Though the mechanism of extra-matrix Ca2+ dependency of mitochondrial Ca2+ uptake has been well characterized both experimentally and mathematically, the mechanism of membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake has not been completely characterized. In this paper, we perform a quantitative reevaluation of a previous biophysical model of mitochondrial Ca2+ uniporter that characterized the possible mechanism of ΔΨ dependency of mitochondrial Ca2+ uptake. Based on a model simulation analysis, we show that model predictions with a variant assumption , that provides the best possible description of the ΔΨ dependency, are highly sensitive to variation in matrix [Ca2+], indicating limitations in the variant assumption (Case 2) in providing physiologically plausible description of the observed ΔΨ dependency. This sensitivity is attributed to negative estimate of a biophysical parameter that characterizes binding of internal Ca2+ to the uniporter. Reparameterization of the model with additional nonnengativity constraints on the biophysical parameters showed that the two variant assumptions (Case 1 and Case 2) are indistinguishable, indicating that the external and internal Ca2+ binding constants for the uniporter may be equal (Case 1). The model predictions in this case are insensitive to variation in matrix [Ca2+] but do not match the ΔΨ dependent data in the domain ΔΨ≤120 mV. To effectively characterize this ΔΨ dependency, we reformulate the ΔΨ dependencies of the rate constants of Ca2+ translocation via the uniporter by exclusively redefining the biophysical parameters associated with the free-energy barrier of Ca2+ translocation based on a generalized, non-linear Goldman-Hodgkin-Katz formulation. This alternate uniporter model has all the characteristics of the previous uniporter model and is also able to characterize the possible mechanisms of both the extra-matrix Ca2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake. In addition, the model is insensitive to variation in matrix [Ca2+], predicting relatively stable physiological operation. The model is critical in developing mechanistic, integrated models of mitochondrial bioenergetics and Ca2+ handling.Mitochondrial Ca Mitochondrial Ca2+ homeostasis is critical for metabolic regulation, mitochondrial function/dysfunction, and cell physiology/pathophysiology 2+ uptake via the uniporter is essential for developing mechanistic, integrated models of mitochondrial bioenergetics and Ca2+ handling that can be helpful in understanding the mechanisms by which Ca2+ plays a role in mediating signaling pathways between cytosol and mitochondria and modulating mitochondrial energy metabolism in health and disease Mitochondrial Ca2+ uptake depends on the catalytic properties of the uniporter and also on the electrochemical gradient of Ca2+ across the inner mitochondrial membrane (IMM), which has been extensively studied both experimentally 2+ dependency of mitochondrial Ca2+ uptake has been well characterized, the mechanism of membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake has not been completely characterized.The kinetics of mitochondrial Ca2+ uniporter  under two different kinetic assumptions  were formulated to characterize the extra-matrix Ca2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake via the uniporter 2+ dependent data 2+ uptake.In the development of our recent model of the uniporter 2+] was unknown from the measurements. For model parameterization, matrix [Ca2+] was fixed at 250 nM. Although the two variant models under Case 2 were able to adequately describe all the available experimental data with appropriate model perturbations as provided by the experimental protocols, it was unknown whether physiological variation of matrix [Ca2+], as seen in the intact myocyte, have significant impacts on the estimates of model parameters and model predicted trans-matrix Ca2+ fluxes via the uniporter. Therefore, it is important to test the robustness of the estimates of model parameters and model predictions subject to such physiological variation.The four variant models of the uniporter 2+] (ranging from 100 nM to 500 nM), suggesting that the model parameter estimates under Case 2 would vary significantly to variation in matrix [Ca2+], and hence can not be robust. This indeed indicates that the Case 2, in which the Ca2+ binding constants for the uniporter at the inside and outside of the IMM are distinct, is physiologically implausible, and hence can not be a feasible representation of the observed ΔΨ dependency of the uniporter-mediated mitochondrial Ca2+ uptake. Furthermore, the Case 2 is associated with negative estimates of the biophysical parameter αx (with αe = 0 fixed)  are indistinguishable from each other, indicating that the external and internal Ca2+ binding constants for the uniporter may be equal (Case 1). The model predictions in this case are insensitive to variation in matrix [Ca2+], but do not match the ΔΨ dependent data In the present paper, we attempt to provide a quantitative reevaluation of our previous model of the uniporter xed) see , which i2+ uptake via the uniporter in the entire ΔΨ domain for which data are available kin and kout of Ca2+ translocation in our previous model of the uniporter βe and βx associated with the free-energy barrier of Ca2+ translocation based on a generalized, non-linear GHK (Goldman-Hodgkin-Katz) formalism  under both the kinetic assumptions (Case 1 and Case 2) is chosen, because both the fully and partial cooperativity binding models (Model 1 and Model 2) are indistinguishable from the available experimental data This section presents the detailed simulation analyses of our previous model of mitochondrial Ca2+ uptake based on our previous model (Model 1) of mitochondrial Ca2+ uniporter The simulation analyses of mitochondrial Ca2+ influx via the uniporter were measured in suspensions of energized mitochondria purified from rat hearts and rat livers following additions of varying levels of extra-matrix Ca2+   (2+] was fairly unknown. Our previous model of the uniporter 2+] of 250 nM. 2+] on the estimates of model parameters and model predicted Ca2+ fluxes via the uniporter.In the experiments of Scarpa and Graziotti ctively) . In the  = 0 mM) ; ΔΨ was 2+ fluxes via the uniporter (lines) as functions of extra-matrix [Ca2+] (ΔΨ = 190 mV) and ΔΨ  over a range of matrix [Ca2+] along with the experimental data 2+] was varied from 100 nM to 500 nM. The simulations corresponding to the model fits to the data are based on matrix [Ca2+] of 250 nM. 2+ fluxes via the uniporter as a function of extra-matrix [Ca2+] for a range of ΔΨ (100 mV to 210 mV) and as a function of ΔΨ for a range of extra-matrix [Ca2+] (10 µM to 150 µM), with matrix [Ca2+] fixed at 250 nM.Specifically, 2+] fixed at 250 nM is able to fit well to all of the available experimental data 2+]  , it is unlikely that physiological variation of matrix [Ca2+] would have any appreciable effects on the experimental measurements and model predictions on Ca2+ fluxes via the uniporter as well as on the estimates of the uniporter model parameters. Furthermore, it is unlikely that in the experiments of Scarpa and colleagues 2+] would have been precisely maintained at 250 nM. Therefore, the present model simulation analyses suggest that the model parameter estimates under Case 2 would vary considerably with variation in matrix [Ca2+] as well as with different initial guesses for the parameters, compared to that under Case 1. In the other words, the model parameter estimates under Case 2 would be ambiguous and not unique (robust), and are expected to be different for different matrix [Ca2+] and different initial guesses for the parameters. Given matrix [Ca2+], the initial guesses for the parameters need to be close to the optimal parameter estimates for the optimization algorithm to converge to the optimal parameter estimates. In this case, the sensitivities of the model to variations in matrix [Ca2+] would also be different for different model parameter estimates with similar fittings of the model to the experimental data.It is apparent from the model simulation analyses in αx (with αe = 0 fixed), which is found to be contributing to the high sensitivities of the model predictions to variation in matrix [Ca2+] and stiff gradients of Ca2+ uptake profiles to variation in ΔΨ. The Ca2+ uptake profiles under Case 2 attaining saturation for a lower level of extra-matrix [Ca2+] is attributed to the lower estimates of 2+] to the uniporter , as in our previous paper on the uniporter 2+ dependent kinetic data of Scarpa and Graziotti α, βe, and βx are arbitrarily chosen to satisfy the two kinetic and thermodynamic constraints of Eq. (S7) as well as to fit these data sets, as these parameters can not be accurately estimated from these data sets in which ΔΨ is constant. These extra-matrix Ca2+ dependent data sets, however, provide accurate estimates of the binding constants α, βe, and βx are estimated from the ΔΨ dependent kinetic data of Wingrove et al. α, βe, and βx. In the final step, with the values of α, βe, and βx fixed, as estimated from the first and second steps, the rate constants 2+ in the experimental buffer).As shown in rter see . To recoαe = αx = α≈0, βe≈0.113, and βx≈0.887. The new model fittings to the data for both the cases (Case 1 and Case 2) are exactly same as those shown in 2+ binding constants for the uniporter were assumed to be distinct  of mitochondrial Ca2+ uniporter, in which αe = αx = α, and βe and βx are functions of ΔΨ and nH (see Eq. 7), are shown in 2+ dependent kinetic data of Scarpa and coworkers 2+ to the uniporter with arbitrary values of α, and nH. This estimated α, and nH) are estimated from the ΔΨ dependent kinetic data of Gunter and coworkers α, and nH fixed, the rate constant The simulation analyses of mitochondrial Ca2+ fluxes via the uniporter (lines) as functions of extra-matrix [Ca2+] (ΔΨ = 190 mV) and ΔΨ  with the variation in matrix [Ca2+] (100 nM to 500 nM) and their comparisons to the available experimental data 2+] = 250 nM. 2+ fluxes via the uniporter as a function of extra-matrix [Ca2+] for a range of ΔΨ (100 mV to 210 mV) and as a function of ΔΨ for a range of extra-matrix [Ca2+] (10 µM to 150 µM), obtained with matrix [Ca2+] = 250 nM. The left, middle, and right plots correspond to the model simulation analyses based on the experimental protocols of Scarpa and Graziotti 2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake via the uniporter in the entire ranges of extra-matrix [Ca2+] and ΔΨ for which data were available. In addition, this alternate uniporter model is insensitive to variation in matrix [Ca2+], making the model physiologically plausible. This characteristic of the model helps provide unique and accurate estimates of the model parameters with different matrix [Ca2+]. It is observed from 2+ uptake profiles, obtained from the present alternate uniporter model, do not have stiff gradients with respect to ΔΨ, and reach saturation for a higher level of extra-matrix [Ca2+], comparable to that obtained under Case 1 of our previous uniporter model  to the extra-matrix