A novel chemogenetic tool that allows intracellular pH modulation.

Abstract

The invention enables the determination of intracellular pH in living cells and tissues with high spatio-temporal resolution. with a new chemogenetic tool that manipulates blood glucose and hydrogen sulfide levels It is related.

Description

1 TARIFF A novel chemogenetic approach that allows intracellular pH modulation. VEHICLE Technical Area Intracellular pH with high spatio-temporal resolution in living cells and tissues. a new method that allows manipulation of levels of hydrogen sulfide This approach has been presented. When substrates are provided to the enzyme, β-Chloro D-alanine is released in sequence. 10 that allow the biochemical conversion of hydrochloric acid or D-cysteine ​​to hydrogen sulfide It is a substrate-based chemogenetic approach. This invention applies to both normal conditions and disease conditions. to study pH- and / or hydrogen sulfide-dependent signaling and metabolic pathways It offers a new vehicle. State of the Art (Prior Art) 15 Intracellular pH levels are tightly regulated. Gene expression, cell Mobility and metabolic processes are many things under the control of local pH fluctuations. These are a few examples of cellular processes. Therefore, cancer, cardiovascular disease, and neurological diseases... Multiple disorders, such as diseases, can be associated with pH imbalances. Intracellular pH The ability to directly monitor and manipulate levels within a single cell, subcellular and lower 20 Understanding organelle processes is crucial for understanding disease diagnosis and developing new therapeutic strategies. It has significant results. Various technologies are being used to investigate the role of pH at the single-cell level. They have been developed, but the application of micropipettes is hampered by the genetic or genetic requirements of proton pumps. chemical manipulation, optogenetic approaches, and small chemical inhibitors, etc. Traditional methods have off-target effects or are less practical. Therefore, acidic 25 allowing manipulation of pH levels with high spatiotemporal resolution in the range Lack of traceable experimental tools, pH imbalance in health and disease, and cellular imbalance This weakens the examination of the relationship between the functions. Previous attempts to manipulate internal pH (pHi) levels have involved ion transporters. overexpression or disruption of drugs, exogenous administration of drugs (e.g., cariporide, 30 This involves the use of EIPA, DMA or amilorid) and micropipettes. A very close At the same time, an optogenetic device is suitable for the precise and specific regulation of pH. It was developed to address the deficiency of the vehicle. Optogenetics and chemogenetics, physiological Optogenetics are very sophisticated tools that allow for the modulation of events at the single-cell level. 2 In this approach, the authors use light-assisted outward stimulation to increase pH within a physiological range. They used the proton pump Archaerhodopsin. Using this method, they observed an increase in pH. They were able to demonstrate the relationship between membrane fluctuation and dynamics. However, this The sophisticated device can only provide an increase in pH. Some conditions, such as neurodegeneration, are problematic. Since disease conditions are associated with a decrease in pH, 5 changes occur in cell physiology and behavior. It cannot cover the even more critical acidic pH. In addition, optogenetic devices have high While they can provide spatio-temporal resolution, chemogenetic tools can penetrate very deep tissues and in specific ultra-local areas (subcellular spaces such as mitochondria, nucleus, cytosol, ER, Golgi, caveolae, etc.) (regions) allow for controlled manipulation. More importantly, optogenetics These approaches require state-of-the-art and expensive facilities and a high level of expertise. This is why they are heard of, which limits their widespread use in basic and translation research. A similar situation applies to H2S. So far, it has not been possible to fully isolate H2S in single cells. There is no way to modulate it. There is no current method to study the role of H2S in cells. solutions, endogenous enzymes (mercaptopyruvate sulfur transferase, cystathionine beta-synthase and knockout / overexpression (like cystathionine gamma-lyase) or H2S-releasing 15 like NaHS It is the provision of medicines. Intracellular pH (pHi) levels and reducing stress are two meticulously regulated mechanisms in the human body. This is a critical parameter. However, precise mechanisms are lacking due to the absence of information technologies. This has been less studied. These tools allow for the real-time monitoring of these parameters in living cells. Because they allow for monitoring, genetically encoded biosensors developed in the last 20 years, 20 This has significantly improved our understanding of these complex biochemical pathways. However, to investigate the underlying mechanisms associated with the development of various pathologies in cells and endogenous and controlled increase in pH or H2S levels in tissues experimentally observable approaches that allow for manipulation and disruption There are none. The current methods for manipulating pH and H2S are completely outdated. It is outdated and incompatible with the current scientific age. In this context, a recent article, Increasing cardiac pH is a non-physiological method developed in 1981. to reduce the temporary change in pH by using NH4Cl and CH3COONa perfusion In another study, they investigated how it affects heart contraction, using a pH fluorescent probe. Its specificity lies in exposing mouse models to 10% CO2, an uncontrolled and radical approach. Intracellular acidification was assessed by leaving pH and H2S at the single-cell level. In the absence of a robust and specific tool for its manipulation, pHi's cellular physiology and It is impossible or unreliable to study its role in pathophysiology. 