Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators . In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train . In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca However, in the case of disinhibition, these channels play a role in effectively increasing the evoked release of ACh [2+ conductivity, are localized at the membranes of motor nerve terminals and can serve as presynaptic Ca2+ inputs [−/−) mice, lacking the pannexin-1-dependent tonic activation of inhibitory presynaptic purinergic receptors, the same activation of presynaptic P2X7 receptors may be accompanied by signaling pathway activation, which includes the activation of calcium-calmodulin-dependent protein kinase type II (CaMKII), the disinhibition of L-type VDCCs and the potentiation of evoked ACh release [2+ entry, directly via the channels of P2X7 receptors itself or via L-type VDCCs, provides Ca2+-dependent facilitation of ACh release and the extent of this facilitation remain unclear. In this context, the aim of the present study was to evaluate the relative contribution of the Ca2+ signal generated by Ca2+ influx via the channels of P2X7 receptors compared with that of Ca2+ influx via L-type VDCCs to the potentiation of evoked ACh release in mouse motor synapses by loading NMJs with Ca2+ chelators and through the selective activation of P2X7 receptors and the blockade of L-type VDCCs. Membrane-permeable forms of Ca2+ chelators with different Ca2+ binding kinetics were used; EGTA-AM was used as a slow Ca2+ chelator and BAPTA-AM was used as a fast Ca2+ chelator. Both Ca2+ chelators were used at the same concentration (50 μM). The use of equimolar concentrations of slow and fast Ca2+ chelators allowed us to compare and estimate the dynamics of the effects of various Ca2+ signals in nerve terminals and the relative distance from the Ca2+ inputs to their targets [To date, several sources of Caescribed ,2,3. Theive zone . In addie of ACh ,6,7,8. F+ inputs . Recentl release . However targets ,12. 2+ buffering on ACh release in the motor synapses of WT mice. First, we investigated the effect of intraterminal Ca2+ chelator  of muscle fibers (–39.6 ± 1.2 mV in the control (n = 22) and –38.4 ± 1.4 mV after EGTA-AM loading ). Neither spontaneous nor evoked ACh release during short rhythmic trains of stimuli was affected after NMJs were incubated with the slow Cachelator . The min WT mice .plateau/EPP1) increased from 0.70 ± 0.02 in the control (n = 16) to 0.93 ± 0.08 after incubation with BAPTA-AM . This shift in the EPP train pattern reflects a decrease in the probability of ACh release in the presence of BAPTA. As a result, the quantal content of the first EPP in the train was reduced, and there was more pronounced facilitation rather than depression along the rhythmic EPP trains, as has already been shown in the CNS synapses [2+ chelator BAPTA-AM was able to suppress the Ca2+ signals mediated by P/Q-type VDCCs, thus triggering and maintaining the Ca2+-dependent evoked release of ACh during EPP trains in mouse motor synapses.The incubation of neuromuscular preparations with 50 μM BAPTA-AM did not lead to any statistically significant changes in MEPP parameters, but, unlike EGTA-AM, BAPTA-AM caused significant decreases in the amplitude and quantal content of EPPs compared to those in the control . In addisynapses . Thus, o2+ influx to nerve terminals, to the regulation of ACh secretion in NMJs. Like we demonstrated recently [plateau/EPP1 ratio increased from 0.69 ± 0.02 in the control (n = 16) to 1.21 ± 0.13 after incubation with BAPTA-AM  and was then decreased almost to the control value during the application of BzATP in the presence of BAPTA   E. Next, ol level F-H. Thus−/− mice. Recently, we found that in the motor synapses of Panx1−/− mice but not in those of WT mice, BzATP induces the potentiation of evoked ACh release, which can be prevented by the inhibition of CaMKII or L-type VDCCs [−/− mice (2+-dependent ACh release [2+ influx as a form of Ca2+ entry (in comparison with Ca2+ influx through L-type VDCCs) in enhancing ACh secretion in Panx1−/− motor synapses remains unknown. We next tested the ability of fast and slow Ca2+ chelators to affect the BzATP-induced potentiation of ACh secretion at the NMJs of Panx1−/− mice. We found that incubation with a slow Ca2+ chelator did not change the parameters of spontaneous or evoked release of ACh in the NMJs of Panx1−/− mice, as in the synapses of WT mice. The mean amplitude of MEPPs was 0.83 ± 0.04 mV in the control (n = 24) and 0.94 ± 0.06 mV after incubation with 50 µM EGTA-AM . The mean quantal content of the first EPP in the short rhythmic train was 23.39 ± 1.58 in the control and did not change significantly when EGTA-AM was loaded into nerve terminals . We found that the application of BzATP in the presence of EGTA did not affect the MEPP amplitude, but the ability of BzATP to provoke a significant increase in the quantal content of EPPs was fully prevented by EGTA; the mean value of this parameter for the first EPP in the train was 25.10 ± 2.21 , which was indistinguishable from the control  to 1.21 ± 0.13 after incubation with BAPTA-AM  (plateau/EPP1 ratio was 0.79 ± 0.03 (n =19) when BzATP was applied to NMJs loaded with BAPTA-AM  C. The suBAPTA-AM .2+ signaling associated with BAPTA activity and ACh secretion. These data suggest that the BAPTA-mediated reduction in intraterminal Ca2+ signaling followed by the suppression of evoked ACh release can be counterbalanced by the activation of P2X7 receptors and the build-up of additional Ca2+ signals provided by Ca2+ influx via P2X7 receptor channels and/or L-type VDCCs. Which of the two Ca2+ inputs triggered upon the activation of P2X7 receptors is more important and contributes more to the compensation of decreased evoked ACh release induced by the fast Ca2+ chelator in motor nerve terminals remains unclear.Thus, the loading of BAPTA into motor nerve terminals allowed us to detect the specific contribution of the tonic activation of P2X7 receptors to presynaptic Ca−/− mice with BAPTA-AM and activated P2X7 receptors in the presence of nitrendipine (1 μM), an inhibitor of L-type VDCCs. Even when L-type VDCCs were blocked, the BzATP-induced activation of P2X7 receptors fully retained the ability to restore the decreased EPP quantal content and the pattern of EPP to the control level despite the presence of BAPTA in the motor nerve terminals  did not affect the parameters of evoked ACh release, while a fast chelator (BAPTA) significantly reduced both the amplitude and quantal content of the EPPs. This finding is consistent with the generally accepted concept that in mouse motor nerve terminals, fast Ca2+ chelators are able to “intercept” Ca2+ influx via P/Q-type VDCCs (the main Ca2+ input) located in close proximity to synaptic vesicles in active zones. In contrast, slow Ca2+ chelators are unable to interfere with this Ca2+ signal, which is transient and tightly coupled to vesicular Ca2+ sensors and therefore does not affect the quantal content of EPPs in mouse motor terminals [We established that in the motor synapses of WT mice, a slow Caerminals ,14.2+ inputs present during the activation of presynaptic P2X7 receptors was our main interest. Recently, we showed that the activation of these receptors by their agonist BzATP does not affect ACh secretion in the motor nerve terminals of WT mice [2+ influx than other presynaptic forms of Ca2+ entry and cause partial saturation of preloaded Ca2+ chelators. Thus, our data suggest that Ca2+ input via activated P2X7 receptors can augment the presynaptic Ca2+ concentration and thereby decrease the buffering capacity of Ca2+ chelators, which is manifested as the restoration of the BAPTA-induced depression of neuromuscular transmission back to control levels.Analyzing the roles of additional Ca WT mice . In the  WT mice ,16. Ther−/− mice. Pannexin-1 knockout is primarily followed by a lack of activation of metabotropic A1- and P2Y13 receptors, which inhibit (silence) L-type VDCCs and their coupling to ACh secretion [−/− mice, the activation of P2X7 receptor function by BzATP becomes more pronounced and is accompanied by the activation of presynaptic CaMKII, leading to the potentiation of ACh secretion due to the activation of L-type VDCCs, which can be blocked by nitrendipine [2+ that enters the nerve terminal via activated P2X7 receptors alone remains unclear. Can this additional Ca2+ signal play a distinct role in neurotransmitter release independent of and/or in parallel with the role of simultaneously activated L-type VDCCs? To answer this question, we studied the effects of Ca2+ chelators and their influence on different Ca2+ inputs in nerve terminals and ACh secretion during the activation of P2X7 receptors in the NMJs of Panx1−/− mice.We previously showed that the supply of endogenous ATP/adenosine to the synaptic terminals via pannexin-1 hemichannels and the activation of various presynaptic purinoreceptors is abolished at the NMJs of Panx1ecretion ,17. As aendipine . However−/− NMJs with Ca2+ chelators induced different changes in evoked ACh release, as in the motor synapses of WT mice; BAPTA reduced both the amplitude and the quantal content of EPPs and changed the pattern of EPP train, while EGTA failed to do so. Hence, it is reasonable to suggest that in Panx1−/− mice, the triggering and regulation of evoked ACh release occurs via the same form of Ca2+ entry as in the motor synapses of WT mice: fast P/Q-type VDCCs.We found for the first time that the incubation of Panx1−/− mice, the activation of P2X7 receptors causes a uniform increase in the amplitude and quantal content of EPPs in short EPP trains by an average of 30% relative to the control. This P2X7-mediated effect can be prevented by nitrendipine, suggesting the involvement of L-type VDCCs in the regulation of ACh release as the final target of P2X7 receptor activity [Our previous data demonstrated that in Panx1activity .In the present study, we found that loading EGTA-AM into nerve terminals, like nitrendipine application, fully prevented the BzATP-induced facilitation of evoked ACh release. In the presence of BAPTA, BzATP also lost its ability to enhance the EPP quantal content above the control level; the activation of P2X7 receptors returned the decreased quantal content of EPPs to the control level but not beyond this level. Of special interest is the fact that nitrendipine (1 μM) was unable to prevent this BzATP-induced potentiation of EPPs to the control level in the presence of BAPTA.2+ entering via the channels of P2X7 receptors. As a result, the buffering capacity of BAPTA-AM becomes insufficient to effectively reduce the Ca2+ signal triggered by P/Q-type VDCC activation, and therefore, both the amplitude and quantal content of EPPs quickly return to the control values upon the stimulation of P2X7 receptors in the presence of the fast Ca2+ chelator. Similar partial saturation of BAPTA and EGTA as a result of P2X7-mediated influx of Ca2+ has been observed in other cells [The most likely reason for the ability of BzATP to enhance evoked ACh release to the control level despite the presence of BAPTA is the partial saturation of the BAPTA buffering capacity by Caer cells ,19.2+ via P2X7 receptors, both fast and slow Ca2+ chelators were able to equally and effectively prevent the potentiation of EPP quantal content above the control level, which was observed in the absence of incubation with Ca2+ chelators upon the stimulation of P2X7 receptors followed by the upregulation of Ca2+ input via L-type VDCCs in the NMJs of Panx1−/− mice. This means that both Ca2+ chelators, even when partially saturated due to P2X7 receptor activity, retained sufficient buffering capacity to effectively “quench” the Ca2+ signal generated by L-type VDCCs, thereby preventing its potentiating effect on the evoked quantal release of ACh. Apparently, this is due to the short-term and relatively weak Ca2+ signals generated by opened L-type VDCCs in motor nerve terminals compared to the long-lasting Ca2+ influx via the channels of tonically active P2X7 receptors [2+ chelators and the activity of P2X7 receptors in regulation of Ca2+-dependent ACh release in mouse NMJs are depicted in It is worth noting that despite the probable partial saturation due to the influx of Caeceptors . Overall−/−) mice of either sex. The characteristics of Panx1−/− mice were described previously [Electrophysiological experiments were performed on isolated neuromuscular preparations of the hemidiaphragms (m. diaphragm – n. phrenicus) of age-matched adult C57BL/6 mice   or pannexin-1 knockout  Liley solution containing 135 mM NaCl, 4 mM KCl, 0.9 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 16.3 mM NaHCO3 and 11 mM glucose. To prevent contraction at the time of recordings of synaptic activity evoked by nerve stimulation, the muscle fibers of the neuromuscular preparations were transversely cut. This procedure allowed both spontaneous MEPPs and multiquantal EPPs to be recorded from the same synapse [All experiments were performed at room temperature 20–22 °C). The left hemidiaphragm with the phrenic nerve attached was dissected quickly and stretched in a 3-mL Sylgard-coated chamber perfused (0.4–0.5 mL/min) with oxygenated . Next, the neuromuscular preparations were washed for 15 min with the calcium-free solution without Ca2+ chelators. Then, the washing solution was replaced with Liley solution with a normal concentration of Ca2+ (2 mM), and the neuromuscular preparations were washed with this solution for 15 minutes, after which recordings of synaptic activity were initiated.After control recordings, the neuromuscular preparations were incubated for 60 minutes in a calcium-free solution of 135 mM NaCl, 4 mM KCl, 0.9 mM NaHt-test or one-way ANOVA. In the case of non-normal distribution, the Mann–Whitney test and the Kruskal–Wallis test were used. To analyze the amplitude and quantal content of EPPs, two-way ANOVA followed by the post hoc Bonferroni correction (when comparing two groups) or Tukey’s test (when comparing three groups) was used. Values of p < 0.05 were considered significant for all analyses.Primary analysis of electrophysiological data was performed using MiniAnalysis software . The values of the RMP of muscle fibers and the amplitude and time course of MEPPs and EPPs in short rhythmic trains were evaluated. To mitigate changes in the driving force caused by a shift in voltage upon RMP changes, the amplitudes of the MEPPs and EPPs were standardized to a –50 mV membrane potential . The EPP2+ chelator (BAPTA-AM), the slow Ca2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne  and Calbiochem Biochemicals , respectively. The P2X7 receptor agonist BzATP was purchased from Sigma-Aldrich . The L-type VDCC inhibitor nitrendipine was obtained from Biomol GmbH . Stock solutions of Ca2+ chelators, A740003 and nitrendipine were prepared using DMSO  as the solvent. BzATP was dissolved in deionized water. The final concentration of DMSO in the working solution did not exceed 0.01% (v/v) and did not affect spontaneous or evoked activity in mouse NMJs.The fast Ca2+ entry via the channels of P2X7 receptors can act as an additional presynaptic source of Ca2+, which can partially compensate for deficiencies in Ca2+ signaling induced by Ca2+ chelators in motor nerve terminals. This Ca2+ input, which is mediated by tonically active P2X7 receptors, can partially downregulate the buffering capacity of Ca2+ chelators and recover the fast chelator-induced decrease in Ca2+ signals entering via P/Q-type VDCCs, triggering direct ACh secretion. This means that P2X7-induced Ca2+ input alone may support presynaptic Ca2+ homeostasis. Moreover, Ca2+ entry through P2X7 receptors may indirectly contribute to the enhancement of evoked ACh release via triggering the activation of CaMKII followed by upregulation of L-type VDCCs [2+ entry is primarily intended to trigger the activation of calcium-dependent enzymes and ultimately upregulate L-type VDCCs, which in turn can potentiate Ca2+-dependent ACh release in mouse motor synapses. The variety of effects revealed by the activation of P2X7 receptors is of practical interest for understanding their specific role in the activity of motor synapses under certain normal and pathological conditions.In summary, it was revealed for the first time that Cape VDCCs . Accordi


A reduction in store-operated Ca2+ entry (SOCE) is shared by numerous FAD-linked mutations, and SOCE is involved in Aβ accumulation in different model cells. In neurons, both the role and components of SOCE remain quite obscure, whereas in astrocytes, SOCE controls their Ca2+-based excitability and communication to neurons. Glial cells are also directly involved in Aβ production and clearance. Here, we focus on the role of ORAI2, a key SOCE component, in modulating SOCE in the human neuroglioma cell line H4. We show that ORAI2 overexpression reduces both SOCE level and stores Ca2+ content, while ORAI2 downregulation significantly increases SOCE amplitude without affecting store Ca2+ handling. In Aβ-secreting H4-APPswe cells, SOCE inhibition by BTP2 and SOCE augmentation by ORAI2 downregulation respectively increases and decreases Aβ42 accumulation. Based on these findings, we suggest ORAI2 downregulation as a potential tool to rescue defective SOCE in AD, while preventing plaque formation.Senile plaques, the hallmarks of Alzheimer’s Disease (AD), are generated by the deposition of amyloid-beta (Aβ), the proteolytic product of amyloid precursor protein (APP), by β and γ-secretase. A large body of evidence points towards a role for Ca The cytAEQ measurements were carried out in mKRB containing CaCl2 (1 mM) at 37 °C. Where indicated, in mKRB, NaCl (100 mM) was substituted by KCl (100 mM)  to set to zero the membrane potential. Agonists and other drugs were added to the same medium, as specified in the figures. The experiments were terminated by lysing the cells with digitonin (100 μM) in a hypotonic Ca2+-rich solution (10 mM CaCl2) in H2O to discharge the remaining unused AEQ pool. Signal detection and analysis was carried out as previously described [Upon transfection, the cells were incubated for 1 h with coelenterazine (5 μM) in modified Krebs-Ringer Buffer  at 37 °C. When indicated, cells were bathed in a Ca2+-free mKRB containing EGTA (0.6 mM) or in a Ca2+-free intracellular solution also containing EGTA (0.05 mM) of the following composition in mM . Cells were analyzed using an inverted microscope  with a Fluar 40X oil objective (NA 1.30). Excitation light  was produced by a monochromator  and passed through a dichroic mirror DRLP (505 ext XF73). The emitted fluorescence was collected by a bandpass filter (500–530 nm). Images were acquired using a cooled CCD camera . All filters and dichroic mirrors were from Chroma Technologies . Images were collected at 0.2 Hz with 200 ms exposure time. Cells were mounted into an open-topped chamber and maintained in mKRB medium. Additions were made in the same or different medium, as specified in the figures. G-CEPIA1er experiments: data are presented as ΔF / F0 or ΔF / Fmin values . H2B-GCaMP6 experiments: data are presented as ΔR values . All other materials were analytical of the highest available grade were purchased from Sigma-Aldrich .n = number of independent experiments or cells, as indicated). Significance was evaluated by the non-parametric Kruskal–Wallis test. Where the Kruskal–Wallis test resulted in the existence of a pair of different populations, differences between means were tested with the Wilcoxon–Mann–Whitney test. Boxplots show median, mean, the 25th and 75th percentile of the distributions, while whiskers indicate the upper and lower extremes. * p < 0.05, ** p < 0.01, *** p < 0.001.Data were analyzed using Origin 8.0 SR6  and Microsoft Excel 2010 . All data are representative of at least 3 independent experiments. Numerical values presented throughout the text are mean ± S.E.M. (


