a definite conclusion .Selective COX-2 inhibitors like celecoxib show less gastrointestinal tract (GIT) toxicity compared to non-selective NSAIDs, with better control of IBD's extraintestinal manifestations (osteoarthritis). However, their long-term use carries the risk of cardiovascular complications like heart failure, hypertension, myocardial infarction, and stroke. These cardiovascular complications are more common with selective COX-2 than non-selective NSAIDs. Thromboxane is a potent vasoconstrictor formed by the COX-1 enzyme and is not inhibited by COX-2 inhibitors, while prostacyclin, which is a vasodilator formed by COX-2 is inhibited by COX-2 inhibitors, this doubles the vasoconstrictor effect of selective COX-2 inhibitors . All theIBD is a sum of diseases characterized by alteration in the immune system leading to abnormal response, causing inflammation of gastrointestinal layers. Most of the inflammatory mediators implicated in this process also help in the progression of colon cancer. Several anti-inflammatory drugs are used in the treatment of acute attack and/or permanent remission of the disease. The mechanisms of these drugs to do so are COX-2 inhibitory and COX-2 non-inhibitory effect. Most of the NSAIDs and aspirin are needed to be taken for an extended period to reach a promising result, which puts the patient at risk for many complications of long-term usage like anemia, gastrointestinal ulceration, myocardial infarction, stroke, bleeding, kidney dysfunction and even flare of IBD. That's why more studies are needed in the future to know the lowest effective dose with the more local action and least systemic absorption.


Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections. Persistent infections with the gastric pathogen Helicobacter pylori (H. pylori) are a significant risk factor for the induction and progression of stomach cancer. Approximately 50% of the human population is infected with H. pylori, which can induce chronic gastritis, duodenal, and gastric ulcers and finally gastric adenocarcinoma or MALT (mucosa-associated lymphoid tissue) lymphoma3. Accordingly, the complex network of cellular and molecular mechanisms of H. pylori–host interactions has been intensively investigated.Gastric cancer is associated with one of the highest mortality rates among all cancerous diseases in humans since efficient treatment options are still not availableH. pylori targets cell surface proteins of infected host cells added an important aspect to the model of H. pylori pathogenesis. During infection, H. pylori secretes HtrA and cleaves off the ectodomain of the cell adhesion protein and tumor suppressor E-cadherin, which was identified as the first HtrA substrate significant for pathogenesis4. E-cadherin is the key molecule of adherens junctions and necessary for establishing and maintaining intact intercellular adhesions between epithelial cells. Loss of E-cadherin function has drastic consequences not only on the epithelial architecture, but also on tumor prevention through the lack of recruitment of cancer-associated signal transduction molecules like β-catenin or p120-catenin6. In fact, E-cadherin ectodomain shedding leads to the disintegration of intercellular adhesion and promotes malignity of gastric, pancreatic, or breast cancer8. Therefore, E-cadherin cleavage serves as a reliable cancer biomarker10.The finding that the serine protease high temperature requirement A (HtrA) expressed by cis and trans interactions of E-cadherin between epithelial cells6. Importantly, these sites have been identified as preferred signature motifs for H. pylori HtrA11. Later studies indicated that the presence of calcium ions efficiently blocks E-cadherin cleavage by interfering with the accessibility of calcium-binding regions representing HtrA cleavage sites12. Additional HtrA substrates, including fibronectin, occludin, and claudin-8, have been described, confirming the capability of HtrA to break open intercellular adhesions and to disrupt the integrity of the epithelial barrier13. As a consequence, HtrA paves the intercellular way for H. pylori to transmigrate across the epithelial layer and to facilitate β1-integrin-mediated delivery of the bacterial oncoprotein cytotoxin-associated gene A (CagA)13.Structurally, E-cadherin is composed of an extracellular domain (EC), a transmembrane domain (TMD) and an intracellular domain (IC). The EC domain consists of the five tandem repeats EC1–EC5 with interspaced calcium-binding motifs, which are required for functional homophilic htrA deletion mutants of Streptococcus pneumoniae, Listeria monocytogenes, Staphylococcus aureus, etc. show a reduced ability to colonize host cells or tissues15. Similar findings have been reported for Shigella flexneri DegP, which is important for the surface exposure of the virulence factor and autotransporter intra/intercellular spread protein A (IcsA)16. Chlamydia trachomatis secretes HtrA from chlamydial inclusions into the host cytoplasm, where it plays a critical role in the chlamydial developmental cycle18. However, HtrA-mediated E-cadherin cleavage appears to be a prevalent mechanism since similar observations have also been made for Campylobacter