Cavely see . These dβe and βx that characterize the ΔΨ dependent factor E(ΔΨ) in the uniporter flux expressions , and hence the ΔΨ dependency of mitochondrial Ca2+ uptake via the uniporter βe and βx are analytical functions of ΔΨ (Eq. 7), while the dotted lines are based on our previous model of the uniporter βe and βx are constants and are numerically estimated. The results show that βe and βx based on the previous model abruptly change their respective values at ΔΨ = 0, resulting in non-differentiability of the factor E(ΔΨ) at ΔΨ = 0. In contrast, βe, βx, and E(ΔΨ) based on the present model are smooth functions of ΔΨ. The ΔΨ dependent factor E(ΔΨ) in the present model has a larger spread, resulting in a better fit of the model to the ΔΨ dependent data The ΔΨ dependencies of the biophysical parameters 2+ uniporter 2+ into energized (respiring) mitochondria, and hence plays an important role in mitochondrial Ca2+ homeostasis. Specifically, the present paper provides an alternate (improved) biophysical model of the uniporter that overcomes the limitations of our previous uniporter model by mechanistically recharacterizing the membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake via the uniporter. This alternate uniporter model is able to accurately characterize the possible mechanisms of both the extra-matrix Ca2+ and ΔΨ dependencies of the uniporter-mediated mitochondrial Ca2+ uptake +/Ca2+ exchanger 2+ handling that can be helpful in understanding the mechanisms by which Ca2+ plays a role in mediating signaling pathways between cytosol and mitochondria and modulating mitochondrial energy metabolism in health and disease.The major contributions of the present paper lies in the improvements of our previous biophysical model of mitochondrial Ca2+ translocation) 2+ to the uniporter, as depicted experimentally 2+ to the uniporter; Case 1 or Case 2: external and internal Ca2+ binding constants for the uniporter are equal or distinct) to several independent experimental data sets from the literature on the kinetics of Ca2+ fluxes via the uniporter 2+ dependent data of Scarpa and coworkers 2+ to the uniporter are distinct (Case 2). Therefore, the mechanistic formulation, thermodynamic feasibility, and ability to describe a large number of independent experimental data sets are some of the notable features of the model 2+ translocation, the present model has all the characteristics of the previous model.Our previous model of the uniporter 2+] was not measured. Our two variant models of the uniporter under two different cases 2+] of 250 nM. Although both the variant models under Case 2 were able to satisfactorily explain all the available experimental data with suitable model perturbations as provided by the experimental protocols, it was not realized whether physiological variation of matrix [Ca2+], as observed in the intact myocyte, will have any appreciable effect on the estimates of model parameters and model predicted trans-matrix Ca2+ fluxes via the uniporter. The present study provides a quantitative reevaluation of the previous uniporter model 2+] ranging from 100 nM to 500 nM. Based on this model simulation analysis, it is found that the two variant model predictions under Case 2 are highly sensitive to variation in matrix [Ca2+]   , attribu2+ uniporter 2+ binding constants for the uniporter were assumed distinct  , and the Eq. B7; . The altvailable . In addi2+ signaling 2+ in distinct region of the cells and maintain spatial Ca2+ concentration low even under strong global Ca2+ mobilization upon cell stimulation is critical for the Ca2+ sensitive signal transduction within the cell 2+ oscillations and waves, generated by the Ca2+ release from the endoplasmic reticulum 2+ oscillations, the mitochondrial Ca2+ uptake has also been shown to influence significantly the nature of Ca2+ oscillations 2+ entry 2+ uptake has considerable effect on the STIM1-Orai1-dependent store operated Ca2+ entry into endothelial cells 2+ uniporter will be crucial for developing an integrated model of intracellular and mitochondrial Ca2+ handling which can be helpful in understanding many aspects of signal transduction mechanisms.Recently, mitochondria have been recognized as one of the key organelles that actively involves in physiological Ca2+ uniporter that accurately characterizes the ΔΨ dependency of mitochondrial Ca2+ uptake via the uniporter kin and kout of Ca2+ translocation  formulation. The expressions for the binding constants Ke and Kx and the equilibrium constants Keq remain the same as in Eqs. (S4) and (S6) of 2+ and ΔΨ dependencies of the uniporter-mediated mitochondrial Ca2+ uptake 2+], making the model physiologically plausible.This section provides the derivation of the present alternate (improved) biophysical model of mitochondrial Ca Eq. S5,  in our pkin and kout of Ca2+ translocation via the uniporter (see Eq. S5) can be expressed in the following form kin and kout and Eq. (S4) for Ke and Kx into Eqs. (S1–S2), the uniporter flux expression is reduced toD = D1 for Model 1 (Eq. S9a) and D = D2 for Model 2 (Eq. S9b). In order to derive the functional form of 2+ transport via the uniporter 2+ transport via the uniporter in the absence of ΔΦ nH, and hence the two rate constants kin and kout in Eq. (1) are fully characterized by only three unknown parameters nH, in contrast to four unknown parameters βe and βx in the previous formulation  and D = D2 for Model 2 (Eq. S9b). By comparing the present uniporter model (Eq. 5) with the previous uniporter model (Eq. S8), we obtain the following functional relationship between the biophysical parameters αe, αx, βe, βx, and nH:βe and βx can be expressed in terms of ΔΦ asβe and βx in the present model of the uniporter become functions of ΔΨ, compared to the previous model of the uniporter βe and βx were constant with respect to ΔΨ. Furthermore, both the parameters are characterized by only one unknown parameter nH.Based on a generalized, non-linear GHK formulation, the rate constants tion see . In stan2+ fluxes via the uniporter subject to the constraints: αe = αx = α≥0 (Case 1). Thus, only four unknown parameters Nexp is the number of experiments and Ndata is the number of data points in a particular experiment, 2+ uptakes and EClick here for additional data file.Table S12+ uniporter.Estimated parameter values in the previous models of mitochondrial Ca(0.09 MB DOC)Click here for additional data file.


In this study, we extend these investigations asking specifically whether the TNF-α effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-α on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-α significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-α were inhibited by the general protease inhibitor α2 macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-α on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF- Proteolytic degradation of the basement membrane and the extracellular matrix (ECM) and movement through it are regarded as essential steps in the cascade of events leading to successful tumour cell invasion and metastasis. Studies on human melanoma have provided evidence that the plasminogen activator system and the matrix metalloproteinase (MMP) enzyme systems play an important role  opposed cell attachment to ECM substrates and cell invasion through fibronectin, both of which would be consistent with TNF-α promoting metastasis and α-MSH providing partial protection against the action of TNF-α.Studies on the effects of TNF-α on melanoma cell migration and degradative enzyme activity.In the current study, we extend our investigations into the role of inflammation in melanoma invasion by studying the effects of TNF-α on melanoma cell general proteolytic enzyme expression and expression/activation of MMPs -2 and -9 and studied the extent to which degradative enzymes are involved in the TNF-α induced cell responses by looking at the effect of the general protease inhibitor α2 macroglobulin on TNF-α stimulated cell migration and invasion.Specifically, we examined the effect of TNF-α and α2 macroglobulin from Roche Diagnostics (Germany); and fluorescent BODIPY® casein (EnzChek® Protease Assay Kit-green fluorescence) from Molecular Probes Inc. . Tissue culture plastics were purchased from Corning Costar Corporation . All other chemicals were of analytical grade.Culture medium, additives and antibiotics were obtained from Gibco BRL  and Sigma Chemicals Ltd. . Foetal calf serum (FCS) was purchased from GlobePharm Limited ; newborn calf serum (NBCS) from Sigma Chemicals Ltd. ; trypsin/ethylenediaminetetraacetic acid (EDTA) and phosphate-buffered saline (PBS) tablets from Oxoid Ltd. . Human fibronectin was purchased from Sigma Chemicals Ltd. ; human recombinant TNF-−3 mol l−1 glutamine, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin sulphate.The human cutaneous melanoma cell line HBL was established in Professor Ghanem's laboratory from a lymph node metastasis of a nodular malignant melanoma , 0.2% (w/v) D-glucose solution, 0.1% (w/v) bovine serum albumin (BSA), 2 × 10−3 mol l−1 glutamine, 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin sulphate and the relevant concentration of TNF-α was added. This serum-free medium was used for the fibronectin invasion assays as described later (serum-free invasion assay medium – SFIAM) and subsequently in all the assays where a 24-h preincubation period was required in order to keep the experimental culture conditions the same.For the experiments where a 24-h preincubation was required, the cell culture medium was changed to RPMI-1640 medium (Gibco) supplemented with 20 ng mlα and α2 macroglobulin on the invasion of the melanoma cells through a layer of human fibronectin. Briefly, a suspension of 1.8 × 105 HBL melanoma cells (contained in 500 μl SFIAM with or without the appropriate drug concentration as required) was added to Nunc™ tissue culture inserts containing a polycarbonate filter with 8 μm diameter pores covered with fibronectin (100 μg ml−1). Cells were incubated for 20 h under standard culture conditions. At the end of the incubation period, the cells that had invaded through the filter (cells attached to the bottom of the well and to the undersurface of the filter) were collected separately from the cells that had not invaded (cells remaining attached to the upper surface of the filter). A combined analysis of the cell counts was used to calculate the percentage of the total population of cells that had invaded through the fibronectin monolayer.A modification of the method we first described in α 200 U ml−1 for the last 24 h before passaging and adding to the Nunc™ tissue culture inserts.When a preincubation period was required prior to the invasion assay, the cells were cultured in SFIAM with or without TNF-α2 macroglobulin was used as a general protease inhibitor after initially confirming its activity against trypsin prior to using it to inhibit proteolytic activity in the cell culture medium during the fibronectin invasion assay.