The objective of the present study was to identify and correlate extracellular Ca2+ concentration, intracellular Ca2+ dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials: Biodentine and ProRoot white MTA. The hDPSCs were exposed to the biomaterials, brought in contact with the overlaying medium, with subsequent measurements of extracellular Ca2+ and pH, and intracellular Ca2+ changes. Messenger RNA expression , cytotoxicity (MTT and TUNEL) and mineralization potential (Alizarin red and Von Kossa staining) were then evaluated. Biodentine released significantly more Ca2+ in the α-MEM medium than ProRoot WMTA but this had no cytotoxic impact on hDPSCs. The larger Biodentine-linked Ca2+ release resulted in altered intracellular Ca2+ dynamics, which attained a higher maximum amplitude, faster rise time and increased area under the curve of the Ca2+ changes compared to ProRoot WMTA. Experiments with intracellular Ca2+ chelation, demonstrated that the biomaterial-triggered Ca2+ dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. In conclusion, biomaterial-specific Ca2+ dynamics in hDPSCs determine differentiation and mineralization outcomes, with increased Ca2+ dynamics enhancing mineralization.Calcium (Ca Several commercial TCS-based cements with subtle modifications in the manufacturing process and composition are available. ProRoot White MTA (WMTA)  and Biodentine  are two representative TCS-based cements with superior clinical success in dentistry. The widespread clinical indications of TCS-based cements are primarily based on their ability to form calcium hydroxide as a by-product of hydration2. The subsequent dissolution of calcium hydroxide to release hydroxide (OH-)and calcium ions (Ca2+) creates a desirable environment to promote healing and repair of soft and hard tissues4. While hydroxide ions create an alkaline environment responsible for antibacterial and anti-inflammatory activity, Ca2+ ions play a comprehensive role as a vital intracellular second messenger that governs diverse cellular processes such as gene transcription, protein expression, cell proliferation, differentiation, apoptosis, and activation of excitatory cell types6.Tricalcium silicate-based (TCS-based) cements are hydraulic bioactive materials widely used as endodontic cements in dentistry and as bone substitutes in orthopedics2+ in the microenvironment causing elevated extracellular Ca2+ concentration and as a consequence transiently increase the intracellular Ca2+ concentration due to calcium-sensing receptor (CaSR) activation8. The dynamic Ca2+ dependent signalling system can affect numerous Ca2+ sensitive enzymes that convert changes in extracellular Ca2+ concentration and intracellular Ca2+ dynamics into well-defined cell actions9. Analysis of the molecular cues embedded in the intracellular Ca2+ dynamics will lead to better understanding of the intracellular signalling mechanisms involved in regulating the bioactivities of cells10. Intracellular Ca2+ dynamics play a dual role by acting both as an initiator and mediator of stem cell differentiation. Thorough knowledge on the role of Ca2+ dynamics in the differentiation of stem cells into a tissue-specific lineage may offer an alternative biotechnological approach to exploit the unique properties of stem cells10.TCS-based cements release Ca9. Although Ca2+ signalling plays an indispensable role in dental pulp and dentin regeneration, there is a lack of information on the Ca2+ dynamics of human dental pulp stem cells (hDPSCs). The aim of the present study was to identify and correlate extracellular Ca2+ concentration, intracellular Ca2+ dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials; ProRoot WMTA and Biodentine. Our work shows significant differences in extracellular and intracellular Ca2+ changes that link to distinct patterns of hDPSCs gene expression, cellular differentiation and mineralization potential.Calcium signalling is multifaceted and depends on the cell type2+ measurements in the cell medium, in response to exposure to TCS-based cements in the absence as well as presence of hDPSCs were performed  , indicating that the cells take up the extra Ca2+ load provided by Biodentine 2+ measur2+ imaging experiments to determine the cellular responses to biomaterial exposure and its associated changes in extracellular Ca2+ were performed. Cells with acetylcholine (1 µM) were challenged to verify their responsiveness. Oscillatory changes in intracellular Ca2+ that are typical for hDPSCs were found    .Finer grained analysis of the Ca01) Fig. A, a redu01) Fig. B and an 01) Fig. C as compups Fig. D. On ave2+ chelator BAPTA-AM were performed. Complete absence of intracellular Ca2+ dynamics upon exposure to the two biomaterials were found   Fig. A. Furthe01) Fig. B and liv01) Fig.  did not Further, the mineralization potential of TCS-based cements was verified. Therefore, hDPSCs cultured in the presence of TCS-based biomaterials were evaluated for 7, 14, 21 and 28 days. Our results show that Biodentine induced faster mineralization (14 days) compared to ProRoot WMTA (21 days). At 21 days, Biodentine showed extensive mineralization while ProRoot WMTA displayed scattered presence of mineral nodules comparable to that seen in the Biodentine group at 14 days Fig. .Figure 614. Potential use of hDPSCs in tissue regeneration by virtue of their ability to differentiate into fibroblasts, osteoblasts, odontoblasts, adipocytes, neurogenic and myogenic tissues in vitro make them a favorable model system for studying Ca2+ signals15. hDPSC derived odontoblasts play an active role in the transport and accumulation of Ca2+ which leads to regeneration of the dentin-pulp complex16. In this study, custom prepared hDPSCs with 96% purity, extracted by an enzyme digestion method from healthy unerupted human third molars were used. Transwell membranes with a pore diameter of 0.4 µm were used to mimic the absence of direct contact between the biomaterial and hDPSCs while allowing for soluble compounds from the biomaterials to reach the cells, similar to clinical conditions17.TCS-based cements are primarily used in dentistry and their interaction with hDPSCs are of great interest2+ in varying concentration, depending on the cement composition. In this study, Biodentine released significantly more Ca2+ than ProRoot WMTA after 30 min in α-MEM medium (p < 0.001). This could be explained by the fact that despite both ProRoot WMTA and Biodentine contain TCS as their main ingredient, the structure and composition of the two TCS-based cements reveal certain differences that could contribute either directly or indirectly to the difference in Ca2+ release. Compared to ProRoot WMTA, Biodentine has a smaller particle size, which is known to increase the Ca2+ release18. In addition to TCS as the major ingredient, ProRoot WMTA contains dicalcium silicate and tricalcium aluminate19. These additional ingredients form minimal or no calcium hydroxide upon hydration leading to a lesser degree of Ca2+ release. The fact that the Ca2+ release was not accompanied by a corresponding rise in pH suggests that the source of this Ca2+ is not only from the formation and dissolution of calcium hydroxide but possibly also from other calcium compounds being present. Biodentine uses calcium chloride as the liquid medium for hydration while ProRoot WMTA uses water. The use of calcium chloride could enhance the Ca2+ release by accelerating the setting reaction, increasing calcium hydroxide formation and by Ca2+ release from unreacted calcium chloride20.TCS-based cements are known to release Ca2+ release from Biodentine was not detectable in the presence of hDPSCs, indicating that Ca2+ is taken-up by the cells. Cellular Ca2+ uptake involves subsequent Ca2+ binding to proteins and uptake into organelles like the endoplasmic reticulum (ER) and mitochondria21. This organelle-based Ca2+ load may affect gene expression and viability/cell death. In fact, the ER-mitochondrial Ca2+ axis is a major player and determinant of cell survival/cell death22 and is also known to affect gene expression23.The larger Ca2+ measurements closely reflect the differences observed between the two TCS-based cements on extracellular Ca2+. The significantly larger Ca2+ release from Biodentine correlates well with the significantly larger area under the curve of intracellular Ca2+ changes (p < 0.01), the significantly higher maximum amplitude (p < 0.0001) and the significantly shorter time to maximum change (p < 0.0001) observed in Biodentine as compared to ProRoot WMTA  channels in stem cells26 or TRPM4 channels27. Increased Ca2+ entry is followed by intracellular cycling between the cytoplasm, ER Ca2+ stores and mitochondria in mesenchymal stem cells, thus leading to the observed Ca2+ dynamics29.Our results obtained from intracellular Casee Fig. . The lar30. The frequency, duration and amplitude of Ca2+ transients are essential for increasing the efficiency and specificity of gene expression21. There is a nonlinear relationship between gene transcription and intracellular Ca2+ dynamics, that periodically exceeds the threshold for activation of gene transcription31. The Ca2+ oscillation frequency hereby differentially controls the activation of different genes and direct cells to specific developmental pathways32. The present experiments did not reveal prominent oscillatory activity, with only 3 elevations occurring per cell over the 30 min recording period for both biomaterials. This makes it possible that amplitude rather than frequency is most important in the observed effects, as is the case in the slow oscillatory activity leading to oocyte activation34. In any case, the results revealed that Biodentine promoted mRNA expression of BGLAP, TGF-β, and BMP2 indicating that the increased Ca2+ load and intracellular dynamics can enhance the expression of genes associated with odonto-/osteogenic differentiation necessary for successful regeneration of the dentine-pulp complex36. Intracellular buffering with BAPTA-AM treatment inhibited the expression of BGLAP and TGF-β, thereby supporting an underlying role of Ca2+ signalling in the upregulation of these genes. Furthermore, addition of BAPTA-AM reduced the upregulation of BMP2 but resulted in overexpression of MMP1. It is well known that an increase in extracellular Ca2+ enhances the expression of BMP2 by activation of the Ca2+ sensing receptor, elevation of intracellular Ca2+ and stimulation of Ca2+/calmodulin-dependent nuclear factor of activated T cells (NFAT) signalling pathways7.Calcium dynamics can activate signalling pathways in both the nucleus and cytoplasm to induce gene expression by different pathways, that can function both as an inhibitor or activator of gene expression38. In the present study, combined MTT and TUNEL assays revealed absence of cytotoxicity after 1 day exposure to the biomaterials. Moreover, BAPTA-AM treatment caused suppression of mitochondrial metabolic activity causing a decrease in the MTT signal in all the groups. Inconsistent results regarding cell death were seen in the literature where Biodentine performed better39, similar43 or worse44 than MTA. Such contradictory results may be related to the type of target cells, method of cytotoxicity assessment, direct