jejuni, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. enterica ser. Typhimurium, Yersinia enterocolitica, and Proteus mirabilis22.HtrA proteins are widely expressed and their role in bacterial pathogenesis is well established. The HtrA proteins of several pathogens have been suggested to process adhesins, as E. coli, which expresses the three homologues DegP, DegQ, and DegS23. DegP proteins in Gram-negative bacteria comprise of an N-terminal signal peptide, followed by a chymotrypsin-like protease domain with the catalytic triad histidine, aspartic acid, and serine residues. Substrate recognition, binding, and HtrA/DegP homo-oligomerization are mainly mediated by the two C-terminally located PDZ  modules24. HtrA/DegP protease activity has been associated with the formation of higher order multimers composed of trimers. DegP is thought to form inactive hexameric oligomers that are converted into proteolytic active oligomers consisting of 12–24 HtrA monomers upon substrate binding27.The structure and regulation of prokaryotic HtrA proteases have already been extensively studied in 29. The development of a substrate-derived peptide inhibitor further emphasized the significance of HtrA activity in H. pylori pathogenesis11. Importantly, bacterial HtrA also functions as a chaperone that refolds and degrades misfolded proteins under stress conditions30. Due to this important function in bacterial physiology, HtrA expression is essential for H. pylori. This is obviously a unique phenotype for H. pylori32 as deletion of the htrA gene from the bacterial genome has not yet been reported to be lethal for other bacteria. The unexpected finding that small molecule inhibitors targeting HtrA can efficiently block H. pylori growth and survival31 indicates that H. pylori HtrA could be an attractive target for screening of pharmacological inhibitors.Computational drug design has identified functional HtrA inhibitors docking either into the active pocket of HtrA or interfering with an allosteric ligand-binding site that is important for HtrA oligomerization++ can directly block the activity of serine proteases and potentiate moderate serine protease inhibitors by chelating the inhibitor to the histidine and serine of the catalytic triad in the active center33. In our study, we found that Zn++ and Cu++ can block HtrA activity and hence, we hypothesize that Zn++ or Cu++ could function as a co-inhibitor of HtrA proteases.In this study, we established a novel fluorescence assay based on Förster resonance energy transfer (FRET) that is suitable for high-throughput screenings and determining the effect of divalent ions on the activity of HtrA. Previous studies have reported that ZnH. pylori HtrA (HpHtrA) has been mainly investigated by casein zymography or Western blot analyses of substrate fragments, which are laborious, slow, and low-throughput methods34. FRET technology represents state-of-the-art methodology and allows continuous assays of protease activity and high-throughput screening of protease inhibitors. To develop a FRET peptide assay containing an optimized short cleavage site for HtrA, we performed global specificity profiling for HtrA using a direct in-gel profiling of protease specificity (DIPPS) assay35. Analyzing HtrA-targeted proteome-derived peptides from MKN-28 cells, we detected 2,479 peptides that were processed by HtrA. These peptides were commonly cleaved after the aliphatic amino acid residues V, I and A in P1 position, while preference for basic amino acid residues such as R and K was observed in P2 position. Specificity of other non-prime binding sites was less pronounced, with R showing the highest enrichment at the P3 position and A at P4. Among the prime sites, the amino acids A and K were shown to be enriched at P1′ position and Y at P2′ position  was synthesized  is similarly inhibited by Zn++ and Cu++, and has also been observed for DegP from E. coli and human HtrA138. Interestingly, the crystal structure of the HtrA homolog A (HhoA) from the photosynthetic cyanobacterium Synechocystis sp. PCC 6,803 revealed that the hexameric structure contains a Zn++ ion within the central channel, which inactivates HhoA in a casein cleavage assay39. These data support our findings that HpHtrA can be inhibited through direct binding of Zn++ and Cu++.The FRET peptide assay is a suitable approach for drug screening to identify and optimize HpHtrA inhibitors. A wide range of non-metalloproteases can be inhibited by Zn11  was incubated with recombinant HpHtrA for 16 h in the presence of various divalent cations as indicated. Fragments of hCdh1 were detected by Western blot analyses using an antibody recognizing the EC5 domain of E-cadherin. Efficient HpHtrA-mediated cleavage of hCdh1 was indicated by the presence of the characteristic HtrA-induced fragmentation pattern11 Fig. B, lane 3++ and Cu++, we performed titration experiments to determine the concentrations of Zn++ and Cu++ that are required for HtrA inhibition using the FRET peptide assay  values calculated for Zn++ were 649.5 nM  after 15 min and 2.57 µM  after 180 min. Cu++ exhibited an IC50 of 295.4 nM  after 15 min and of 937.5 nM  after 180 min, indicating efficient HpHtrA inactivation. The inhibition