α-stimulated cells (incubated with TNF-α 200 U ml−1 for 24 h) were detached from the tissue culture flask and were lysed using cell extraction buffer  for 15 min at 4°C. The cell conditioned medium was also collected for gelatin zymography analysis (following centrifugation at 2000 g for 5 min to remove any cellular debris).Cell extraction of MMPs was performed when cell cultures were 70–80% confluent. Control and TNF-3 cells per well with or without TNF-α  for 24 h. At the end of the preincubation period, the conditioned medium was collected for measurement of proteolytic activity, and extraction from the cells was performed according to the protocol described above for extraction of MMPs.For the purpose of extraction of general proteolytic activity, the melanoma cells were cultured in a 24-well plate at a density of 50 × 10s-indacene-3-propionic acid (BODIPY® FL) . BODIPY® FL casein is a conjugate consisting of casein labelled with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-μg ml−1 was used in each experiment to demonstrate proteolytic degradation of BODIPY® casein. The above concentration was selected after initial experiments to confirm that the fluorescence emitted by the cleaved substrate was proportional to the concentration of trypsin. The following controls were also included in each assay: BODIPY® casein alone, fresh culture medium (SFIAM) with BODIPY® casein and extraction buffer with BODIPY® casein. Cell extracts were diluted 1 : 20 in assay buffer before being added to the fluorescent substrate as experimentation showed this to be the optimum dilution to minimise the effect of the extraction buffer itself on the fluorescent BODIPY® substrate.As an internal control for the assay, trypsin at a concentration of 3.3 α treated cell extracts (24-h cell preincubation in SFIAM containing TNF-α 200 U ml−1 as described above) and conditioned medium (collected at the end of the 24-h preincubation period). Proteins were separated in nonreducing, nondenaturing conditions using a 10% resolving polyacrylamide gel containing 0.5% gelatin substrate. Internal human purified MMP-2 and MMP-9 standards  were included at concentrations of 20 ng per lane. After electrophoresis, the zymographs were developed, fixed and stained according to the protocol described by Zymography was carried out on control (nontreated) and TNF-A modified ‘scratch wound’ migration assay was used for the assessment of the migration of the melanoma cells. The method was previously described by 5 cells per ml per well and were incubated for 24 h under standard culture conditions. After 24 h, when the cells had attached to the bottom of the tissue culture dish, the confluent cell monolayer was scratched with a plastic pipette tip to create a cell free zone in the well. The culture medium was removed and was replaced with an equal volume of fresh SFIAM with or without TNF-α 200 U ml−1 or α2 macroglobulin. The cells were incubated for a further 24 h under standard culture conditions. During this period, photographs of the scratched cell monolayer were taken at predetermined time points  using an Apple McIntosh video microscope (Open Lab 3.0.4 software) and measurements of the distance between the two edges of the scratch were recorded for each different time point. The percentage reduction of the distance between the scratch edges at the different time points represented the migration rate of the melanoma cells. Triplicate wells were used for each experimental condition.Cells were plated in a 24-well culture plate in serum containing Ham's F10 culture medium at a density of 2 × 10vs stimulated cell samples were analysed using Student's paired t-test for fibronectin invasion assays, migration assays and for general protease activity assays.Control α ranging from 100 to 500 U ml−1 on HBL melanoma cell invasion through fibronectin over 20 h. Unstimulated (control) cells showed a mean invasion rate of 24.6±2.7% (n=4 experiments). Stimulation with TNF-α 100 U ml−1 significantly increased the invasion rate of the cells to 33.3±3.5% (P<0.05), which represents an increase of +35% above the control level of invasion. Stimulation with higher concentrations of TNF-α (250 and 500 U ml−1) was less effective and the increases in invasion did not reach statistical significance.Figure 1Aα, cells were preincubated with TNF-α for 24 h prior to the invasion assay and were also subsequently stimulated with TNF-α again during the period of the invasion assay (a further 20 h). Preincubation with TNF-α resulted in a 43% increase in invasion above control level . Further addition of TNF-α to the invasion assay did not increase any further the invasiveness of these preincubated cells (results not shown). On the basis of these data, we concluded that the biological effects of TNF-α on invasion had occurred fully within 24 h.To determine the optimum period of exposure for maximum response to TNF-α on melanoma cell migration. Cells stimulated with TNF-α 200 U ml−1 (cytokine present during the 24-h assay) had a significantly higher migration rate compared to the control (nonexposed to TNF-α) cells. The migration rate (represented by the percent reduction of the scratch width) of the TNF-α stimulated cells was 53.2±4.4% by 24 h, whereas the migration rate of the control cells at the same time point was 43.8±2.9%. This represented a 21% increase in migration as a result of TNF-α stimulation, P<0.05 based on eight experiments (as illustrated in α stimulated) at 8 or 24 h.A ‘scratch wound’ model was used to examine the effect of TNF-α 200 U ml−1for 24 h prior to cell extraction and collection of cell conditioned medium for gelatin zymography . α2 macroglobulin at a concentration of 2 U ml−1 was used for future experiments as this achieved 84% inhibition of trypsin activity.To confirm that the fluorescence emitted by cleavage of the BODIPY® casein substrate was representative of proteolytic enzyme activity, initial experiments used serial dilutions of trypsin  and the lower chamber (invading cells) was sampled at the end of the 20-h assay period and the fluorescence of the sample, representing protease activity, was measured. TNF-α did not increase general protease activity in the conditioned media in these experiments (results not shown).To measure noninvasively whether cell surface bound proteolytic activity was modified by TNF-α on proteolytic activity of HBL melanoma cells, it was still conceivable that levels may have been beneath the level of detection of these biochemical assays and that proteases played some role in the invasion of these cells through fibronectin. Accordingly, we used α2 macroglobulin, an inhibitor of all classes of endoproteases, to examine its effect on control and TNF-α stimulated HBL melanoma cells. α2 macroglobulin inhibits proteases present in the cell conditioned medium or on the cell surface, but it cannot enter the cell or penetrate rapidly into the extracellular matrix due to its large size (720 kDa tetrameric glycoprotein).Although we had failed to show any effect of TNF-α2 macroglobulin (2 U ml−1) was added to the cell suspension in the upper chamber with or without TNF-α 200 U ml−1 for the 20-h culture period of the assay. Controls included cells with no stimulants and cells where only TNF-α was added during the invasion assay. In three separate experiments, α2 macroglobulin had no significant effect on cell invasion through fibronectin (−6% of the control level of invasion), as summarised in Figure 4AP<0.05) in invasion observed following stimulation with TNF-α (+21% of the control) was completely inhibited by the addition of α2 macroglobulin.Using the model of fibronectin invasion, α2 macroglobulin (2 U ml−1) was added to cells alone or in combination with TNF-α (200 U ml−1) for the 24-h duration of the migration assay. The migration rate of the non-TNF-α stimulated cells decreased by −26%  when α2 macroglobulin was added  was completely inhibited by the addition of the protease inhibitor  insults. As such, it is invariably associated with the early phases of wound healing and persists to a certain degree, until regeneration of the injured tissues and restoration of function have been achieved. .vs narrow margin excisions were compared  and significant inhibition by α-MSH in all the cell types examined  within 24 h of TNF-α exposure for the HBL melanoma cells  expression by the malignant cells . Upregulα stimulation, we examined the expression and activation of MMPs -2 and -9 using gelatin zymography, and the general protease activity of the melanoma cells using a quenched fluorescent substrate assay. As maximal upregulation of cell invasion was achieved with TNF-α between 100 and 250 U ml−1 and previous data from our group demonstrated maximal activation of NFκB in these cells with 200 U ml−1 of TNF-α  in SK-MEL-109 melanoma cells. This suggests that TNF-α may be a potent regulator of degradative enzyme activity during malignant cell invasion. More importantly, this study demonstrates that the TNF-α cell response can be modulated (both upregulated and downregulated) at a postreceptor level via activation of different second-messenger systems suggesting that the effect of TNF-α on various target cells can be modified inside the cell to produce the cell's final response to the cytokine.The literature also reports that TNF-α on the functional biology of different human malignancies including melanoma (Although our current study reports observations made on a single cell line, our data are in agreement with previous reports from the literature on the effects of TNF-melanoma  and exteα upregulates malignant melanoma invasion and migration in vitro. Taken together, the results of our current study combined with the data we have previously published (α may exert its proinvasive effect on HBL melanoma cells via an integrin-dependent mechanism as well as a modest upregulation of degradative enzyme activity not readily detected in general protease assays.In conclusion, we report that the proinflammatory cytokine TNF-