contact of cells with the materials and concentration of materials. Three-dimensional culture of dental pulp stem cells in direct contact to Biodentine and MTA revealed higher cell viability compared to ProRoot MTA after 1, 3 5 and 7 days45. In the study by Daltoe et al., serial dilutions  of extracts from Biodentine and ProRoot MTA showed no significant differences in cell viability after 1 and 2 days in any of the concentrations tested46. The cytotoxic effect of Biodentine and ProRoot MTA on hDPSCs were concentration dependent as cell viability was higher at 1:100 concentration compared to 1:10 or 1:1 concentration. Similar results were obtained when hDPSCs were cultured in medium conditioned with Biodentine or ProRoot MTA47. When transwell membrane was used to avoid direct contact between the biomaterials and hDPSCs, there was no significant difference in cell viability and cell migration between ProRoot MTA and Biodentine48.Calcium plays a significant role in molecular processes responsible for mediating cell survival and death, including defence and programmed cell death mechanisms, such as cell cycle, apoptosis, and autophagy50. The increased mineralization potential of Biodentine is in conformity with its larger material-linked Ca2+ discharge, the larger intracellular Ca2+ response and the upregulation of BMP2, BGLAP and TGF-β observed in our study. As Ca2+ is deposited by osteoblasts and/or odontoblasts, the present results also highlight the osteogenic and/or odontogenic differentiation potential of hDPSCs in the presence of Biodentine and ProRoot WMTA. In agreement with the findings of the present study, previous literature suggests that in comparison to ProRoot MTA, Biodentine demonstrated significantly better cell survival and proliferation of osteoblasts and periodontal ligament cells51.Further, the effects of TCS-based cements on the mineralization potential of hDPSCs were investigated using the Alizarin red and Von Kossa staining technique. Biodentine induced faster and increased mineralized nodule formation compared with the other groups, consistent with previous literature2+ signalling, but their applications have certain drawbacks. Special conditions are required for loading of the cells, bleaching may occur because of extended imaging periods and intracellular dye accumulation may increase cytoplasmic Ca2+ buffering52.Limitations to simulate the complex biological conditions of the clinical situation exist and hence the results obtained from the present study must be observed with caution for direct correlation with clinical scenarios. Calcium sensitive dyes are widely used for evaluating Ca2+ regulates cellular functions9. A typical characteristic of Ca2+ signalling is the manner by which different Ca2+ signals can be translated into specific cell functions depending on the type of signal6. The wide range of functions executed by Ca2+ is attributed to the versatility in speed, amplitude, duration and spatiotemporal pattern of Ca2+ signals as well as by interactions between Ca2+ and other signalling pathways most likely mediated by different cellular processes53. Further research on the versatile patterns of Ca2+ signals is essential for studying the mechanisms lying beyond the cellular functions5.It is essential to expand our knowledge on the numerous pathways by which Ca2+ released from TCS-based biomaterials without provoking cell death. The larger Biodentine-linked Ca2+ load was reflected in altered intracellular Ca2+ dynamics, which consequently resulted in differential gene expression, cellular differentiation and mineralization potential of hDPSCs stimulated with TCS-based cements.Our work demonstrates that hDPSCs take up the Ca54. Written informed consent was collected from all patients and ethical approval was obtained from the Ethical Committee of University hospital, Ghent, Belgium according to laws of ICH Good Clinical Practice (GE11-LM-go-2006/57). The tooth crown was cleaned with iodine and 70% ethanol. The tooth was then cut with a bone cutter at the cemento-enamel junction to remove the pulp tissue and digested with type 1 collagenase and dispase. Cell suspension was cultured in a 25cm2 flask in Alpha modified Eagles medium  with 10% fetal bovine serum and antibiotics (100U/ml Pencillin and 100 mg/ml streptomycin) at 37 °C and 5% CO2. Flow cytometry analysis was performed to identify the purity of the stem cell culture obtained. Purity of the custom prepared stem cells were determined by mesenchymal stem cell markers CD90, and CD105. Custom prepared hDPSCs were grown to subconfluence and attained a purity of 96%  were extracted from unerupted human third molars by enzyme digestion2+ release, pH, cytotoxicity and mineralization assay), in a 6 well plate at a density of 5 × 105 cells/well (for qRT-PCR), or in 35 mm glass bottom dishes  at a density of 1 × 105 (for live Ca2+ fluorescence imaging). For intracellular buffering 1 µM of BAPTA-AM  was added to α-MEM medium. All groups were maintained at 37 °C and 5% CO2 for 1 day.For the experiments, hDPSCs were seeded in a 24 well plate at a density of 40,000 cells/well  or height 2 mm and diameter 8 mm (for qRT-PCR). Although the size of samples was different, the ratio of the exposed surface area of the sample to the volume of the surrounding medium (mm2/ml) was maintained as a constant in all experiments. The samples were allowed to set for 3 h in 100% relative humidity at 37 °C and sterilized by ultraviolet radiation for one hour. Samples were placed in transwell inserts of 0.4 µm pore size  to avoid direct contact of the biomaterial with hDPSCs  and ProRoot White MTA (WMTA)  were mixed according to manufacturer’s instructions and condensed in teflon moulds of height 1 mm and diameter 5 mm . To reduce interferences and the formation of refractory oxides, 40 µl ionic strength adjuster  was added to 1 ml of all sample solutions. The electrode was calibrated using a standard calibration line. The pH of the solutions was measured with a pH glass electrode . Calcium release from the biomaterial and pH were measured both in the presence and absence of hDPSCs.Calcium release was measured by use of a Ca2+ imaging, hDPSCs were loaded with 5 µM of Fluo-3-AM  and 0.01% pluronic F127  at room temperature in the dark and incubated for 1 h. The cells were then washed and de-esterified for 15 min in HBSS  – HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (0.14 g/l cacl2.2H2O (1.8 mM) containing 0.2 g/l MgSO4.7H2O (0.81 mM), 0.032 g/l Na2HPO4.2H2O (0.18 mM), 0.4 g/l KCl (5.36 mM), 0.06 g/l KH2PO4 (0.44 mM), 1.0 g/l D-glucose (5.55 mM), 5.95 g/l HEPES (25 mM) with PH 7.4) with a Ca2+ concentration of 1.8 mM similar to α-MEM. Cells were superfused with HBSS-HEPES buffer for 2 min and were then challenged with acetylcholine (ACh) 1 µM for 8 min. After recording the ACh induced Ca2+ oscillatory activity, superfusion was switched back to HBSS-HEPES for 1 min. The biomaterials were inserted with transwell inserts (0.4 µm) using a special holder after 1 min and recorded for 30 min. Calcium imaging was performed on an inverted fluorescence microscope with an EM-CCD camera and FluoFrame imaging Software. Peak amplitude, time to maximum amplitude, number of Ca2+ transients of individual cells and area under the curve of the Ca2+ dynamics were calculated with Matlab 8.0  software.For live cell CaRpl13a, Eif4b, B2m, Actb, or Gapdh as described previously55. Details of specific primers used for gene expression analysis are provided in Table qRT-PCR analyses were done after 1 day. cDNA was produced and amplified using the Reverse Transcriptome kit . Target cDNA was amplified using specific primer pairs. qRT-PCR was performed using Sensimix SYBR No-ROX Kit  on Light cycler 480 System . Samples were normalized using qBasePlus  against at least three of the following genes: Live/dead staining was performed using calcein acetoxymethyl (AM) ester  / propidium iodide . The samples were rinsed with 500 µL phosphate buffered saline (PBS). 500 µL PBS, 1 µL PI and 1µL calcein AM  was added to each well and after 10 min incubation in a dark room, samples were evaluated with a fluorescence microscope .56:To quantify cell viability, the colorimetric 3-- 2,5-diphenyl-2H-tetrazolium bromide  assay was performed. The cell culture medium was replaced by 0.5 mg/ml MTT reagent and cells were incubated for 4 h at 37 °C. After removal of the MTT reagent, lysis buffer (1% Triton X-100 in isopropanol/0.04 N HCl) was added and incubated for 30 min at 37 °C on a gyratory shaker (70 rpm). The dissolved formazan solution was transferred into a 96-well plate and the optical densities (OD) were measured spectrophotometrically at 580 nm . Cell viability was calculated according to the following equation57.TUNEL assay kit  was used for staining TUNEL positive cells and DAPI nuclear stain was used for staining viable cells. The TUNEL positive and DAPI cells were visualized in fluorescence microscope  and images were analysed using Fiji Image J softwareThe cells were seeded on plastic coverslips of 13 mm diameter  in an adhesive 24 well plate. Osteogenic medium was prepared by supplementing standard culture medium with 10 mM β-glycerophosphate (Sigma-Aldrich), 100 µM L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 100 nM dexamethasone (Sigma-Aldrich). Sample extracts were prepared at a non-toxic dilution as determined by MTT assay. For both Alizarin Red and Von Kossa staining, either pure extract with standard culture medium or extract supplemented with osteogenic medium was added to the cells. At 7, 14, 21 and 28 days, Alizarin Red  and Von Kossa  staining were performed. After rinsing with PBS, the cells were fixed with neutral buffered formaldehyde. The reaction was accelerated by adding formaldehyde-sodium carbonate solution . Unreduced silver ions were removed by Farmer’s solution  containing 10% potassium ferrocyanide and 90% sodium thiosulfate. All images were captured with a microscope  equipped with Xcellence software .p < 0.05 using Statistical Package for Social Sciences (SPSS) v25.0  and GraphPad Prism .All data were subjected to statistical analysis by unpaired t-test with Welch's correction, analysis of variance (ANOVA) and individual comparisons were performed by Tukey post-hoc at a significance level of Parts of this study has been presented at the 6th Belgian Symposium on Tissue Engineering, 2018.Supplementary Figures.


Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca2+ and SOCE regulation.Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca In Homo sapiens, two STIM homologs exist: stromal interaction molecule−1 (STIM1) and −2 (STIM2)3. Upon ER luminal Ca2+ store depletion, human STIM1 undergoes structural changes that promote oligomerization and translocation to ER-plasma membrane (PM) junctions6. At these junctions, STIM1 interacts with Orai1 proteins, which are the pore-forming subunits of Ca2+ release activated Ca2+ (CRAC) channels9. The direct interaction of STIM1 with Orai1 facilitates gating of the CRAC channels to induce the influx of extracellular Ca2+ into the cytosol12, otherwise known as store operated Ca2+ entry (SOCE)14. The human STIM2 paralog similarly regulates SOCE; however, STIM2 is less efficient than STIM1 in this cellular process18. Instead, STIM2 is more intimately involved in the regulation of basal Ca2+ levels15. Interestingly, in lower order eukaryotes such as Drosophila melanogaster and Caenorhabditis elegans, only one STIM gene product has been identified3.Stromal-interaction molecules (STIM)s are endoplasmic reticulum (ER) membrane-inserted calcium  coiled-coils are the highly conserved cytosolic domains that couple to and open Orai1 channels12. Compared to STIM1, the STIM2 EF-SAM domain has a higher stability that suppresses the extent and rate of oligomerization17, and the SOAR domain binds to Orai1 with lower affinity16, making STIM2 less efficient at mediating SOCE. Nevertheless, several structural and mechanistic differences between the STIM homologs allow STIM2 to more effectively regulate basal Ca2+  ~0.4–0.8 mM]21 compared to STIM1 (Kd ~0.2–0.6 mM), which more closely matches resting ER Ca2+ levels of ~0.4–0.7 mM19. Consequently, STIM2 becomes activated after smaller decreases in luminal Ca2+ levels, and a higher proportion of STIM2 molecules are active at resting ER Ca2+ levels15. Second, the C-terminal polybasic domain of STIM2 has a stronger affinity for PM phosphatidyl 4,5-bisphosphate (PIP2) compared to STIM1, which is necessary for STIM2 ER-PM localization23. Third, compared to STIM1 SOAR12, STIM2 SOAR is less sequestered, placing the coiled-coil domains that interact with Orai1 closer to the PM24. Thus, in addition to mediating SOCE, STIM2 also functions as an efficient regulator of basal Ca2+.The highly conserved EF-hand and sterile α-motif (EF-SAM) domains of STIMs are the core luminal protein machinery that sense ER Caa2+ Fig. . First, 25. For example, oxidative stress causes S-glutathionylation within the STIM1 luminal domain, leading to constitutive Ca2+ entry26. Additionally, we recently demonstrated that two Cys residues within the variable N-terminal region of STIM1 (Cys49 and Cys56) can undergo S-nitrosylation, stabilize the luminal domain of STIM1 and inhibit SOCE28. While these two Cys residues are conserved between STIM vertebrate homologs (Cys53 and Cys60 in STIM2), STIM2 contains an additional Cys, non-existent in STIM1, within its variable N-terminal region (Cys15)29, which may serve as an added regulatory input site after thiol modifications 3, highlighting the functional importance of this residue , enhancing the stability, perturbing the structure and inhibiting the Ca2+-regulating function of STIM2. Our data suggest that all three Cys residues within the variable N-terminal region of STIM2  can be modified by GSNO. S-Nitrosylation of these residues thermally and thermodynamically stabilizes the luminal domain via altered interactions of the variable N-terminal region with an isolated face of the Ca2+-sensing EF-SAM core. Remarkably, substitution of the unique Cys (Cys15Ser) attenuated S-nitrosylation-induced stabilization. Congruently, promoting S-nitrosylation in HEK293T cells significantly decreased resting cytosolic Ca2+ levels and SOCE. Unexpectedly, mutating the conserved Cys residues in the luminal region resulted in a loss of STIM2 function, underscoring the sensitivity of the protein to variations at these Cys positions. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly affect the Ca2+ sensing function of the molecule in the regulation of basal cytosolic Ca2+ and SOCE.Here, we show that the luminal domain of STIM2 is indeed sensitive to 2+ from the EF-hand concomitant with EF-SAM structural destabilization are hallmarks of STIM mediated SOCE initiation20. Recent evidence indicates that S-nitrosylation of the variable N-terminal region of human STIM1 enhances the stability of the full luminal domain and inhibits SOCE28. Therefore, we sought to determine whether S-nitrosylation also affects the stability of the human STIM2 luminal domain. Using a pET-28a vector and Escherichia coli, we expressed and purified the full human STIM2 luminal domain corresponding to residues 15–217 , which contains three modifiable Cys. S-Nitrosylation of these Cys was promoted by exchange from a dithiothreitol (DTT)-containing buffer to a GSNO-containing buffer using ultrafiltration. The ability of this approach to efficiently promote GSNO-mediated modification of the STIM1 luminal Cys residues was previously confirmed by solution nuclear magnetic resonance (NMR) spectroscopy28. Here, we assessed the migration of STIM2 15–217 by SDS-PAGE to verify that S-nitrosylation rather than disulfide bond formation was favoured using this same protocol. Ultrafiltration exchange of STIM2 15–217 into a buffer containing neither DTT nor GSNO resulted in enhanced migration on SDS-PAGE gels compared to the same protein in a DTT-containing buffer, indicative of intramolecular disulfide bond formation. In contrast, exchange into a GSNO-containing buffer minimally altered migration, consistent with the small mass changes caused by S-nitrosylation  signal at 225 nm as a function of temperature. Indeed, excess GSNO significantly increased the apparent midpoint of temperature denaturation (Tm) of Ca2+ loaded wild-type STIM2 15–217 by ~+4.1 °C compared to the control thermal melts acquired in the presence of 1 mM DTT, indicating a pronounced stabilization  were extracted from the chemical denaturation curves via the linear extrapolation method34 using a two state, folded to unfolded model. Consistent with the thermal melt data, our urea experiments showed that S-nitrosylation enhanced the thermodynamic stability of Ca2+ loaded wild-type STIM2 15–217 by +0.63 kcal mol−1  and deactivating (Ca2+-loaded) conditions.Collectively, our thermal and chemical denaturation assessments reveal that the full human STIM2 luminal domain is susceptible to S-Nitrosylation of both Cys49 and Cys56 in STIM1 is required to measurably stabilize the full luminal domain of STIM127. STIM2 not only contains Cys residues at similar and conserved positions (Cys53 and Cys60), but also has an additional Cys residue at the N-terminus (Cys15), which we posit is also susceptible to S-nitrosylation. To determine whether the S-nitrosylation induced stabilization observed for STIM2 15–217 occurs in a Cys-specific manner, we created a series of Cys to Ser mutant STIM2 15–217 proteins. In total, five different mutant proteins were expressed and purified: Cys15Ser, Cys53Ser, Cys60Ser (single mutants), Cys53Ser/Cys60Ser (double mutant) and Cys15Ser/Cys53Ser/Cys60Ser (triple mutant). For all variants, protein expression levels and purities were similar to wild-type; however, the triple and double mutant proteins tended to aggregate and precipitate more readily compared to the other preparations.i.e. 1 mM DTT) and in the presence of Ca2+, the Cys15Ser and Cys53Ser single mutant proteins marginally increased the Tm by ~+0.2 and ~+0.5 °C, respectively, compared to wild-type , indicating that the unique Cys15 of STIM2 15–217 plays a key role in the observed S-nitrosylation induced thermal stabilization , the STIM2 15–217 1H-15N-HSQC spectrum exhibited highly dispersed amide 1H(15N) crosspeaks ranging from ~6.5 to 11 ppm in the 1H dimension, suggesting a well-folded conformation. After ultrafiltration exchange from DTT into a GSNO containing buffer, the spectrum showed numerous 1H(15N) chemical shift perturbations (CSP)s, indicative of structural changes . Most 1H(15N) resonance signals of STIM2 15–217 matched those observed for EF-SAM in isolation, suggesting EF-SAM adopts a very similar conformation in the context of the full luminal domain.Having uncovered a Cys-dependent synergism in the ges Fig. . To dete1H and 15N CSP caused by S-nitrosylation for each EF-SAM 1H(15N) resonance identified within the STIM2 15–217 spectra  and monomeric cherry fluorescent protein (mCh) tagged STIM2 (mCh-STIM2). Control cells co-overexpressing mCh and eGFP-Orai1 exhibited a mean basal cytosolic Ca2+ level of ~118 ± 6 nM. Consistent with past studies15, cells co-overexpressing wild-type mCh-STIM2 with eGFP-Orai1 showed an ~2-fold increase basal cytosolic Ca2+ compared to the control cells  was used to block the sarco/endoplasmic reticulum Ca2+ ATPpase pumps. We induced SOCE by adding 2 mM of Ca2+ back into the extracellular medium to elicit Ca2+ influx through Orai1-composed PM channels. As anticipated, cells overexpressing wild-type mCh-STIM2 and eGFP-Orai1 displayed significantly higher levels of SOCE compared to control cells co-overexpressing mCh and eGFP-Orai1 ] induced by CRISPR/Cas9 gene editing35. In contrast to HEK293T cells with unaltered endogenous STIM genes, eGFP-Orai1 and mCh-STIM2 co-expression did not significantly enhance basal cytosolic Ca2+ in the HEK293 STIM1/2(−/−) cells compared to vehicle-transfected controls, and GSNO treatment had no effect on this basal Ca2+  cells with or without GSNO treatment  cells significantly enhanced peak SOCE compared to vehicle-transfected controls; further, GSNO significantly suppressed wild-type mCh-STIM2-mediated peak SOCE  cells expressing the Cys15Ser mutant  cells were unable to induce SOCE, as observed for our Cys15Ser/Cys53Ser/Cys60Ser triple mutant both in the HEK293T and these HEK293 STIM1/2(−/−) cells. GSNO had no effect on the double and triple mutant SOCE responses  retains a well-folded structure in contrast to the Ca2+-depletion induced structural destabilization of STIM1 EF-SAM (residues 58–201)43. Moreover, previous urea-induced kinetics of unfolding studies indicate that Ca2+ loaded STIM1 EF-SAM unfolds>3-times faster than STIM2 EF-SAM44. In agreement with these phenomena, we found that under reducing conditions, the full luminal domain of STIM2 (residues 15–217) displays a larger Δ2+ loaded state, the STIM2 15–217 Δ−1 larger than STIM1 23–213 (i.e. Δ−1), and in the Ca2+ depleted state, Δ−1 (Table 2+-loaded state (Δ−1) and remains larger for STIM2 15–217 compared to STIM1 23–213 in the Ca2+-depleted state (Δ−1) states  and -deplete (ΔΔ−1 versus ΔΔ−1) conditions  also significantly attenuated the GSNO-mediated STIM2 15–217 stabilization; however, the remaining modifiable Cys53 and Cys60 residues were able to promote an ~+0.5 kcal mol−1 stabilization  peaks undergo large CSPs due to alterations in chemical environment  peaks17 that cluster on one face of the EF-SAM core were also affected. A control NMR titration of GSNO into an isolated STIM1 EF-SAM sample showed no CSPs  peaks were largely undetectable and severely broadened (not