of HpHtrA wt detected in the FRET peptide assay was also compared with in vitro cleavage experiments using hCdh1 or casein as substrates. As a control, we included a proteolytically inactive HpHtrA mutant (SA)40, which does not process hCdh1 or casein. Zn++ clearly inhibited HpHtrA-mediated hCdh1 fragmentation at a concentration of 50 µM 42. However, in combination with serine protease inhibitors, zinc has been reported to be a potent intensifier of moderate small molecule inhibitors43. Even though not all serine proteases contain typical Zn++-binding sites, which are characterized by two histidine residues with their imidazole side-chains as ligands for Zn++, it has been suggested that Zn++ and Cu++ bind in the active pockets of thrombin33, trypsin33, and kallikrein36 and bridge the inhibitor to the active site leading to an efficient inhibition. These studies demonstrated that lower concentrations of both, inhibitor and Zn++, were sufficient to strongly potentiate inhibitory effects. Since we found that Zn++ blocks HpHtrA activity, it is tempting to speculate that moderate HtrA inhibitors combined with a low concentration of Zn++ ions may synergistically function as highly potent zinc-mediated serine protease inhibitors.The ICE. coli HtrA homologue DegP, which is converted into proteolytically active oligomers upon substrate binding44. HtrA proteases harbor several domains that are crucially important for assembly and activity. DegP/HtrA proteases contain an N-terminally located signal peptide (SP), followed by the protease domain, which is characterized by the catalytic triad histidine (H), aspartic acid (D), and serine (S). The C-terminally located domains PDZ1 and PDZ2 are important for substrate recognition and oligomerization44. Several regulatory loops  have been investigated for E. coli DegP, which share sequence similarities with HpHtrA  placed between the PDZ1 domain and the serine protease domain has been identified in the 2.6 Å X-ray structure of HpHtrA that functions as an important protein–protein interface , glutamic acid (E), or cysteine (C), which can bind Zn++ or Cu++ equally well47. Therefore, we wanted to investigate whether Zn++ and Cu++ could target the aspartic acids exposed in this loop, which have previously been suggested as ligand-binding motifs48. The individual amino acids S164, D165, S166, and D168 were mutated to alanine and analyzed for oligomer stability  (Table ++ could be detected for HpHtrA wt (Kd = 23 µM) and HpHtrA S164A (Kd = 55 µM) with signal-to-noise ratios of 6.3 and 6.0, respectively. Although binding curves were also fitted for HpHtrA D165A, S166A, and D168A . Our results thus provide support for the hypothesis that HpHtrA D165 and D168 are involved in the interaction with Zn++ as a putative binding site. Since the mechanism of HpHtrA activity has not yet been intensively investigated, it is still unknown whether oligomerization is required for proteolytic activity or whether HpHtrA forms active oligomers in the zymogram after renaturation. But based on our data in this report, we conclude that the allosteric loop is important for the stability of oligomeric HpHtrA. Interaction with Zn++ might drastically change the conformation of HpHtrA, leading to an increase in oligomer stability, but also to an efficient inhibition of the proteolytic activity or substrate recognition. The detailed mechanism of HpHtrA oligomerization and activity will be further investigated in the future.Since the allosteric loop is important for HpHtrA regulation and ligand binding, we further investigated whether Znity Fig. . HpHtrA T) Table . Binding68A Fig. , the sig++ differ considerably from those obtained with Zn++. HpHtrA wt was only slightly stabilized at low Cu++ concentrations, as analyzed by Coomassie-stained SDS-PAGE under non-reducing conditions , its isogenic inactive mutant (HpHtrA S221A), as well as HpHtrA mutants S164A, D165A, S166A, and D168A was performed as previously described40. Briefly, transformed E. coli BL21 were grown in terrific broth (TB) medium to an OD600 of 0.7 and expression of glutathione S-transferase (GST)-HtrA was induced by addition of 100 µM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 h at 30 °C. The bacteria were pelleted and lysed by sonication in phosphate-buffered saline (PBS). The lysate was cleared by centrifugation and recombinant GST-tagged HpHtrA proteins were bound to glutathione sepharose beads . The GST tag was removed by addition of PreScisssion protease  for 16 h at 4 °C. After purification, the proteins were dialyzed against 50 mM tris(hydroxymethyl)aminomethane (Tris)/HCl (pH 7.5) and 150 mM NaCl. Purity of proteins was routinely determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie Brilliant Blue G250 .Recombinant human E-cadherin  was obtained from Sino Biological . According to the manufacturer’s protocol, the lyophilized hCdh1 was reconstituted in sterile water to give a concentration of 250 ng/ml. Casein was obtained from Carl Roth  and reconstituted in water. Purification of HtrA wild type (wt) from the 35. Briefly, 150 µg of the cellular proteome of MKN-28 cells was separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250. Protein lanes were