The geometry optimization and vibrational frequency analysis of all the chemical species and transition states (TS) were performed with B3LYP along with a basis set of 6–311 +G, and Gaussian 09 software was used to perform the study. The rate constants were calculated by KiSThelP according to the conventional transition state theory (TST), and the Wigner method was applied to acquire the tunneling correction factors. Then the rate constants were fitted to the modified Arrhenius expression in the temperature range of 800–2000 K. As for the barrierless reactions calculated in this paper, the rate constants were selected from the relating references. Through the energetic and kinetic calculations, the most favorable reaction pathway for benzene formation from methane was determined, which were mainly made of the unimolecular dissociation. The conversion trend from C1-species to C4-species is mainly guided by a strong tendency to dehydrogenation and the pathways from C4-species to C6-species are all presumed to be able to produce C6H6 molecule.A density functional theory (DFT) study was employed to explore the mechanism of the conversion of methane to benzene in chemical vapor infiltration (CVI) based on the concluded reaction pathways from C It is one of the most important ways to prepare high performance C/C composites using methane as precursor10, and the pyrolysis in the gas phase has been reported to play a significant role in the formation of C/C composites11. To fully explain the process of the gas-phase reactions and therefore help to understand and optimize the production process of C/C composites, it is essential to study the mechanism of the homogeneous reactions involved in the gas phase.Chemical vapor infiltration (CVI) is a widely used method for the production of carbon/carbon (C/C) composites, which are mainly employed in the manufacture of solid rocket motor nozzles, brake discs of military and commercial aircraft, and spacecraft heat shields because of their excellent thermal and mechanical properties, like high specific modulus, high specific strength, and wear resistance at high temperature12 proposed a detailed reaction scheme for pyrocarbon deposition from methane, which illustrated the reaction pathways from methane to C6-species and was used to simulate the pyrocarbon deposition kinetics. Huttinger13 presented a simplified reaction scheme for the deposition process from methane which helped to explain the deposition chemistry and kinetics. Kahle et al.14 studied the reaction pathways of the conversion of methane and concluded that coke precursor was produced from methane. Hiblot et al.15 performed an experimental and modeling study for the pyrolysis of methane and found that the methyl radical formed from methane mainly combined with another methyl radical to form C2-species and the H2 molecule could strongly limit the formation of C2-species. Although the above researches studied the decomposition of methane, they were unable to illustrate the nature of the mechanism of the homogeneous reactions during the pyrolysis of methane, and these results could not be explained from the view of the molecular level. Besides, the presence of the species formed during the decomposition process could not easily be detected and distinguished using the online analysis instruments, which made it quite difficult to explore the corresponding mechanisms.There are some researches that have been performed to explore the mechanism of the decomposition of methane. Becker and Huttingeret al.16 calculated the complete dehydrogenation of methane on platinum catalysis by DFT method and found that species like CH3 and CH2 were stabilized at edges and corners of the particles, which helped to study the surface reactivity of extended transition-metal terraces and nanoparticles. To further elucidate the reaction mechanism of the conversion of methane on the single iron sites embedded in a silica matrix, Guo et al.17 simulated the reaction profile of CH3 radicals with DFT and displayed a simplified reaction pathway from CH3 to C6H6. Huang et al.18 presented a DFT study on the sequential process of methane decomposition on iron oxides, which could provide a detailed explanation of the reaction mechanism at the atomistic for the design of more efficient oxygen carriers. Besides, in order to understand the activity of heterogeneous catalysis affected by nanostructure, Kozlov et al.19 calculated the complete methane dehydrogenation on nanostructured palladium by DFT. Despite the plenty of theoretical studies of the decomposition of methane published already, there are few DFT researches concerning the detailed conversion pathways of methane to benzene in CVI, and it is of great necessity to figure out the mechanism from the molecular level.Nowadays, density functional theory (DFT) has become a powerful method to study the mechanisms of the chemical reactions for its convenience and accuracy, and it has also been applied to the research of the decomposition of methane. ViCes 1-species to C6-species. The energy profiles of each reaction were calculated and the corresponding rate constants were obtained subsequently, then the most favorable reaction pathway was proposed.During the decomposition of methane in CVI, benzene is a basic element of pyrolytic aromatic hydrocarbons which can further form the pyrocarbon, thus the present work is performed by limiting the reaction route only to the first cyclic ring formation. In this work, a DFT study was employed to explore the mechanism of the conversion of methane to benzene in CVI based on the concluded reaction pathways from C20 was performed to prove that each TS was connected to the desired reactant and product. To get the IRC results with high quality and complete curve, the max points of each IRC calculation were set to be 150 coupled with the default step size of 10. The relative energies of the species and TS were applied to figure out the corresponding reaction energies and activation energies which were used for the energy profiles. All the quantum calculations were performed with Becke three-parameter exchange and Lee, Yang and Parr correlation function (B3LYP)22 along with a basis set of 6–311 +G, and Gaussian 0923 software was used to study all the DFT calculations.All the chemical species involved in the study were optimized with different spin multiplicities followed by vibrational frequency calculations to obtain the stable molecular structures that provided the lowest energy and correct geometry. In order to verify the structure of the transition states (TS) of all the chemical reactions, the frequency analysis was conducted after the geometry optimization, which turned out that each stable TS was equipped with only one imaginary frequency. Besides, the intrinsic reaction coordinate (IRC)24 according to the conventional transition state theory (TST)25. The parameters required by TST such as molecular energy, spin multiplicity, and vibrational frequencies were obtained from the Gaussian output files. As for the quantum tunneling effect, the Wigner method27 was applied to acquire the tunneling correction factors for all the elementary reactions. Besides, the total reaction degeneracies of each case were taken into account by multiplying a symmetry number of each reaction by the number of equivalent abstractable hydrogen atoms28. Finally, the rate constants were fitted to the modified Arrhenius expression28 in the temperature range of 800–2000 K. The formula is expressed as:a is the barrier height, and n is the temperature exponent indicating the deviation from the standard Arrhenius equation. In addition, due to the fact that KiSThelP is only programmed for the reactions with TS, and for the barrierless reactions calculated in this paper, the rate constants were selected from the relative references published before.The rate constants as a function of temperature for the reactions involved in the study were calculated by using the kinetic and statistical thermodynamical package (KiSThelP)40. By studying and summarizing the related researches published previously, detailed reaction schemes for benzene to be prepared using methane as precursor are summarized, as shown in Fig. 11, so most of the concluded reactions are radical ones. As depicted in Fig. 2-species, C4-species and C6-species, respectively.The primary goal of this section is to obtain the main reaction pathways of the decomposition of methane during CVI by distinguishing the pathways collected from the previous researches6H6 that has four different chemical structures. The reaction pathways proposed in the following are all involved with the dominant species mentioned above.The molecular structures of all the reactants and products studied in this work are displayed in Fig. 1-species to C2-species, the reaction profiles containing every reaction pathway and their potential energies are displayed in Fig. 42. The reactions contributing to the same product have been divided into the same column, which are arranged from left to right in substantially the order of the formation of hydrocarbon shown in Fig. For the discussion on the mechanism of reaction pathways from C4 molecule, which decomposes directly into CH3 radical and H atom with the endoergic energy of 463.7 kJ/mol, and this is consistent with the results calculated before44, which proves the accuracy of this calculation method. With H atom generated, it can combine with CH4 molecule to produce CH3 radical and H2 molecule through TS1 with the activation energy of 40.1 kJ/mol. At the second column, two methyl can bond together to produce an ethane molecule, which is a barrierless process with the exothermic energy of 383.9 kJ/mol. The methyl can also react with methane to yield ethane and hydrogen atom by overcoming the energy barrier of 212.1 kJ/mol, and the total energy of the products is 79.7 kJ/mol higher than that of the reactants. At the column of C2H5, C2H6 molecule can be abstracted by an external H atom to form C2H5 radical and H2 molecule by conquering the energy barrier of 27.8 kJ/mol corresponding to TS3, and the energy of products lies 15.8 kJ/mol below the reactants. C2H6 molecule can also undergo H-elimination reaction forming C2H5 radical and H atom with the barrierless endothermic energy of 444.7 kJ/mol. As for the formation of C2H4 molecule, it can come from both C2H6 molecule and C2H5 radical. After overcoming the high energy barrier of 541.2 kJ/mol, C2H4 molecule and H2 molecule can be produced from C2H6 molecule with the reaction energy being 161.4 kJ/mol, indicating an endothermic reaction. C2H5 radical can decompose into C2H4 molecule and H atom by the dehydrogenation reaction, of which the activation energy and reaction energy are 178.5 kJ/mol and 176.8 kJ/mol, respectively. This column also involves the abstraction of H atom from C2H5 radical by an external H atom to form C2H4 molecule and H2 molecule with the barrierless reaction energy lying 283.3 kJ/mol below the reactants. The fifth column shows two endothermic reactions to the formation of C2H3 radical. By losing one H atom of C2H4 molecule, C2H3 radical can be produced with the high barrierless reaction energy being 484.1 kJ/mol. And with an extraneous H atom attacking C2H4 molecule, the corresponding products are C2H3 radical and H2 molecule with the energy barrier and reaction energy being 48.4 kJ/mol and 23.7 kJ/mol, respectively. Finally, the rightmost column of Fig. 2H2 molecule. The most complicated pathway is the formation of C2H2 molecule and H2 molecule from the dehydrogenation of C2H4 molecule, which gets the reaction energy of 208.7 kJ/mol after overcoming the energy barriers of 326.3 kJ/mol and 472 kJ/mol corresponding to TS8 and TS9, respectively. Another endothermic reaction is the H atom elimination of C2H3 radical, which overcomes the energy barrier of 191.5 kJ/mol and results in the energy of products being 184.2 kJ/mol. The last reaction is exothermic and barrierless, C2H3 + H → C2H2 + H2, and the energy of products is 275.9 kJ/mol below than that of reactants.The initial step of the whole reaction system is the dehydrogenation of CH2-species to C4-species, and the corresponding potential energies of the possible and reasonable pathways are shown in Fig. 4H7 radical to C4H2 molecule.The aim of this section is to deal with the different reaction pathways from C4H7 radical, which