shown). The degenerate NMR spectrum could be caused by conformational exchange (i.e. multiple conformations) and/or higher order protein assembly, which are both indicative of some fundamental structural change. Additionally, previous studies showed that both Ca2+ binding affinity and EF-SAM stability are integral to dictating the distinct functions of human STIM1 and STIM217. Hence, changes in Ca2+ binding affinity may underlie the inhibited function. New interactions between the mutated STIM2 luminal domain and the N-terminal mCh or other endogenous proteins may cause the loss of function. Interestingly, the Cys49Ala/Cys56Ala STIM1 double mutant was shown to have a severely compromised ability to induce SOCE in mouse embryonic fibroblast with endogenous STIM1 knockout40. This phenotype was in contrast to Cys49Ala/Cys56Ala STIM1 expression in DT40 STIM1 knockout cells, which induced constitutive Ca2+ entry26. Thus, cell/environment-specific differences may contribute to inhibition versus activation phenotypes. Nevertheless, our in cellulo data unequivocally demonstrate an extraordinary sensitivity in STIM2 function to modifications of the Cys residues within the luminal region. This sensitivity is consistent with work showing that swapping the STIM2 N-terminal region (residues 15–69) with the STIM1 N-terminal region (residues 23–66) leads to a switch in the function of the resulting full-length STIM2 chimera, which enhances resting Ca2+ and SOCE compared to wild-type STIM218.Several explanations may underlie the loss of function observed for the double and triple mutant mCh-STIM2. Similar conformations may be induced by these mutation sets that prevent the specific protein interactions necessary for STIM2 activation. This notion is supported by our data showing the Cys53Ser/Cys60Ser and Cys15Ser/Cys53Ser/Cys60Ser mutations both destabilized the CaIn vivo, STIM2 isoforms have been identified with long 87 residue and short 14 residue signal peptides ; thus, it is interesting to speculate that S-nitrosylation of Cys15 (Cys88 in the long isoforms) could add another layer of regulation to STIM2 by modulating ER targeting and processing efficiency. Once in the ER, STIM2 is an integral regulator of basal cytosolic Ca2+ and SOCE36, as was reinforced in the present study. With replete ER Ca2+ stores, active STIM2 at ER-PM junctions trap STIM1 at the same sites, enhance the coupling of STIM1 with Orai1 and augment cytosolic Ca2+ entry38. Consistent with this mechanism, we found that mCh-STIM2 co-expression with eGFP-Orai1 in HEK293 STIM1/2(−/−) cells did not enhance basal cytosolic Ca2+, in contrast to HEK293T cells with intact endogenous STIM genes, which showed enhanced basal cytosolic Ca2+  cells increased peak SOCE, indicating that STIM2 can regulate SOCE independent of STIM1 in this cell type.des Fig. . The N-t46). For example, within the vascular system, STIM2 (but not STIM1) protein expression is upregulated in pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension47. Interestingly, ≥sub-μM concentrations of NO donors have been shown to indirectly inhibit SOCE in platelets through the activation cGMP dependent protein kinase and the possible phosphorylation of STIM248. Biophysically, our data reveal that S-nitrosylation stabilizes the full luminal domain of STIM2 in a Cys-specific manner, whereby a unique Cys residue present in the Ca2+ sensing region of STIM2 (but not STIM1) is involved in a synergistic stabilization. Structurally, S-nitrosylation induces changes to the full luminal domain of STIM2, which are predominantly localized to one face of the EF-SAM core that is involved in destabilization-induced activation  corresponding to residues 15–217 was cloned into a pET-28a vector (Novagen) using NheI and XhoI restriction sites and expressed with an N-terminal hexahistidine tag. Cys15Ser, Cys53Ser, and Cys60Ser single-, Cys53Ser/Cys60Ser double- and Cys15Ser/Cys53Ser/Cys60Ser triple-mutants were introduced into separate plasmids by PCR-mediated mutagenesis. Wild-type and mutant proteins were expressed in BL21(DE3) codon plus E. coli cells and purified under denaturing conditions as described in the nickel-nitrilotriacetic acid agarose beads manufacturer protocol . Protein was refolded by overnight dialysis in ~65 volumes of 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 5 mM CaCl2, pH 8. The N-terminal hexahistidine tag was removed through overnight incubation with ~2 units of bovine thrombin  per mg of protein. Proteins were further purified via size-exclusion-chromatography using a Superdex 200 10/300 GL . Concentrations of each protein were estimated using UV extinction coefficients at 280 nm (ε280 nm). The extinction coefficients were ε280 nm = 1.2944 mg mL−1 cm−1 for wild-type STIM2 15–217, ε280 nm = 1.2953 mg mL−1 cm−1 for Cys15Ser, Cys53Ser or Cys60Ser single mutant STIM2 15–217, ε280 nm = 1.2961 mg mL−1 cm−1 for Cys53Ser/Cys60Ser double mutant STIM2 15–217, and ε280 nm = 1.2970 mg mL−1 cm−1 for Cys15Ser/Cys53Ser/Cys60Ser triple mutant STIM2 15–217 proteins. STIM1 EF-SAM was expressed and purified as recently described27.The full luminal domain of 2+ depletion condition was achieved by overnight incubation with 50 mM EDTA followed by a 20 × 20 × 20 × 20-fold exchange by ultrafiltration into nominally Ca2+ free buffer. GSNO was prepared as described previously49, and the concentration was estimated using ε335 nm = 0.92 mM−1 cm−150. Proteins were exchanged into a buffer containing 1 mM GSNO by ultrafiltration using a 20 × 20 × 20 × 20-fold total buffer exchange.The Ca−1), an 8 second averaging time and 1 nm bandwidth. Thermal melts were acquired by monitoring the change in negative ellipticity (mdeg) at the 225 nm peak minima as a function of temperature using a 1 °C min−1 scan rate. The apparent midpoints of temperature denaturation (Tm) were extracted from the thermal melts using Boltzmann sigmoidal fitted curves in GraphPad Prism.Data were acquired on a Jasco J-815 CD Spectrometer (Jasco Inc.) equipped with a PTC-423S temperature controller using a 0.1 cm path length quartz cuvette in 1 nm increments (20 nm minex) = 280 nm and emission wavelength (λem) =339 or 337 nm for the Ca2+-loaded and -depleted conditions, respectively. Protein thermodynamic stability parameters were extracted from the equilibrium chemical denaturation curves using a two-state unfolding model and the linear extrapolation method51.Protein samples were diluted to 5 μM and were incubated overnight in the presence of 0–6 M urea at 25 °C. A Cary Eclipse spectrofluorimeter (Varian/Agilent Inc.) was used to detect intrinsic fluorescence intensity for each sample using an excitation wavelength . S-Nitrosylated and reduced spectra were compared using two approaches: first, by acquiring spectra on two samples separately containing 1 mM GSNO or 1 mM DTT, and second, by acquiring a spectrum on one sample containing 1 mM GSNO, and re-acquiring a spectrum on the same sample after supplementing with 15 mM DTT. 1H−15N HSQC spectra of STIM1 EF-SAM were similarly compared using the two approaches; however, the NMR data was acquired using 32 transients, 64 increments in the nitrogen dimension, 8,000 Hz 1H sweep width, and 1,800 Hz 15N sweep width at 20 °C.(−/−) cells (a gift from M. Trebak35) were seeded in a 96-well plate. All cells were cultured in Dulbecco’s modified Eagle’s medium (Wisent) supplemented with 10% (v/v) fetal bovine serum (Sigma) and 100 μg mL−1 penicillin-streptomycin (Wisent) at 37 °C and 5% CO2 in an air humidified incubator. pCMV6 vectors containing wild-type or mutant mCh-STIM2 variants were co-transfected with eGFP-Orai1 into cells at ~70–80% confluency using PolyJet transfection reagent (SignaGen Laboratories) according to the manufacturer’s protocol. The pCMV6-eGFP-Orai1 vector has enhanced green fluorescent protein  fused in-frame to the N-terminus of human Orai1 , interceded by a Gly-Thr linker. The pCMV6-mCh-STIM2 vector was previously described17. From the N- to C-terminus, the construct contains the human N-terminal STIM1 signal peptide  fused in frame to mCherry (PCRed from mCherry-pRSET-B52), a Gly-Thr linker and human STIM2 . To promote S-nitrosylation in cellulo, the growth medium of select wells/dishes was supplemented with 250 μM GSNO at 8 h post-transfection and incubation was continued overnight. All plasmid DNA was suspended in water (vehicle).HEK293T cells were seeded in 35 mm dishes while HEK293 STIM1/22. The cell suspensions were equilibrated for 10 minutes at 22.5 °C before fluorescence assessments using a Cary Eclipse spectrofluorimeter (Varian/Agilent). Fluorescence emission at 510 nm was measured at alternating excitation wavelengths of 340 and 380 nm  in 1 second intervals. After the initial basal Fura-2 F340/F380 ratios were determined, 1.0 mM EGTA (0.5 mM molar excess over Ca2+) was added to the cell suspension. Subsequently, 2 µM TG and 2.5 mM CaCl2 (2.0 mM excess over EGTA) was sequentially added to the extracellular solution to gauge SOCE. The data are plotted as a normalized F/F0 ratio, where F is the F340/F380 ratio, and F0 is the average F of the first 10 data points (50 s) before the addition of TG. Basal cytosolic Ca2+ concentrations were calculated from F as previously described54 using a Ca2+ equilibrium dissociation constant of 225 nM. The Ca2+ saturated and depleted conditions were calibrated by adding 0.2% (v/v) Triton X-100 and 10 mM EGTA to the cell suspensions, respectively. The same calibration values were applied to all experiments.HEK293T cells were lifted from 35 mm dishes by pipetting and subsequently incubated with 2 µM Fura-2-AM (Alfa Aesar) for 45 minutes in the dark at 37 °C. Cells were washed twice with HEPES-buffered saline solution  before being resuspended in 1.2 mL HBSS buffer containing 0.5 mM CaCl(−/−) cells in a 96-well plate were incubated with 1 µM Fura-2-AM for 45 minutes in the dark at 37 °C and subsequently washed twice with 200 μL HBSS before being covered in 100 µL HBSS containing 0.5 mM CaCl2. The adherent cells were equilibrated for 10 minutes at ambient temperature before fluorescence assessments using a FlexStation 3 Multi-Mode microplate reader . Fluorescence emission was measured at alternating excitation wavelengths in 1 second intervals. The FlexStation automated pipettor was used to sequentially deliver 1.0 mM EGTA, 2 μM TG and 2.5 mM CaCl2 to the wells. The data are plotted as F340/F380 ratios, where F340 and F380 are the 510 nm fluorescence emission intensities at excitation wavelengths of 340 and 380 nm, respectively. The baseline (F0) was taken as the mean F340/F380 ratios measured for 80 s before the addition of TG and in the presence of EGTA. Relative basal cytosolic Ca2+ levels were calculated as the difference between the mean F340/F380 ratios measured for 30 s before the addition of EGTA  and baseline . Relative peak SOCE was taken as the difference between the mean F340/F380 ratios after the 2.5 mM CaCl2 addback (FSOCE) and baseline (FSOCE-F0).HEK293 STIM1/2t test was used to compare two independent groups, and a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to compare three or more groups. All data are reported as means ± SEM.An unpaired Student’s Supplementary Information.