sliced, cut into pieces, and destained twice with 150 μl 50% acetonitrile in 25 mM ammonium bicarbonate for 30 min at 25 °C and 1,200 rpm. The gel pieces were dehydrated with 100% acetonitrile and vacuum‐dried. The dehydrated pieces were incubated with 1 µM HpHtrA in 100 mM Tris (pH 8.0) and 50 mM NaCl for 2 h at 37 °C. Following in‐gel proteolysis, the generated peptides were extracted from the gel and analyzed by LC‐MS/MS using an Orbitrap LTQ Velos mass spectrometer coupled to EASY-nanoLC II HPLC unit (Thermo Fischer Scientific). Database searches were performed using the MaxQuant software package version 1.6.3.451 and UniProt derived human reference proteome , as described previously35. The cleaved peptides were identified with a length‐limited unspecific database search and aligned to generate a substrate specificity profile of the studied protease. Visualization of relative ratios as iceLogos52 provided unambiguous insight into the extended cleavage preferences of HpHtrA.Direct in-gel profiling of protease specificity (DIPPS) has been described previously55 was introduced into the sequence AQRVAF: the fluorophore  was coupled at the N-terminus, whereas the quencher  was positioned at the C-end. The FRET activity of this peptide was first confirmed by measuring the fluorescence emission in the range 400–450 nm before and after the trypsin-mediated cleavage of the peptide bond R_V  with a filter setting of 320 nm/425 nm (Ex/Em). Statistical analysis was performed with GraphPad Prism software (Vers. 8.0.2). One-way ANOVA was used to statistically compare changes in fluorescence between samples treated with HpHtrA wt or HpHtrA S221A with those treated with trypsin or with increasing concentrations of ZnCl2 or CuCl2, or the untreated control. Three independent experiments containing three technical replicates were analyzed for every sample. Significance is indicated as non-significant (ns) for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.For the establishment of a FRET-based assay for the quantification of the HpHtrA activity, the 2-Abz/3-nitrotyrosine FRET pairR_V Fig. . The mea2, MgCl2, MnCl2, ZnCl2, BaCl2, CuCl2, NiCl2, CoCl2, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) were added to the cleavage reactions. The proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G250 . Alternatively, 50 ng recombinant hCdh1 were incubated with 250 ng HpHtrA wt or inactive mutant for 16 h at 37 °C in combination with different chlorides as indicated. Proteins were separated using SDS-PAGE and blotted onto a nitrocellulose membrane. Human Cdh1 was detected by an antibody recognizing the EC5 domain of hCdh1  and a polyclonal serum was used to detect HpHtrA22. Independent experiments were repeated at least three times.For in vitro cleavage experiments, 10 µg casein were incubated with 250 ng HpHtrA wt  or inactive mutant in 20 µl 50 mM HEPES buffer (pH 7.4) for 16 h at 37 °C. Where indicated, 1 mM of CaCl2, MgCl2, MnCl2, ZnCl2, BaCl2, CuCl2, NiCl2, CoCl2, EDTA, or EGTA in 50 mM HEPES buffer (pH 7.4) for 60 min on ice. Non-reducing sample buffer without β-mercaptoethanol was added and the samples were separated by SDS-PAGE containing 0.1% casein  as a substrate. The gel was incubated for 60 min in renaturing buffer (2.5% Triton X-100) and equilibrated for 30 min in developing buffer  at room temperature under gentle agitation. Afterwards, the gel was incubated for 24 h at 37 °C in developing buffer under gentle agitation. To visualize caseinolytic activity, the gel was stained in 0.5% Coomassie Brilliant Blue R250 . For detection of multimers by SDS-PAGE, HpHtrA was separated by SDS-PAGE under non-reducing conditions. Proteins were visualized by staining with Coomassie Brilliant Blue G250. Independent experiments were repeated at least four times.HpHtrA wt was incubated with 1 mM of CaCl164A, D165A, S166A, D168A) in the presence of increasing concentrations of ZnCl2 or CuCl2. A change in the melting temperature in the presence ZnCl2 or CuCl2 indicates stabilization or destabilization of the protein and is therefore evidence for binding of Zn++ or Cu++ to HpHtrA. Recombinant HpHtrA proteins  in 50 mM HEPES buffer (pH 7.4) were mixed with SYPRO Orange  to final concentrations of 4 µM HpHtrA and 7 × SYPRO Orange in a 96-well PCR plate. Where indicated, increasing concentrations of ZnCl2 and CuCl2 were added. Fluorescence data was collected using an Applied Bioscience StepOne Plus PCR cycler  during a temperature ramp from 25–95 °C (0.5 °C per min) with a 4 min hold at 25 °C and 95 °C. SYPRO Orange binds to exposed hydrophobic regions within the denatured HpHtrA proteins, which increases the fluorescence of the dye58. The fluorescence emission signal can then be used to determine the melting temperature of the protein, which is located at the infliction point of the fluorescence signal curve. The experiment was performed in triplicates.Thermal shift assays were performed to determine the melting temperatures of recombinant HpHtrA proteins