comes from the combination of C2H3 radical and C2H4 molecule, and the reaction energy of this exothermic process is 140.3 kJ/mol with the energy barrier of 13.6 kJ/mol. At the second column of C4H6 molecule, three distinct reactions are calculated. The bonding of C2H2 molecule and C2H4 molecule results to the formation of C4H6 molecule with releasing 176.9 kJ/mol, after undergoing TS11 with the energy barrier of 296.3 kJ/mol. C4H6 molecule can also be formed by the dehydrogenation of C4H7 radical, which requires the activation energy of 154.3 kJ/mol, and the energy of products is 143.3 kJ/mol higher than that of reactant. Another reaction leading to the appearance of C4H6 molecule is the radical association of two C2H3 radical, which is observed to be a barrierless process with the exothermic energy being 480.1 kJ/mol. With C4H6 molecule brought into being, the C4H5 radical can be generated consequently. As displayed at the third column of Fig. 4H6 molecule, the C4H5 radical is raised with the energy of the products being 488.6 kJ/mol. On the other hand, by attacking C4H6 molecule with an external H atom, the C4H5 radical can be formed as well with another product of H2 molecule. This step is slightly endoergic (28.2 kJ/mol) and presents the energy barrier of 51.9 kJ/mol corresponding to TS13. Besides, C4H5 radical can also be brought out by the chemical integration of C2H3 radical and C2H2 radical, which loses 172.6 kJ/mol after conquering the energy barrier of 24 kJ/mol corresponding to TS14. At the fourth column demonstrated in Fig. 4H4 molecule is analyzed by two reactions. Like the H-abstraction of C4H7 radical, C4H5 radical can decompose into C4H4 molecule and H atom by dehydrogenation, which is calculated to be endoergic (176 kJ/mol) and needs to overcome the energy barrier of 189.3 kJ/mol. Another reaction is the combination of two acetylene molecules, which goes through one intermediate of C4H4b radical and two transition states of TS16 and TS17. This complicated process needs to overcome a small energy barrier of TS16 to form C4H4b, the energy of which is 228.7 kJ/mol lower than that of the reactants, and then C4H4b radical is transformed into the more stable structure of C4H4a molecule after overcoming the barrier energy of TS17. As can be observed from the structures of C4H4a molecule, C4H4b radical and TS17, which are illustrated in Figs. 4H4a molecule is transformed from C4H4b radical by moving one H atom to the end of the carbon chain. At the fifth column of Fig. 4H3 radical and H atom, which is calculated to be barrierless and endothermic. Another endothermic reaction is the interaction of C4H4a molecule and H forming C4H3 radical and H2 molecule with the energy of 22.6 kJ/mol, which undergoes TS18 of 56.4 kJ/mol. The last part of this diagram is the formation of C4H2 molecule, which is produced by abstracting the H atom of C4H3 radical, as can be seen from the structure of TS19. The energies of TS19 and the products are 178.5 kJ/mol and 157.2 kJ/mol, respectively.As is depicted at the first column of Fig. 4H6 molecule, C4H5 radical, C4H4 molecule, and C4H3 radical, respectively. All the reactions analyzed in the following are multistep which are more complicated than the reactions from C1-species to C4-species, and they are divided into five columns for the convenience of discussion, as shown in Fig. 4H5 → C6H6.This part discusses the reaction pathways leading to benzene, which is originated from C4H6 molecule, and four transition structures can be found. This process is initiated by the chemical combination of C4H6 molecule and C2H3 radical that brings C6H9a radical into existence after overcoming the small energy barrier of 20.5 kJ/mol. Then C6H9a radical undergoes a ring closure reaction after overcoming the activation energy of TS21 to form C6H9b molecule, which lies 203.2 kJ/mol below the reactants. And for the reaction to proceed further, benzene can arise from the dehydrogenation of C6H9b molecule followed by subsequent decomposition of C6H8c molecule into C6H6 molecule and H2 molecule, which goes through two TS configurations of TS22 and TS23. At the second column, the interactions between C4H5 radical and C2H2 molecule or C2H3 radical are calculated separately. The addition of C2H2 molecule takes place on C4H5 radical to form C6H7a radical, which turns out to be a barrierless and exothermic process with the energy below than that of the reactants by 148.7 kJ/mol. Then C6H7a radical undergoes a ring closure step after overcoming a small energy barrier of TS24 to produce a more stable structure of C6H7b radical, which lies 388.5 kJ/mol below the group of C4H5 radical and C2H2 molecule. After that, the C-H bond scission happens to C6H7b radical to generate benzene molecule and H atom, and TS25 is observed during this endothermic step with the energy of the final products being 273.3 kJ/mol lower when compared to the initial reactants. As for the addition of C2H3 radical to C4H5 radical, the whole process is very similar to the addition from C2H2 radical to C4H5 radical. At first, C6H8a molecule is produced with its energy lower than the reactants by 437.3 kJ/mol after the chemical integration of C2H3 radical and C4H5 radical, which is proved to be barrierless and exothermic. And C6H8a molecule is then transformed into C6H8b molecule by the ring closure step, which needs to conquer the energy barrier of TS26 and release energy to achieve the stable structure of C6H8b molecule with its energy lying 521.7 kJ/mol below the reactants. Further, in the third step of this route, two neighboring hydrogen atoms of C6H8b molecule ruptured from C−H bonds tend to form a dihydrogen molecule leaving a stable benzene molecule, which needs to overcome the high activation energy of TS27.As depicted in the left column of Fig. 4H4 molecule and C2H2 molecule resulting in benzene is calculated, which undergoes three transition structures TS28, TS29 and TS30, respectively. The first step of this route is exothermic (−102.7 kJ/mol) and presents the activation energy of 136.3 kJ/mol, which brings the C6H6b species out. Then there are two steps of H atom migration on C6H6b species leading to benzene molecule eventually after overcoming the energy barriers of TS29 and TS30, respectively. And the energy of the benzene molecule is 446.5 kJ/mol lower than that of the beginning reactants showing the exothermicity of this process. As for the fourth column of Fig. 4H4 molecule to benzene molecule. It begins with the addition of C2H3 radical to C4H3 radical forming C6H6a species by releasing 476.6 kJ/mol without overcoming the energy barrier. Then C6H6a species is converted to C6H6b species by conquering the activation energy of TS31. From the next step on, the following process is exactly the same as the conversion from C6H6b species to benzene molecule described in the third column except for the absolute values marked in Fig. 4H3 radical to the benzene molecule. Actually, this process is composed of two separate reactions, which is connected by the C6H5b radical. At first, the chemical bonding of species C4H3 radical and C2H2 molecule takes place resulting in the presence of C6H5a radical, which releases 177.2 kJ/mol without any activation energy. Then C6H5a radical is converted to a phenyl radical after overcoming the slight energy barrier of TS32. Due to the instability of phenyl radical, it is very possible for it to bond with an external H atom to form benzene molecule, which is calculated to be barrierless and releases the energy of 490.3 kJ/mol. Because the external H atom is added during this process rather than in the beginning, it makes sense to draw the energy profiles of these two reactions separately to illustrate this process more clearly and correctly.As displayed in the middle column of Fig. To figure out the reactions among those discussed above that play important roles during the conversion of methane to benzene, reaction kinetics of the elementary reactions were calculated and used for kinetic simulations, as illustrated below.1–C6 in the temperature range of 800–2000 K which covers the possible formation temperature of the species studied in this work, and for the benefit of the analysis of the mechanisms of these reactions the rate constants of the corresponding reactions in the temperature of 1353 K are listed individually because this temperature is very special during the decomposition of methane by using CVI method9. As is shown in Table −22 cm−3 mol−1 s−1 of C2H2 + C2H4 → C4H6, and the highest reaction rate in 1353 K is 1.52 × 1012 cm−3 mol−1 s−1 of C6H6c → C6H6d, which shows a difference of 34 orders of magnitude between these two reaction rates.Table 1–C6. Because the KiSThelP is unable to calculate the rate constants of the barrierless reactions, the corresponding rate values are taken from the literatures by searching the NIST chemical kinetics database45. To the best of our knowledge, there are no relevant theoretical or experimental results about the rate constants for the reaction C4H3 + C2H3 → C6H6a, and as a result, this specific reaction is disappeared in Table 2H3 + C2H3 → C4H6 whose rate constant is elected at 298 K because of the lack of its kinetic study at high temperatures. Like Table −21 cm−3mol−1s−1 of reaction CH4 → CH3 + H, and the largest value is 13.70 cm−3mol−1s−1 of reaction C2H6 → C2H5 + H, which indicates that all the rate constants of the barrierless reactions are commonly low.Table 2H2 + C2H3 → C4H5 whose rate constants decrease gently with the increase of temperature, illustrated in Fig. 6H6c → C6H6d in Fig. 2H6 → C2H4 + H2, C2H4b → C2H2 + H2, C6H8b → C6H6d + H2 and C6H8c → C6H6d + H2. Obviously, the dissociation of these 4 reactions is quite different from the previous four reaction classes, and the difference of the orders of magnitude among these four reaction rates are too large to be plotted in one figure. However, the rate constants of these reactions still increase with the increase of temperature.To better understand the variation tendency of the rate constants of the reactions listed in Table 2H6 molecule in Fig. 3 + CH3 → C2H6 has an advantage over the reaction CH3 + CH4 → C2H6 + H because of its exothermicity. On the other hand, the reactions with higher rate constants are more favorable. For instance, between the two reactions forming C2H5 radical in Fig. 2H6 → C2H5 + H is preferential rather than the reaction C2H6 + H → C2H5 + H2 because the rate constant of the former reaction is 11 orders of magnitude higher than that of the latter one. And by adopting the two rules into all the reactions, the concluded main pathway is drawn in Fig. 4-species to C6-species, each reaction route is composed of at least three steps and exhibits similar potential energies and rate constants, which makes it quite difficult to distinguish the most favorable route among these reactions. Consequently, it is inferred that C6H6 molecule can be possibly produced through all these routes shown in Fig. It is widely accepted that all the reactions proposed above are possible pathways for the overall process of the conversion of methane to benzene in CVI. Furthermore, based on the above analysis, the most preferred pathway can be identified by comparing the potential energies and rate constants of the reactions resulting in the same products, concluded in Table 4 molecule which is the original material of the process, even though this step takes a lot of energy to proceed and has a low rate constant. Besides, the dehydrogenation effect plays a significant role in the whole process. The C2H4 molecule, C2H3 radical, and C2H2 molecule produced during the dehydrogenation can help the reactions of C4-species to perform further, and C2H3 radical and C2H2 molecule can also take part in the production of C6H6 molecule. The multistep reactions forming C6H6 molecule are mainly attributed to the participation of C4H3 radical, C4H4 molecule, C4H5 radical, and C4H6 molecule. From Fig. As depicted in Fig. 1-species to C6-species were obtained and the most favorable route was proposed. The conversion trend from C1-species to C4-species is mainly guided by a strong tendency to dehydrogenation, which is found to play an important role in this route. And the main reaction routes are made of the unimolecular dissociation. In addition, the pathways from C4-species to C6-species are all presumed to be able to produce C6H6 molecule.Based on the concluded pathways from methane to benzene, the DFT study was performed to explain the reaction mechanisms and corresponding kinetics, which presented the calculation results of the potential energy and Arrhenius parameters involved in the process. The reaction profiles and rate constants of each pathway from C