SOCE sensors, STIM1 and STIM2, can activate Orai, TRP channels and AMPA receptors, and inhibit voltage-gated channels in the plasma membrane. However, the link between STIM, SOCE, and NMDA receptors, another key cellular entry point for Ca2+ contributing to synaptic plasticity and excitotoxicity, remains unclear. Using Ca2+ imaging, we demonstrated that thapsigargin-induced nSOCE was inhibited in rat cortical neurons following NMDAR inhibitors. Blocking nSOCE by its inhibitor SKF96365 enhanced NMDA-driven [Ca2+]i. Modulating STIM protein level through overexpression or shRNA inhibited or activated NMDA-evoked [Ca2+]i, respectively. Using proximity ligation assays, immunofluorescence, and co-immunoprecipitation methods, we discovered that thapsigargin-dependent effects required interactions between STIMs and the NMDAR2 subunits. Since STIMs modulate NMDAR-mediated Ca2+ levels, we propose targeting this mechanism as a novel therapeutic strategy against neuropathological conditions that feature NMDA-induced Ca2+ overload as a diagnostic criterion.Neuronal Store-Operated Ca This ratio represents the emission intensities at 510 nm obtained after excitation at 340 and 380 nm. To measure Ca2+ response via NMDAR, neurons were stimulated with 100 µM NMDA + 10 µM glycine in the presence of 2 mM CaCl2, 5 μM NM, and 30 μM CNQX and in the absence of Mg2+. The low Ca2+ medium (Ca2+-free solution) contained 0.5 mM EGTA in the standard buffer. At the end of all experiments, 50 mM KCl in the presence of 2 mM CaCl2 was added to assess which cells were neurons. Cells that responded with rapid, high [Ca2+]i rise were identified as neurons and only these cells were analyzed in the experiments. Approximately 60% of cells responded to KCl. Cells that responded with a delay or did not respond at all were assumed to be non-neuronal glial cells, most likely astrocytes. Data processing was performed using Olympus Cell^R software.The i response is sensitive to STIM proteins, we monitored [Ca2+]i in neurons with increased levels of STIM1 and STIM2 protein expression using transfection of cortical neurons with plasmids encoding YFP-tagged STIM1 or YFP-tagged STIM2. As shown in 2+ increase characterized by a sharp increase in signal and a sustained plateau (blue line). Overexpression of either STIM1 or STIM2 reduced cytosolic [Ca2+] after NMDA stimulation by 50% and 30%, respectively Ibd mice . After N neurons c,d, whic neurons a,b. Thes2+ ions and supplemented with 10 µM glycine was used. After obtaining stable whole-cell configuration, the agonist solution (100 µM Na-glutamate in Mg2+-free ACSF) was applied locally above the patched neuron with the use of an automated perfusion system  for 10 s. To isolate NMDAR-mediated currents, action-potential dependent activity was blocked by the application of 1 µM tetrodotoxin, and AMPARs and VGCCs were blocked by 10 µM NBQX and 10 µM nifedipine, respectively. Application of NMDA evoked robust inward currents that were significantly diminished by an NMDAR blocker, D-AP5 (100 µM), and almost entirely blocked by joint application of 100 µM D-AP5 and 20 mM Mg2+ (Tg(Stim1)Ibd transgenic mice  using established methods i induced by NMDA. Using different SOCE inhibitors, such as 30 µM 2-APB, 3 µM SKF96365 and 100 µM La3+, Baba et al. found altered slow, but not fast, exponential [Ca2+]i decay coefficient of NMDA responses i, the same drugs do not inhibit NMDAR currents using electrophysiology  and thus contribute to elevated levels in the cytosol (2+ ejection and buffering mechanisms are weakened in neurons after STIM silencing. Downregulating STIM2 [2+ overload and destabilization of hippocampal mushroom spines in AD or aged neurons. Downregulating STIM2 proteins was also observed in cells from AD patients [2+ overload in AD neurons [2+ levels induced by NMDA treatment (2+ responses, which suppressed intracellular Ca2+ overload.STIM and NMDAR abnormalities have been implicated in the pathogenesis of neurodegenerative diseases ,21. HoweD and AD ,42,43,65 neurons , as stimm the ER ,56,57,58tes SOCE  (Figure o the ER b as in P cytosol b. It is ng STIM2 ,66 or STng STIM2 ,68 can ipatients . In turn neurons ,66,68,70reatment c. We sus2+ sources for nSOCE [2+ elevations in cortical neurons, as is known for VGCC activity [2+ homeostasis. Thus, our results advance our knowledge on the pathophysiological mechanisms of neurodegenerative diseases. In conclusion, our results demonstrate that NMDAR, in addition to Orai, TRPC, VGCC, and AMPAR, comprise putative Caor nSOCE ,31. Moreactivity ,28. We p


Amycolatopsis mediteranei (AML) with potential applications in process biotechnology was recently cloned and examined in this laboratory. In the present study, the 3D structure of AML was elucidated by comparative modelling. AML lacked the ‘lid’ structure observed in most true lipases and shared similarities with plastic degrading enzymes. Modelling and substrate specificity studies showed that AML was a cutinase with a relatively exposed active site and specificity for medium chain fatty acyl moieties.An extracellular lipase from Amycolatopsis enzymes are capable of degrading a wider range of plastics than is generally recognised. The potential for application of AML in the bioremediation of plastics is discussed.AML rapidly hydrolysed the aliphatic plastics poly(ε-caprolactone) and poly extended with 1,6-diisocyanatohexane under mild conditions. These plastics are known to be slow to degrade in landfill. Poly(L-lactic acid) was not hydrolysed by AML, nor was the aromatic plastic Polyethylene Terephthalate (PET). The specificity of AML is partly explained by active site topology and analysis reveals that minor changes in the active site region can have large effects on substrate preference. These findings show that extracellular  They belong to the α/β hydrolase superfamily with the signature α/β fold and a catalytic triad formed by three residues  at the active site Amycolatopsis mediterannei (AML) with biotechnologically useful properties In previous work, we identified an interesting extracellular lipase from Amycolatopsis is capable of digesting a wider range of polyesters than is generally recognised. The significance of an extracellular plastic degrading cutinase in Amycolatopsis species is discussed.The structural characterisation of AML along with substrate specificity studies show that this enzyme is more appropriately described as a cutinase. A BLAST search of the α/β hydrolase database found that AML was homologous to several known plastic degrading enzymes. When tested for this activity, AML was found to be capable of the degradation of certain aliphatic, but not aromatic, plastics. This interesting specificity is discussed in terms of the active site structure and homology with other plastic degrading enzymes. The ability of AML to degrade aliphatic plastics underlines the fact that 22.1pET-49b (+) plasmid was from Merck Novagen®; Macherey-Nagel™; Protino® Glutathione Agarose was obtained from Fisher Scientific and all other chemicals were from Sigma Aldrich.2.2Escherichia coli BL21(DE3) cytosol as described by Tan et al. . A starter culture  on ice using 30–60 s bursts at a setting of 30%-40% amplitude. The sonicated mix was pelleted at 12,000 × g for 15mins. The supernatant was loaded on a Macherey-Nagel™ Protino® Glutathione Agarose column and AML was cleaved off from the tag using GE Healthcare PreScission® Protease.AML was produced using the recombinant system pET-49b (+) in 2.3Amycolatopsis mediterranei U32 lipase GenBank accession no. ADJ49206). The sequence was inputted into the interactive web interface of SWISS-MODEL (https://swissmodel.expasy.org/interactive). The 3-D template coordinates of Streptomyces exfoliatus lipase  database (2.4http://servicesn.mbi.ucla.edu/SAVES/), including VERIFY3D https://prosa.services.came.sbg.ac.at/prosa.php; http://mordred.bioc.cam.ac.uk/≃rapper/rampage.php; http://www.compbio.dundee.ac.uk/jpred/; The quality of the model coordinates was evaluated using the QMEAN score of the SWISS-MODEL server and the tools of the Structure Analysis and Verification Server (SAVES) (2.5http://www.led.uni-stuttgart.de/) and ESTHER database  to classify AML according to the family system in each database respectively.The amino acid sequence of AML in FASTA format (GenBank ID: ADJ49206) was searched through the BLAST tool 2.6All the protein and ligand structures were prepared for docking using Biovia Discovery Studio 4.0 2.7The turbidity assay for polyester degradation was carried out as outlined in Masaki and colleagues, 2.82O, and then ethanol and left in a 37 °C incubator to dry overnight (18hrs). Subsequently, the sample was centrifuged at 17,000 × g for 10 min. The films were coated with a 6 nm layer of gold/palladium (Au/Pd) and analysed using a Hitachi SU6600 Field Electron Microscopy (FESEM) instrument employed at a 5.0 kV voltage acceleration, with a 7.1 mm working distance and beam current of 17µA.The ability of AML to degrade PET was tested using the procedure adapted from Austin and co-workers The supernatant of AML-treated PET film was removed for HPLC and fluorescence spectroscopy analysis. Terephthalic acid (TPA) and bis(2-Hydroxyethyl) terephthalate (BHET) released from PET degradation were quantified using HPLC. The HPLC analysis was performed using a Waters Alliance e2695 Separations Module equipped with ZORBAX Eclipse XDB C-18  HPLC column from Agilent Technologies. The mobile phase was neat methanol/50 mM phosphate-citric acid buffer (60:40% (v/v), pH3.8). Standard