Transient mesoscale oceanic eddies in Eastern Boundary Upwelling Systems are thought to strongly affect key regional scale processes such as ocean heat transport, coastal upwelling and productivity. Understanding how these can be modulated at low-frequency is thus critical to infer their role in the climate system. Here we use 26 years of satellite altimeter data and regional oceanic modeling to investigate the modulation of eddy kinetic energy (EKE) off Peru and Chile by ENSO, the main mode of natural variability in the tropical Pacific. We show that EKE tends to increase during strong Eastern Pacific (EP) El Niño events along the Peruvian coast up to northern Chile and decreases off central Chile, while it is hardly changed during Central Pacific El Niño and La Niña events. However the magnitude of the EKE changes during strong EP El Niño events is not proportional to their strength, with in particular the 1972/1973 El Niño event standing out as an extreme event in terms of EKE increase off Peru reaching an amplitude three times as large as that during the 1997/1998 El Niño event, and the 2015/2016 El Niño having instead a weak impact on EKE. This produces decadal changes in EKE, with a similar pattern than that of strong EP El Niño events, resulting in a significant negative (positive) long-term trend off Peru . It has been shown that the predominant direction of propagation of mesoscale eddies is westward5 and the total transport contribution can be comparable to that from large-scale wind and thermohaline driven circulation6. They can in particular rectify the mean circulation forming quasi-zonal jets or striations8, which appears conspicuous in eastern boundary current systems owing to the relatively weak mean circulation11. In the Southeast Pacific, eddy activity is also suspected to have a significant contribution to the heat budget3 with potential feedback to the tropical Pacific climate system considering the sensitivity of large scale winds to sea surface temperature (SST) in the far Eastern Pacific, in particular at El Niño-Southern Oscillation (ENSO) timescale13. The inability of low-resolution global climate models to account realistically for the eddy heat flux has been invoked as one of the reasons why these models have a persistent warm bias in EBUS14. Eddy-induced circulation was also shown to be essential for shaping the mean structure and seasonal variability in the oxygen minimum zones (OMZs) for the Southeast Pacific16 and Northeast Pacific17, with also probably important feedbacks to the climate system through their role on the biological pump. Understanding how eddy activity is modulated at climatic timescales is thus important for improving our knowledge of the circulation in EBUS and their role in the climate system, and particularly in the Southeast Pacific that is sensitive to tropical Pacific variability19 and that hosts one of the largest OMZs in the world20.In Eastern Boundary Upwelling Systems (EBUS), where the mean circulation is relatively weak, mesoscale eddies tend to play an important role in transporting water masses properties, from heat content to dissolved oxygen22, principally by the Peru–Chile Undercurrent (PCUC) in the Southeast Pacific22. There, climatologically forced regional oceanic models were shown to reproduce reasonably well the observed eddy activity as measured by mean eddy kinetic energy (EKE)3, indicating that it is dominantly associated with transient propagating eddies impacting the circulation at intraseasonal timescales of variability although eddies themselves can live longer than a few months24. This is consistent with a modeling study showing a relatively weak sensitivity of eddy activity to intraseasonal equatorial forcing25 despite the efficient oceanic teleconnection at such a timescale26. This is because equatorially forced intraseasonal fluctuations in coastal currents act as a high-frequency white noise to the instability process, and not as a background change over which instability characteristics (i.e. growth rate and frequency) can vary. At interannual frequency however, there are some evidences that mean EKE could be modulated by the equatorial variability. Chaigneau et al.23 show that eddy activity (and the number of generated eddies) has significant interannual variations off the coast of Peru and Chile, although they do not correlate to the NINO3.4 index, i.e. SST anomalies averaged in the region , the most commonly used index to measure ENSO strength. Conversely, the modeling study by Combes et al.27 found that the generation of subsurface anticyclonic eddies off central Chile is significantly correlated with the ENSO equatorial signal, which is associated with an increase of the PCUC transport. On the other hand, the modeling study of Dewitte et al.28, focused on the Peru region, suggested that eddy activity may be influenced by the ENSO amplitude modulation associated with the low-frequency changes in the frequency of occurrence of the two types of El Niño, that is the Eastern Pacific (EP) El Niño with maximum SST anomalies in the eastern equatorial Pacific, and the Central Pacific (CP) or Modoki El Niño with maximum SST anomalies concentrated in the central equatorial Pacific32. Besides their few numbers, the different methodological approaches and regional focus of these studies call for further investigation of the relationship between eddy activity and climate variability