Heterojunctions or heterostructures based on ultrathin 2D materials have attracted increasing attention due to the integrated merits of 2D ultrathin components and the heterojunction effect on the separation and transfer of charges, resulting in important potential values for catalytic applications. Furthermore, 2D/2D heterostructures with face‐to‐face contact are believed to be a preferable dimensionality design due to their large interface area, which would contribute to enhanced heterojunction effect. Here, the cutting‐edge research progress in 2D/2D heterojunctions and heterostructures is highlighted with a specific emphasis on synthetic strategies, reaction mechanism, and applications in catalysis . Finally, the key issues and development perspectives in the applications of 2D/2D layered heterojunctions and heterostructures in catalysis are also discussed. In recent years, the family of 2D ultrathin layered materials has grown appreciably. Beyond the currently used 2D ultrathin nanosheets, such as graphene, transition metal dichalcogenides, and noble metals,A number of excellent reviews on 2D layered materials focusing on synthesis, physicochemical properties, and applications have been published in the past few years.2Since 2D materials successfully synthesized by mechanical exfoliation, an increasing number of synthetic methods, like chemical exfoliation and chemical vapor deposition (CVD) methods, have been developed to obtain 2D materials with various nanostructures. For example, there are more opportunities to obtain 2D materials with high crystallinity but small‐scale production through mechanical exfoliation method; chemical exfoliation methods such as electrochemical2.1The “ex situ assembly methods” are mainly applied to engineer 2D/2D layered heterostructures based on presynthesized 2D layered materials, which are mechanically/chemically exfoliated from bulk materials or isolated from the synthetic/grown 2D layers, and then manually stacked to form 2D/2D heterostructures, whose 2D ultrathin components are combined together based on weak van der Waals (vdW) interlayer force.2/WSe2 heterostructure through stacking MoS2 and WSe2 triangular layers and subsequent thermal treatment.2 and MoS2 monolayers with few tens of micrometers are first prepared by CVD on c‐plane sapphire substrates.2; PMMA layer was spin‐coated on MoS2/substrate, and then dipped in NaOH together to be released from the substrates; and PMMA was finally removed after the PMMA supported‐MoS2 was transferred onto WSe2 flakes /poly(vinyl acetate) (PMMA/PVA) double layers, which are subsequently removed by acetone washing. MoTe2 and MoS2 2D ultrathin crystals are respectively stabilized by strong covalent bonds, while they are stacked together by relatively weak van‐der‐Waals‐like forces. The interlayer infrared excitation first demonstrated in the as‐prepared MoTe2/MoS2 monolayer heterostructures should be attributed to the type‐II heterojunction induced by the interlayer interactions between MoTe2 and MoS2 monolayer.In another work, Zhang et al. developed a MoTe2/MoS2 layered heterostructures by ex situ assembling resynthesized WS2 and MoS2 monolayer materials.2 and MoS2 monolayers are studied by photoluminescence (PL) spectroscopy, resulting that both the input excitation power and the separation distance play the key role. In this work, insulating h‐BN layers were produced by CVD and introduced between WS2 and MoS2 layers in heterostructures to control the separation distance between WS2 and MoS2 layers  exists as lamellar template for the further growth of graphene and h‐BN in the temperature range of 600–750 °C. In another example, the direct growth of graphene/Mo2C vertical layered heterostructures also can be realized by CVD techniques.Without 2D layer as template, 2D/2D heterostructures also can be obtained by direct synthetic method.3Besides the influence of compositions and dimensionality of constituent 2D materials, the highly coupled interface, especially based on ultrathin 2D materials, always plays key role in facilitating the catalytic performance of 2D/2D heterostructures via electron relocalization induced i) ultrafine nanostructure or interfacial defects and thus novel active sites; ii) band bending and thus enhanced redox power of active sites; iii) high‐speed electron transfer path at interface.3.1Heterojunctions with various geometrical and energy band alignments would exhibit electronic properties or functionalities which are macroscopically different from their constituent materials.FigureThere are generally three types of band alignments at the interface of S–S heterojunctions Figure6a–c: tWhen different dopants are introduced into a semiconductor and even different semiconductors, it is also possible to construct a p–n junction Figure d, result3.23.2.1In the research field of functional materials, engineering the structures, including size, surface area, porosity, crystal facet, and defects, has always been an effective strategy to enhancing catalysts' performance and generating novel active sites. The interfacial effect of 2D/2D heterostructures has been applied to engineer the mesoscale structure of 2D/2D heterojunctions to introduce a rich amount of accessible active sites for specific catalytic reactions.2‐CPs) composed of tiny MoS2 nanosheets and hybrid carbon papers (CPs).2, as a cheap alternative of platinum with noble‐metal‐like catalytic activities, exhibits significantly higher electrocatalytic performance for HER compared with that of bulk phase, because the catalytic active sites mainly lie at the edges of 2D layers. In this work, the small size (<15 nm) and few‐layer thickness (2–3 layers) of the as‐formed patched MoS2 nanosheets ensure the abundance of exposed edges and defects as electrocatalytic active sites  of MoS2 nanosheets, which are beneficial for catalytic performance, should be attributed to presence of CP supports and thus resulting in interfacial effect of highly coupled MoS2 nanosheets and CPs.Ye et al. successfully construct a highly efficient hydrogen evolution reaction (HER) electrode materials with heterostructure  of the C3N4. As shown in Figure3N4 after the introduction of GS boosts the activation of the substrates. The charge‐transfer interactions at heterojunction interface can be confirmed by the significant decrease in photoluminescence intensity as compared with that of g‐C3N4. Most importantly, such an M–S dyad is a mild catalyst to boost the selective oxidation of cyclohexane into ketones without formation of overoxidation products.Metal‐free catalysts, owing to the advantages of low cost, high abundance, and excellent stability, attract increasing research interests. Although a series of metal‐free catalysts as green catalysts have been developed, the catalytic efficiency is still incomparable to that of metal‐based catalyst and limits their practical applications. Li et al. developed a series of metal‐free composite catalysts (GSCN) composed of graphene sheet (GS) and g‐CIn another work, an atomic‐scale heterostructure (h‐BN‐C/G) composed of a boron nitride monolayer and graphene is developed and successfully promoted the activation of molecular oxygen for oxidative coupling of amines to imines.3.3Besides the direct influence of structure or defects on catalytic active sites, the transfer and separation of charges in catalysts play quite important role in the efficiencies of photo/electrocatalysis or photo‐electrocatalysis. Constructing 2D/2D heterojunction interface with strong interaction has been proved to be a feasible way to facilitate the high‐speed electron transfer in catalyst system, so as to enhance their catalytic performance.3N4 and monoclinic BiVO4 for the efficient photo‐electrochemical oxygen evolution reaction (OER).4 with a moderate bandgap and a positive enough valence has been proved to be an efficient catalyst for photocatalytic oxidation reactions, such as decomposition of organics in waste water and water splitting for O2 production, under visible‐light irradiation. However, its potential application in photo‐electrochemical water oxidation is limited by low electron–hole separation efficiency.3N4 nanolayers are highly coupled with BiVO4 nanolayers to act as a “pump” to extract the photogenerated electrons from BiVO4, which effectively promotes the incident photon‐to‐current conversion efficiency and the electron–hole separation under visible light irradiation. The choice of few‐layer g‐C3N4 in this work is quite critical, owing to that the ultrathin thickness comparable to the exciton Bohr radius at one dimension (4–8 nm) lifts the restriction of ultrahigh intrinsic resistance of g‐C3N4, and thus contributes to the successful extraction of electrons from BiVO4.Recently, Zhang et al. developed a highly coupled 2D/2D heterostructures (BV/CN) composed of few‐layer g‐C4Although the materials in their 3D forms have long been used as catalysts, the materials with 2D layered structure exhibit considerable changes in their electronic structure and offer new opportunities for chemical/structural modification and catalytic reactions.4.1Facing the energy shortage and environmental pollution of human society, semiconductor‐based photocatalysis has great potential to assure the long‐term and sustainable development, due to the direct utilization of green solar energy both for producing valuable chemical fuels and degrading organic pollutants.