solutions of TPA and BHET over a concentration range of 0.05 – 1.0 mM were prepared as the reference for sample detection. The elution was operated at the flow rate of 1.0 ml/min in isocratic mode. The eluent peaks were detected at 241 nm using a Waters Alliance 2998 Photodiode Array Detector.eλ = 315 nm, λem = 425 nm) formed by the reaction of TPA and hydrogen peroxide. The reaction was performed by adding 2 ml of hydrogen peroxide into 1 ml of the reaction aliquot. The mixture was heated at 90 °C for 30mins and the reading was taken after the samples had cooled to room temperature. A calibration curve was plotted using the reading from standard solutions of TPA  dissolved in 0.05 M NaOH solution.The analysis was performed as per Silva and Cavaco-Paulo 33.1Streptomyces exfoliatus sharing 75% sequence identity, which is, therefore, suitable as a template for modelling studies The template for comparative modelling of AML was selected through a BlastP search against the Protein Data Bank (PDB) database. A phylogenetic tree was constructed to find the available templates with the closest evolutionary relationship to AML . AML wasStreptomyces exfoliatus (PDB: 1JFR) template with the SWISS MODEL server. The developed AML model was evaluated through a variety of validation tools including RAMPAGE, Verify3D, ERRAT and ProSA  and findings are summarised in A comparative model of AML  was constructed using the selected Ramachandran plots Through sequence alignment with the template, the catalytic triad and the AML oxyanion forming residues were identified as Ser178, Asp224, His256 and Phe110, Met179 respectively. Ser178, Asp224, and His256 are the triad responsible for the hydrolysis of the acyl group while the amide backbones of Phe110 and Met179 help to stabilise the enzyme-substrate intermediate via H-bonding with the carbonyl oxygen of the substrate The AML model has the typical α/β hydrolase structure – with a central β sheet surrounded by α-helices see A. FurtheThe experimentally determined preference of AML for medium chain length fatty acids  matches p-nitrophenyl acetate (C-2), p-nitrophenyl octanoate (C-8), and p-nitrophenyl palmitate (C-16) were generated using flexible docking in Biovia Discovery Studio 4.0. The binding poses were visualised using PyMOL and the ligand protein interactions are shown in a 2D map generated with Biovia Discovery Studio  required for tetrahedral intermediate stabilisation during catalysis p-nitrophenyl acetate has the acyl group pointed towards the less hydrophobic part of the binding pocket. This resulted in a suboptimal interaction of the carbonyl group with the oxyanion residues and the catalytic His225, which is important for the release of an alcohol group during the hydrolysis process p-nitrophenyl acetate .Candida rugosa LIP4 Candida antartica lipase A The main hydrophobic residues that interacted with the fatty acid chain of the acyl group are Gly109, Phe110, Met179, Trp203, and Val226. While Gly109 and Trp203 interact with C1 of the acyl group, Phe110 and Met179 interact with C2 and C3 of the acyl group and the position of their backbone amide is also important for intermediate stabilisation. Val226 interacts with C4 (C-8 substrate) and C5 (C-16 substrate), indicating that the mutation of Val226, to a bulkier hydrophobic residue, could shift the substrate preference of AML towards shorter acyl chains. Such engineering of a lipase substrate binding site for short acyl length preference has been previously demonstrated for 3.2AML was found to be homologous to several plastic degrading cutinases see . The abi3.3Amycolatopsis species have previously been described as having PLA degrading activity Poly is composed of lactic acid monomers. It is a widely used plastic and is the fastest growing market of all bioplastics The effect of degradation over time was also examined. Extensive hydrolysis by AML was observed with PCL and PBSc-D as substrates when the hydrolysis of polyester plastics was carried out for an extended time period (22hrs) at a modest temperature (30 °C): these polymers were degraded by 90 and 80% respectively 3.4T. alba has been recently reported T.alba cutinase Est119) has a sequence similar to AML . In view of the observed lack of PLA degradation by AML, its active site topology compared to Est119 was examined. The objective was to try to understand the differences between these related plastic degrading enzymes at a structural level.The mechanism of PLA degradation by a cutinase from Thermobifida cellulosilytica, were aligned to examine the difference in key substrate binding residues between them . However, when the wider substrate binding domain was considered  some subAmycolatopsis has been widely reported as having polylactide degrading activity the main enzymes responsible are reportedly proteases – not lipases Amycolatopsis are proteases It was clear that under these mild conditions that AML showed considerable activity towards PBS and PCL but showed no activity toward PLA. While 3.5The most persistent plastics in the environment are the petroleum derived aromatic polyesters, such as PET. These are a significant problem as pollutants and an enzyme capable of degrading this plastic is highly desirable PET is known to be highly resistant to enzymatic degradation and it is not easily prepared as an emulsion. Therefore, the same analysis could not be used as for the aliphatic polyesters, instead, degradation of the standard PET film was examined via scanning electron microscope (SEM). Degraded PET products  and bis-) in the reaction buffer were explored using high performance liquid chromatography (HPLC) and fluorometric analysis was performed to detect the release of TPA following PET degradation.Following an extended (96hrs) incubation of AML with PET film, at a moderate temperature (30 °C), there was no observable physical difference in the PET film as determined by SEM . SimilarIdeonella sakaiensis  T.fusca cutinase  Ideonella sakaiensis  4-methyl terephthalate [HEMT] – a PET analogue) PET degrading enzymes are generally divided into two groups; Type I and Type II based on their binding pocket sequence Ideonella sakaiensisA recent paper closely examined the key residues for degradation of PET by a cutinase from A report on the commercial application of cutinases in PET recycling published during the study reported here identified leaf-branch compost cutinase (LCC) as highly efficient at PET hydrolysis https://led.biocatnet.de/), Streptomyces exfoliatus as the structural representative. The family has 945 protein entries with 1,099 sequence entries and 21 structural entries. As the family also includes most of the reported polyester degrading cutinases and with all the members sharing > 60% sequence homology, structural comparison (besides the substrate binding site) reveals differences between the lipases/cutinases in their ability to degrade different plastic substrates. These include electrostatic and hydrophobicity variations In the latest update of the Lipase Engineering Database v4.0 (4Amycolatopsis species indicating that it is probably a key extracellular enzyme of these organisms. Modelling of the 3D structure of AML revealed that the enzyme lacked the lid structure seen with some lipases. This, along with substrate specificity studies, suggested that this enzyme is more appropriately named a cutinase. The catalytic triad residues in AML were identified and homology with known plastic degrading enzymes was observed.Homologs of AML were found in several A. meditarranei was capable of hydrolysing PCL and PBSc-D but not PLA or PET. A previous report identified that some Amycolatopsis species could degrade PLA, PCL and PBS but specific enzymes involved were not isolated Amycolatopsis orientalis spp. showed that the extracellular PLA degrading enzymes of these species were proteases and not lipases or cutinases single extracellular enzyme is likely responsible for the degradation of PCL and PBS by Amycolatopsis spp. It is clear that Amycolatopsis species are capable of degrading a wide range of polyesters and further exploration of this organism in plastics remediation is warranted based on the findings of this study.When plastic degrading activity was examined it was clear that the extracellular cutinase from Sequence comparison of AML with similar plastic degrading enzymes showed remarkable similarities between these cutinases. The most notable difference was where Phe110 was substituted by a Tyrosine residue in the PLA and PET degrading enzymes examined. Further work is needed to determine if this residue alone plays an important role in binding of PET and PLA.The specific substrate preferences of AML may be particularly interesting for enzymatic degradation of plastics where recovery of specific monomers is required. Thus, these studies show that PBS and PCL can be preferentially degraded even in the presence of PET and/or PLA under relatively mild conditions. This is potentially useful for degrading mixtures of plastics. Applications in controlled release drug formulations can also be envisaged.Yeqi Tan: Conceptualization, Methodology, Software, Validation, Visualization, Investigation, Data curation, Writing - original draft. Gary T. Henehan: Conceptualization, Methodology, Supervision, Writing - review & editing. Gemma K. Kinsella: Conceptualization, Methodology, Software, Validation, Supervision, Writing - review & editing. Barry J. Ryan: Conceptualization, Methodology, Validation, Supervision, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.