in this region. Here our objective is to revisit the relationship between ENSO, the dominant climate mode in the tropical Pacific, and mean EKE along the coast of Peru and Chile, taking into account the so-called ENSO diversity that broadly refers to the existence of at least two classes of El Niño events, with distinct dynamics33 and oceanic teleconnection in the Southeast Pacific34. We take advantage of 26 years of satellite altimeter data (1993–2018) that has now sampled two strong El Niño events (1997/1998 and 2015/2016), which allows addressing the impact of extreme events and that spans almost three decades, providing also insights on decadal variability. Through high-resolution regional oceanic modeling (ROMS), we are able to expand the time period of investigation over five decades (1958–2008)28 and address the modulation of ENSO diversity, which is an expression of the modulation in ENSO amplitude (i.e. moderate vs strong El Niño events) and patterns (CP vs EP) at decadal timescales. Regional model simulations, which include a Control Run (CR) and a sensitivity experiment to boundary forcing (Kelvin), are also used for investigating processes at work and forcing mechanisms.Eddies in the EBUS are generated by the instability of the coastal current systemt test. On the other hand, the instantaneous conditional correlation between EKE and the C index when C is negative (i.e. during La Niña events) reaches − 0.6 southward of the Pisco region (box 2), which is significant at the 95% level based on a student t test . Noteworthy a similar feature is found for the Kelvin simulation, which indicates that this originates from the equatorial oceanic forcing and could be related to specific ocean boundary conditions affecting the stability of the coastal current during the 1972/1973 El Niño (see below).In this section, model results are analyzed. We provide in the supplementary information the material for the assessment of the realism of the simulation used here to extend the observational analysis and investigate processes at work. Overall, our results indicate that despite a tendency of the model to overestimate mean EKEThe long-term simulation offers the opportunity to document the decadal EKE variability, and supports the interpretation of the one evidenced from observations which only span 26 years. The dominant mode of decadal EKE variability for both altimetric observations and model simulations are comparable Fig. . The strWhile the 1972/1973 El Niño event can be considered as a strong event since it has a comparable value of the NINO3.4 index than other strong El Niño events  that have been recorded in the last five decades, it did not have an Eastern Pacific warming as high as in the 1982/1983 and 1997/1998 El Niño events, with an E value less than half of those years Fig. . Still i36 (not shown). With such limitation in mind, from our results, we can support the fact that the 1972/1973 El Niño event was associated with a large increase in EKE off Peru, suggesting a non-linear relationship between the El Niño strength as measured by SST indices and the magnitude of eddy activity changes off Peru and northern Chile  and central Chile (37°S–25°S). In the Peru region, the PeKe appears to be the dominant EKE generation term whereas in central Chile the three energy conversion terms have a comparable contribution. To document their low-frequency variations, an EOF analysis is performed separately over these two regions, which provides coastal profiles as dominant mode patterns for both interannual and decadal timescales  region, with PC1-PCUC ahead PC1-PeKe by ~ 3–5 months (maximum correlation larger than 0.5)  Fig. a. The pho38 Fig.  indicateo38 Fig. , S6 are 28, overcoming in particular limitations in atmospheric forcing products40. The study also takes into account the ENSO diversity33 that refers to the existence of two types of El Niño events with distinct spatial pattern and amplitude31. It is shown that the interannual EKE variability is mostly associated with strong EP El Niño events. The latter yields an enhanced (reduced) mesoscale activity off Peru and northern Chile . On the other hand, during CP El Niño (La Niña) events, the mesoscale activity is hardly changed, with a weak increase (decrease) off Peru and a reduction (increase) southward of the Pisco region (15°S). While model results are consistent with our findings from altimetry, they also indicate that the EKE-ENSO relationship arises mostly from the oceanic teleconnections since the model simulation with steady atmospheric forcing (Kelvin) yields comparable results. This is consistent with ENSO-related equatorial Kelvin wave propagating along the coast modulating coastal current stability and stratification. In particular, this oceanic teleconnection is distinct for EP and CP El Niño events, which explains why CP El Niño events are not influential in EKE. First the oceanic teleconnection during CP El Niño event is much less energetic than during EP El Niño which is reflected into the magnitude of SST anomalies along the coast of Peru28. This is due to the fact that the energy of the equatorial Kelvin waves during CP El Niño is less than during EP El Niño and they cannot