−–h+); iii) migration and recombination of photogenerated e−–h+; and iv) redox reactions at the surface of photocatalysts. Among them, the recombination of e−–h+ would create useless heat, which plays negative role in photocatalytic performance. Developing properly engineered 2D/2D heterostructures has been proved to be one of the most promising ways for enhancing photocatalytic activity, due to that the heterojunction interfacial effect can promote the separation and thus prolong the lifetime of photogenerated e−–h+ in catalyst, which directly or indirectly participates in the redox reaction in photocatalytic H2 production or organics degradation. Numerous efforts have been devoted to tune the 2D component or strengthen the interfacial acting force to develop highly efficient 2D/2D photocatalysts.Normally, there are four steps in a photocatalytic process: i) light absorption; ii) formation of photogenerated electron–hole pairs (e4.1.12 electrode under ultraviolet (UV) light irradiation,2 production. Photocatalytic H2 production is a process that produces H2 (and O2) through reducing or oxidizing the adsorbed water molecules by photogenerated electrons and holes at the surface of semiconductor catalysts. Due to the rapid recombination of the photogenerated e−–h+ in catalysts, the developed and reported hydrogen production efficiency was still far from the requirement of practical applications. 2D/2D layered composite photocatalysts with properly engineered heterojunctions are quite promising for enhancing hydrogen production efficiency through promoting the separation of photogenerated e−–h+.Since in 1972 Fujishima and Honda discovered that the water splitting took place on a TiO2 production through in situ growth of ZnIn2S4 ultrathin nanosheets on sheet‐like g‐C3N4 surfaces.3N4 is one of the most promising photocatalysts due to its high stability, nontoxic nature, unique electronic structure, and low cost, its photocatalytic activity is limited by poor light‐harvesting efficiency and fast recombination of photogenerated charge carriers.Compared with UV light with a small proportion of solar radiation, visible light (about 50% of solar radiation) driven photocatalysts are more promising for highly efficient sunlight utilization and photocatalytic activity. Zhang et al. developed a kind of “sheet‐on‐sheet” hierarchical heterostructures for visible‐light‐driven photocatalytic H2S4 ultrathin nanosheets, the above drawbacks are overcome based on heterojunction contact interface, which can induce efficient interfacial transfer of photogenerated e−–h+ from g‐C3N4 to ZnIn2S4 and hinder charge recombination based on the measurement results of surface photovoltage and photoluminescence of ZnIn2S4/g‐C3N4 heterostructures. Both the suppressed charge recombination on g‐C3N4 nanosheets and the increased photogenerated charge carriers in ZnIn2S4 nanosheets contribute to the remarkable enhancement on the photocatalytic activity of ZnIn2S4/g‐C3N4 heterostructures for H2 production. The enhanced photocatalytic performance should also be attributed to the increased surface active sites and extended light absorption through the combination of ZnIn2S4 nanosheets.Through coupling visible‐active ZnIn2 and O2 simultaneously, but it is still a big challenge.+/H2 ) and the valence band maximum should be more positive than the oxidation potential of O2/H2O (1.23 V). Because of the overpotential associated with the charge transfer and gas evolution, the minimum band gap of photocatalysts for effective water splitting is always larger than theoretical value (1.23 eV). Liao et al. design and predict MoS2/AlN(GaN) ultrathin layered heterostructures as highly efficient visible‐light photocatalysts for overall water splitting.2 monolayers act as electron donor and electron acceptor in this heterojunction photocatalysts, respectively. Pristine MoS2 with a direct band gap of 1.9 eV is a promising visible‐light‐driven photocatalyst, and is proved to be inefficient for water splitting. The group III nitrides (AlN or GaN) monolayers with good chemical/thermal stability and high thermal conductivity are demonstrated to be a good choice to construct heterostructures based on MoS2 monolayers so as to enhance photocatalytic activity. In addition, there is only around 2% lattice mismatch among the hexagonal crystal structure of AlN(GaN) and MoS2 monolayers, which is a big advantage for the construction of heterostructures. The MoS2/AlN and MoS2/GaN heterostructures are predicted to be efficient photocatalysts under visible‐light irradiation due to the proper band gaps for water splitting and good optical absorption.Overall water‐splitting photocatalysts also can be developed for the production of H2N) composed of aza‐conjugated microporous polymers (CMP) and C2N nanosheets as 2D polymer‐based Z‐scheme systems for efficient photocatalytic overall water splitting.2N heterostructures were prepared through mixing and subsequently annealing of the CMP and C2N nanosheets. The as‐obtained stacked nanosheets of aza‐CMP/C2N with abundant overlapped areas can be revealed by transmission electron microscopy (TEM) images in Figure2N nanosheets  are simultaneously produced from pure water with aza‐CMP/C2N heterostructures as photocatalysts under visible light (>420 nm) irradiation, while both aza‐CMP and C2N components are inactive. Furthermore, aza‐CMP/C2N heterostructures with the mass ratio of 1:1 exhibit the optimum photocatalytic performance   and thus enhanced the photocatalytic performance.Recently, polymer is believed to be one of the promising alternatives for photocatalytic overall water splitting due to its tunable molecular structures.s Figure b. The st) Figure c. Figure3N4, black phosphorus (BP), boron nitride (BN), and boron carbide (BC), is highly preferred to be explored due to their high abundance, low cost, and excellent stability.2 production remains a big challenge. Zhu et al. successfully developed a 2D layered heterostructures (BP/CN) composed of black phosphorus and graphitic carbon nitride (CN) as metal‐free photocatalysts for H2 evolution in visible to near‐infrared (NIR) region for the first time.2 evolution reached 1.93 µmol under >420 nm light irradiation and 0.46 µmol under >780 nm light irradiation for 3 h  has become a severe threat of water resource and human health worldwide.3 and g‐C3N4 nanosheets (AgIO3/g‐C3N4‐NS) were successfully synthesized for photocatalytic wastewater treatment under visible‐light irradiation.3N4‐NS) as a polymeric organic semiconductor material exhibit good visible light response. The photocatalytic activity of AgIO3/g‐C3N4‐NS heterostructures is significantly higher than single AgIO3 or g‐C3N4 nanosheets toward the degradation of organic dyes. Notably, the reaction rate constant of rhodamine B (RhB) degradation over the as‐prepared AgIO3/g‐C3N4‐NS sample is almost 22.86 times higher than that of heterostructures composed of AgIO3 and bulk g‐C3N4 (AgIO3/g‐C3N4‐B), which indicates the importance of 2D dimensionality for the construction of heterojunction photocatalysts  and CdS with different dimensionality (RGO/CdS) for the photocatalytic degradation of methyl blue (MB).2) based on P‐doped ZnO nanosheets with large surface area and 2D MoS2 for efficient photodegradation of organic dyes.2 heterostructures with different loading amounts of MoS2  and commercial P25 are used as photocatalysts for the photodegradation measurements, which are carried under the same experiment conditions  would significantly enhance the photocatalytic activity compared with that of pure ZnO nanosheets. When the loading amount of MoS2 is increased from 0.01 to 0.1 wt%, the photocatalytic activity of ZnO/MoS2 is further increased, but decreased after the loading amount of MoS2 is further increased to 1 wt%. A large loading amount of MoS2 would block the sunlight, which is applied to drive the photodegradation of MB, and thus reduce the photocatalytic activity. A photocatalytic mechanism of ZnO/MoS2 heterostructures is proposed in Figure 2, the photogenerated electrons would transfer from the CB of ZnO to the CB of MoS2, which significantly improve the separation of carriers and thus enhance the catalytic performance. The enhanced transfer and separation of photogenerated electrons and holes induced by interfacial effect can be confirmed by the increased photocurrent density of MoS2/ZnO heterostructures after the introduction of MoS2 components. The photogenerated charges react with O2 and H2O after transfer to the surface of catalyst, and the as‐produced highly reactive radicals  further degrade the dye molecules. Besides the heterojunction effect, the phosphorous doping induced defects in ZnO nanosheets also promote the photocatalytic performance because they can improve the light absorption through the introduction of energy level between band gap.In some cases, the photocatalytic performance of pollutant degradation may be enhanced by a synergetic effect between heterojunctions and other structure engineering. Liu et al. developed a 2D/2D nanocomposite photocatalyst  is the core reaction in fuel cells and metal−air batteries;In general, advanced electrocatalyst with the features of good electrical conductivity, high activity, and long‐term stability is still urgently needed for practical applications.2 nanosheet decorated carbon paper (MoS2‐CP) through facile two‐step synthetic process: i) direct transformation of biomass bacterial cellulose films into nitrogen‐doped carbon papers, and ii) solution‐phase deposition of MoS2 nanosheets.2‐CPs heterostructures exhibit superb catalytic activity and stability when used as electrode for HER in both acid and basic media  between MoS2 and graphene could significantly influence the hydrogen binding energy and thus enhancing the performance of electrocatalytic HER of MoS2/graphene. It was demonstrated that the electrocatalytic activity at the Mo‐edge of MoS2 would be determined by the interaction with graphene support, which can be controlled by modifying the electronic structures of graphene. The electronic structures of graphene would be varied with different defect density,Tsai et al. investigated the effect of graphene support on the electrocatalytic HER of MoS2P 2D/2D heterostructure as bifunctional electrocatalyst for efficient electrocatalytic water splitting was successfully prepared through three‐step synthetic method composed of hydrothermal, sulfurization, and phosphorization process.2P, NiS/Ni2P heterostructures exhibited extremely higher catalytic performance  under the same conditions. A shift of the X‐ray photoelectron spectroscopy (XPS) peaks assigned to Ni 2p3/2 and Ni 2p1/2 to lower binding energy after merging NiS with Ni2P components compared with those of NiS indicates the charge redistribution induced by strong electronic interactions between NiS and Ni2P, which would significantly influence the chemical properties and thus the catalytic activity. Strong electronic interactions, abundant active sites, and low interfacial resistance all merit the electrocatalytic activity of NiS/Ni2P heterostructures.The development of bifunctional electrocatalysts with high activity for catalyzing both HER and OER is an important research topic for electrocatalytic water splitting, due to not only the requirement of further enhanced efficiency but also the high cost induced by different catalysts (such as different equipment and processes).Figure−2) of Ni‐Fe LDH‐NS@DG catalyst results in better performance. Compared with a series of state‐of‐the‐art non‐noble metal bifunctional electrocatalysts,Jia et al. developed a bifunctional 2D/2D composite electrocatalyst (Ni‐Fe LDH‐NS@DG) composed of Ni‐Fe layered double hydroxide nanosheets (Ni‐Fe LDH‐NS) and defective graphene (DG) with rich defects through electrostatic for efficient alkaline water splitting.4.3Organic synthesis, especially the ones related to the production of chemicals closely linked to our life, is in urgent need of clean, safe, and environmentally benign reaction routes for sustainable development of human society, due to the pollution resulting from the traditional synthesis.2S4 nanosheets onto mesoporous P‐doped graphite carbon nitrogen (P‐C3N4) nanosheets, P‐C3N4/ZnIn2S4 2D/2D heterostructures were rationally developed for the reduction of 4‐nitroaniline to the corresponding 4‐phenylenediamine under visible light irradiation.2S4 with a narrow band is widely studied visible‐light responsive catalyst, and also exhibits stability toward light. P‐C3N4 nanosheets are proved to possess the characteristics of enhanced light harvesting, shorter diffusion distance of charge carrier pairs, and reduced interior defects due to the structure design of mesoporous nanosheets and phosphorus doping. The as‐prepared P‐C3N4/ZnIn2S4 heterostructures exhibit superior catalytic activity due to not only the above‐mentioned merits of two components, but also efficient charge transfer and separation‐induced heterojunction interfacial effect, which could be evidenced by the enhanced photocurrent response, smaller semicircle and lower the PL intensity of P‐C3N4/ZnIn2S4 compared with those of single P‐C3N4 or ZnIn2S4. In another work, CdS/g‐C3N4 2D/2D heterostructures were developed for a coupled system of reducing nitrobenzene to aniline and selective oxidizing aromatic alcohols to aromatic aldehydes under visible light illumination.3N4 with 10 wt% CdS exhibits the optimal catalytic efficiency under visible light illumination.Through immobilizing ZnIn3+, Mn2+, Ca2+, Ni2+, Cu2+, Fe2+, Co2+, and Zn2+) . Two different probe reactions of aerobic oxidation of alcohol and anaerobic reduction of nitro compound are applied to compare the photocatalytic activity of CdS‐(GR‐M) and CdS‐GR under visible light irradiation  into the interface between GR and CdS. Furthermore, the weight addition ratio of GR is increased from ≤5% to 30% after the modification of metal ions, which is a breakthrough of the limitation of shielding effect in GR‐based composite photocatalysts. The enhanced catalytic activity should be mainly attributed to the optimization of heterojunction interfacial effect induced by introducing metal ions as “mediator” for optimized transfer pathway, improved transfer efficiency, and prolonged lifetime of photogenerated charge across the interface. Furthermore, the introduced metal ions can partially offset the “shielding effect” of GR, which is a weakened light absorption due to its opacity resulting from high weight addition.Xu and co‐workers' research proved that the optimization of interfacial composition may significantly boost the catalytic performance for light‐driven organic synthesis through introducing some mediators  into the interfacial layer matrix between the two components in 2D/2D heterostructures.+) Figure15a. Thx heterostructures with different carbon contents were prepared for comparison by changing the weight percentage of glucose in the mixture of reactants  with fixed amounts of urea. h‐BN‐C/G‐15 was proved to be the optimized sample and offered the highest turnover frequency (TOF) value (1.58 mmol BA g−1 h−1) for the coupling reactions of amines to imines under the same reaction conditions as shown in FigureDue to the advantages of low cost and excellent stability, metal‐free 2D/2D heterostructures also have been developed for catalyzing light‐driven organic synthesis.5In summary, this review highlights the general strategies and recent progress in the 2D/2D heterojunctions and heterostructures, which are excellent candidates for fundamental research and potential catalytic applications due to their unique structural physicochemical and electronic properties: i) integrating the merits of their components, such as 2D ultrathin structure, large surface area, and physicochemical/electronic properties; ii) the separation or transfer of charges can be promoted for desirable properties or functions; and iii) versatile options  can be designed to tune properties and thus impact application performance. Driven by the above‐mentioned advantages and potential values of 2D/2D heterostructures, increasing amounts of remarkable achievements have been made in the past few years. Nevertheless, in light of the challenges remaining in practical applications of catalysis, e.g., catalytic efficiency, selectivity associated with yields and pollution, environmentally friendly, and cost, there is still much work to pursue with the aims of developing desired catalyst and reaction system for practical applications.Catalysts hold the key for boosting the efficiency of catalytic reaction process, which is the threshold of commercially feasible applications needed to overcome. 2D/2D heterostructures as photocatalysts have to be designed with the aim to promote the light harvesting, separation/transfer of charge carrier, redox reactivity, etc. Besides adequately taking advantage of 2D components through the fine tuning of the structural, compositional, band gap and surface reaction sites, interfacial engineering at nanoscale of 2D/2D heterostructures is expected to further facilitate the photocatalytic performance. Although many opportunities exist for 2D/2D heterostructures as electrocatalysts, this field is also faced with many challenges and research space: i) synthetic methods are further needed to be optimized to satisfy the industrial and commercial requirements; ii) characterization techniques should be more advanced for the structural, physicochemical properties and performance for such ultrathin heterostructures; and iii) clearer understanding of the work mechanism, especially the reaction intermediates, of 2D/2D heterojunction electrocatalysts is still desired. Besides the above‐mentioned optimization scheme of catalyst design, high product yield/selectivity and the development of new organic synthesis system are all the keys in further research.Thanks to the rapid advances and rich knowledge accumulated in 2D materials and heterostructures in the past decade, one may expect that 2D/2D heterostructures would play an important role in solving energy and environmental challenges.The authors declare no conflict of interest.


The auxin‐inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin‐binding receptor, TIR1, to target the AID‐tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the β‐estradiol expression system. This is a fast‐acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and β‐estradiol systems results in a tightly controlled and fast auxin‐induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of β‐estradiol preincubation. We conclude that TIR1 protein is a rate‐limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects. Ideally, this approach should result in a fast and specific repression to minimise secondary and off‐target effects. The auxin‐inducible degron  was driven by a galactose‐inducible (GAL1–10) promoter or the constitutive ADH1 promoter. The galactose‐inducible expression system can result in metabolic perturbations caused by the shift from glucose to galactose as carbon source , encoded by a codon‐optimized sequence from the Kanemaki lab, under the control of a strong (Padh1–701) or weak (Padh1–409) promoter  zinc finger DNA‐binding domains that recognise a specific promoter, ZnEVpr. In the absence of estrogen (β‐estradiol), the Hsp90 chaperone complex inhibits ZnEV by interacting with its ER domain. Binding of β‐estradiol to ER releases it from the Hsp90 complex, allowing ZnEV to transcriptionally activate ZnEVpr, through direct interaction between the zinc‐finger array of ZnEV and its target DNA sequence within ZnEVpr , yeast codon‐optimized TIR1 from pMK200, and HIS3‐flanking sequences for genomic insertion. To generate plasmids pURA3‐AID*‐6FLAG, pURA3‐AID*‐9myc, and pURA3‐AID*‐6HA, the HPH marker in pHyg‐AID* plasmids was replaced with the Kluyveromyces lactis URA3 gene flanked by a 142 nt repeat sequence from its 5’UTR, which allows selective pop‐out of the URA3 sequence by growth in 5‐fluoroorotic acid. pZTRL was constructed by Gibson Assembly with the following DNA parts: pRS415 (CEN/ARS LEU2), Z4EVpr from pMN10 . The plasmid map displayed in Figure pMK200  was sourced from the Yeast Genetic Resource Centre . pHyg‐AID* plasmids were a gift from the Ulrich lab . PADH1–701‐TIR1 was made by inserting StuI‐linearized pMK200 (containing Padh1–701‐OsTIR1 URA3 expression cassette) into the ura3–1 locus of W303, whereas PADH1–409‐TIR1 was made by inserting the 4398 bp product of PmI1‐linearized pBRT1 (which contains natMX Padh1–409‐OsTIR1 expression cassette) into the his3–11,15 locus of W303. PZ4EV‐NTIR1 was created by inserting pKanMX‐Z4EVpr (from pMN10) directly upstream of the start codon of APE2, as described by McIsaac et al. . This was done by replacing the nonessential APE2 gene in the Z4EV‐expressing strain YMN3 , Prp2, another essential splicing factor with similar abundance (211 copies/cell), and the more highly expressed decapping enzyme, Dcp1  expression cassettes  or without (PZ4EV‐TIR1) NLS on the N‐terminal of TIR1  strain compared with genomic NLS‐TIR1 (PZ4EV‐NTIR1) or plasmid‐encoded TIR1 strains, indicating that TIR1 is better expressed when it is genomically‐integrated and lacks an NLS. Interestingly, Prp22, which is a nuclear localized protein, was quickly depleted irrespective of the presence or absence of the NLS in the N‐terminus of TIR1 protein. This suggests that, for depletion of nuclear proteins, an SV40 NLS need not be added to TIR1. Notably, targeted depletion was slower in the plasmid‐encoded TIR1 strain compared with the other two strains, reaching 15% compared with 2% of Prp22 initial values 30 min after auxin addition, probably due to a lower expression of TIR1 in this strain.We observed that the β‐estradiol‐induced level of TIR1 protein was about threefold higher in the genomic 4Arabidopsis thaliana leads to an auxin‐response phenotype .Notably, the plasmid‐encoded, β‐est AID (pZTRL) depleted the target protein more slowly than genomically integrated TIR1 Figure , likely We targeted only nuclear proteins for depletion, which is why we tested the effect of an NLS sequence at the start of the TIR1 protein. However, as previously reported for budding yeast  either without auxin (left panels) or with auxin addition at a previously determined optimal time . Data represent western blot quantifications relative to initial values.Click here for additional data file.