propagate far into the eastern equatorial Pacific, because they encounter a density front near 120°W along the equator associated with the sloping thermocline and dissipate there41. Second, the energy of equatorial Kelvin waves during CP El Niño events peaks in the intraseasonal frequency band  so that they are trapped along the coast, conversely to equatorial Kelvin waves at interannual timescales that radiate as extra-tropical Rossby waves once they reach the South-American coast26. This trapping makes that they are much less influential on the baroclinic instability process25. This interpretation is confirmed by the results of the EKE budget that indicates that the baroclinic instabilities are modulated at interannual to decadal timescales. The results also suggest a significant non-linearity in the relationship between the amplitude of ENSO  and the EKE changes. For example, the model suggests that the 1972/1973 El Niño yielded the strongest EKE change off Peru, while the altimeter observations indicate that EKE was weakly impacted during the strong 2015/2016 El Niño, although these two events have a comparable amplitude in terms of the E index  conditions. Interestingly, it also was associated to a relatively weak negative EKE anomaly off Peru  yields a long-term negative trend in EKE off Peru. Similarly, the observational record evidences a significant decreasing trend in EKE due to the large positive anomaly during the 1997/1998 El Niño event. This indicates that strong EP events have the ability to produce decadal changes in EKE not necessarily related to natural mode of decadal variability in the Southeast Pacific like the IPO. We find a weak correlation between the IPO index and the PC-1 timeseries associated with decadal variability in EKE  larger than over 1978–2008 over the Peru domain in CR (Kelvin) simulation), which is consistent with this hypothesis. We also note that in the regional simulations, the PCUC transport changes during the 1972/1973 El Niño are twice as large as during the 1997/1998 El Niño event , the zonal pressure gradient across the equator is enhanced, which is associated to an overall increase in the equatorial trade winds51. This tends to increase the EUC transport which variability is correlated to the PCUC anomaly at seasonal and interannual timescales37. However the EUC is not directly connected to the PCUC. Montes et al.52 show that in fact the PCUC is predominantly fed by two narrow eastward subsurface currents  at ~ 3–4°S (primary) and 7–8°S (secondary), which dynamics are highly non-linear53. The recharge process also involved non-linear processes54 and can be influenced by atmospheric high-frequency noise55 so that the relationship between both the ENSO recharge and the EUC, and between the EUC and the PCUC may deviate from linearity.In this paper, we documented the modulation of mesoscale activity in the Southeast Pacific at ENSO timescales through observations and long-term numerical simulations. The satellite altimeter data have now sampled two strong El Niño events, which allows getting insight into the relationship between ENSO and eddy activity. Here we address this issue aided by a previous modeling work dedicated to producing a realistic regional long-term simulation of the Southeast Pacificdex Fig. a and had region 1°S. While region 1°S. Whileent Fig.  and is aent Fig. , althoug56 and in-situ observations57 suggest that subsurface eddies or “Intrathermocline Eddies” (ITEs), also called Puddies58, exist in the Southeast Pacific which tend to be anticyclonic. These eddies have a weaker surface signature and are thus not well observed by altimetry, although they represent a significant source of natural variability in the euphotic layer. They are also thought to be connected to the variability of the PCUC56, which suggests that their activity could be also modulated by strong EP El Niño events. In particular we find that in the model, EKE off central Chile increases at 200 m during strong EP El Niño  product to estimate the surface geostrophic EKE. This data set is based on altimetric data from DUACS (Data Unification and Altimeter Combination System) of AVISO . Data are globally gridded from 1993 to present as daily values with a spatial resolution of 1/4°. We also use sea level anomaly (SLA) data and SST anomalies to validate the model simulation over the period prior to the satellite era. SLA data from tide gauge was provided by the UHSLC (University of Hawaii Sea Level Center), while SST data was obtained from HadSST361 , which provides monthly uninterpolated SST anomalies in 5° × 5° grid box for 1850-present. The anomalies are relative to a 30-year climatology spanning 1961–1990. HadSST3 is based on in-situ measurements of SST from ships and buoys.We use sea surface height from the GEKCO62  model is used over the 1958–2008 period developed in Dewitte et al.28. This model configuration, which is used as a Control Run (CR) simulation, has a horizontal resolution of 1/12° at the equator, covering the domain extending from 12°N to 40°S, and from the coast to 88°W. 5-daily mean oceanic outputs from SODA  provide the open boundary conditions (OBC) for temperature, salinity, horizontal velocity and sea level. To force the regional model at the air/sea interface, wind speed and wind stress from the downscaled product39 were used, which was shown to provide realistic forcing from atmospheric reanalysis that generally do not resolve properly near-shore winds63. Atmospheric fluxes were derived from the bulk formula using the air temperature from COADS64 (Comprehensive Ocean–Atmosphere Data Set) 1° monthly climatology. The reader is invited to refer to Dewitte et al.28, Vergara et al.34, Pizarro-Koth et al.65, and to supplementary information for more details on the model description, skill and validation. To evaluate the impact of equatorial forcing onto EKE variability, a sensitivity experiment to atmospheric forcing was performed. This experiment, called Kelvin, consists in using the same oceanic boundary conditions than CR simulation but using a wind forcing corresponding to the 2005 year repeated each year. This 2005 year is selected as a “normal” year in terms of surface atmospheric circulation in the SEP despite the development of a CP El Niño event in 2004. This Kelvin simulation allows assessing the role of the equatorial oceanic forcing on the EKE modulation off the Peru–Chile coasts.A long-term simulation with the ROMS31 are used, that are based in the principal components time series associated with the first two EOF modes of the monthly mean SST anomalies in the tropical Pacific  product of NOAA  over the 1958–2018 period. The two uncorrelated indices are well suited for describing the so-called ENSO diversity33 by accounting for the variability of Eastern Pacific El Niño (E-index) and Central Pacific El Niño and/or La Niña (C-index). In addition, the Tripole Index for the Interdecadal Pacific Oscillation (IPO) was obtained from NOAA/ERSST.v3b over the same period , which was used as an OBC in the regional model.The two El Niño indices defined by Takahashi et al.see Fig. a. The SSsee Fig. a. The in68. While these studies provide key information on eddy activity, methodological69 and observational70 limitations remain. Here, for simplicity and to avoid comparing different methodological approaches, mesoscale activity is measured from the estimate of EKE (estimated as EKE = (u′g2 + v′g2)/2), which represents a physically-based metrics that obeys a tracer equation and thus can be objectively interpreted from theory. The EKE was obtained from 5-days detrended surface geostrophic currents anomalies , which are derived from sea surface height. Interannual anomalies were computed from 5-days mean outputs removing the monthly climatology interpolated at 5-days temporal resolution. Then, mean EKE over 3-months running windows followed by a monthly average is estimated, providing series of monthly “mean EKE”, which we refer as just EKE. The seasonal cycle from monthly EKE was further removed to derive EKE anomalies. Intraseasonal anomalies in the velocity field were also considered to derive EKE anomalies to filter out the contribution of changes in the circulation at interannual timescale. The intraseasonal anomalies consist of the departure from the monthly mean following previous studies72. In either case, a 1-year running mean filter is further applied on the resulting anomalies to filter out remaining high-frequency variations associated with the low-frequency modulation of the seasonal cycle. The obtained interannual EKE field is then analyzed through regressing on indices and/or EOF decomposition. For addressing decadal EKE variability, a butterworth filter (order 5 with a 10 years cut-off period) is used.Most of the existing literature on mesoscale activity in the Southeast Pacific has used geometric methods to count the number of mesoscale structure either directly from dynamic height data or estimate of vorticity25: wind work  into EKE, and PeKe corresponds to the baroclinic conversion of eddy potential energy (Pe) into EKE. From the horizontal equation of motion, one can derive the expressions:x,τy), velocity field  and density (ρ) variables with prime stand for the anomalies relative to the mean climatology (overbar) calculated over the 1958–2008 period. Since we are interested here in the modulation of these terms at interannual timescales, the brackets stand for the mean over a 3-month running windows to capture transient eddy effect on EKE rate of change. For methodological consistency, anomalies were then calculated in a similar manner than EKE. The constant variables are the gravitational acceleration (g = 9.8196 m/s2) and the reference water density (ρ0 = 1025 g/L). KmKe and PeKe were integrated over the surface layer depth (H = 100 m), where the instabilities in the Southeast Pacific are confined25 and can be directly comparable to wind work.The contribution of each EKE generation process is evaluated from the regional model based on the tracer equation for EKE. We focus here on the energy conversion terms relevant to nearshore following previous studiesThe integrated poleward transport of the PCUC was calculated considering the average of along-shore southward current over the area coinciding with the mean position of the PCUC, as inferred from cross-shelf sections along the coasts see Fig. . We consSupplementary Information.