3 Brief Description and Objectives of the Invention The invention allows for understanding molecular mechanisms at ultra-local levels in cells and tissues. It allows for selective and ultra-precise manipulation of pH and H2S levels. This multifunctional chemogenetic approach uses stDCyD (D-) derived from Salmonella Typhimurium. It is based on a previously described enzyme called cysteine ​​desulfhydrase. This 5 technology, cardiovascular dysfunction, neurological disorders, immunological pathologies, various pathologies such as (but not limited to) cancer and metabolic diseases It can be used to develop a new animal model system that will mimic. Furthermore, this the approach potentially for the treatment of diseases or the development of therapeutic strategies It is useful. 10 The aforementioned chemogenetic tool analyzes high spatiotemporal levels of internal pH (pHi) and H2S. in specific tissues, cell types that allow for precise manipulation with resolution and can be expressed in subcellular regions. These manipulations can be performed on a single cell and This can be done in vivo. Since the tool in question is genetically encoded, the enzyme... It can be fused to any fluorescent protein, allowing for real-time detection. 15 In addition, stDCyD optimizes substrate concentration depending on the biological question. It can produce HCl and H2S in a dose-dependent manner (Figure 1e), meaning it can be converted to H2S. Another advantage of the approach is that stDCyD is silent without substrates (βCDA or D-cysteine). The problem is that it remains. Therefore, the chemogenetic approach involves removing these substrates from the cells after... It no longer produces HCI or H2S. Furthermore, its substrates are 20% effective in mammalian cells. because it is not found (except for D-cysteine, which can be found in a few tissues such as the liver and brain), The chemogenetic approach has no non-specific effects, and the enzymatic reaction... The amount of byproducts is negligible (Table 1, Figure 4). Important application areas for such a new chemogenetic approach could include the following: i.) Both are related to cardiovascular diseases, neurodegenerative diseases, and core 25 intracellular pH and H2S levels are relevant to numerous pathologies, including cellular biochemistry. Basic research aimed at investigating its role. ii.) The effectiveness of different drugs in relation to changing pH levels or hydrogen sulfide signaling. Development of a new animal model system that can serve the research. The aim is to mimic the relevant phenotypic changes in health and disease. 30 It is able to do so and find therapeutic targets for different pathological conditions. iii.) Currently, proton transfer inhibitors and other cellular inhibitors are used for cancer treatment. The inclusion of acidifiers is being improved. Therefore, our chemogenetic tools, 4 as an alternative to traditional therapeutic approaches, pH or H2S levels in tissues As a recombinant protein that manipulates blood vessels, it can be used for therapeutic strategies. This approach provides the following: i) Subcellular structures such as mitochondria, nucleus, cytosol, lysosomes, and endoplasmic reticulum. 5 existing traditional methods for manipulating and investigating the role of pH levels in regions Manipulation of intracellular pH levels is nearly impossible with these approaches. This is a critical feature. The approach we propose involves fusion to target sequences and is specifically designed for this purpose. genetically determined, which can be expressed and localized to desired compartments within the relevant cell. Because it is an encoded enzyme, it can address this deficiency. ii) 10 of the existing methods for manipulating pHi and H2S levels in cells Some use chemicals to inhibit ion transporters. For example, NHE1 and V-ATPase inhibitors have been used to lower pH; however, other molecules have also been used. And because they exchange protons at the expense of ions, these types of carriers are inherently certain targets. They have shown external effects; therefore, these methods reveal the role of pH under physiological settings. It lacks the necessary specificity required for investigation. On the other hand, chemogenetics 15 Our approach has nonspecific responses ranging from low to zero. stDCyD enzymatic The byproducts of its activity are pyruvate and ammonium, and the increase in their concentration is physiological. It falls within an acceptable range. Explanation of the Figures Describing the Invention 20 Figure 1. Characterization of pH Control. a) Schematic representation of the pH-control pathway and Simultaneous visualization with the pH-sensitive biosensor SypHer3s. b) DsRed-stDCyD Representative confocal images of HEK293T cells expressed together. Scale bar = 20 µm. c) In response to 13.4 mM βCDA in WT cells (n=3 / 39) or DsRed-stDCyD Real-time SypHer3s cytosolic pH traces in cells expressing (n=3 / 32). d) Bars, 1 25 After administration of mM βCDA, WT DsRed-stDCyD (n=4 / 21) and mutated and SypHer3s biosensor responses in cells expressing dysfunctional DsRed-stDCyD (n=4 / 18) (e) The left panel shows the response to various βCDA concentrations as shown in the figure. The right panel shows a representative curve of SypHer3s. The specified βCDA Upon application of concentrations, enzyme-free (green curve) or stDCyD expression 30 The red curve shows a concentration-response curve in HEK293T cells. N=3 and n=8-49 individual cells for all experiments. f) Rods, βCDA (n=3 / 29) or βCLA (n=3 / 29) shows the selectivity test of the treated cells. g) As stated (n=3 / 17) SypHer3s in response imaging medium with different pH levels and 1 mM βCDA. Representative real-time traces of HEK293T cells expressing pH control together. h) 10 or SypHer in DRG neurons expressing pH-Control in response to 1 mM βCDA. Representative real-time traces of signals. In addition, co-expression of SypHer3s and pH-Control. This shows representative confocal images of DRG neurons from a viral infection. i) Eight days later, dorsal root ganglion 5 expressed both ASAP2 and pH-Control. Representative confocal images of neurons. Scale bar = 50 µm. j) Representative real-time The curve shows the pH in response to high potassium (50 mM) and 10 mM βCDA, as indicated. ASAP2 voltage sensor signals in DRG neurons that express control together This shows (similar results were obtained from 4 different experiments and eleven separate cells). Student t-test was administered. 10 Figure 2. Mutant enzyme that remains inactive in the presence of βDCA in living cells. Figure 3. Production of H2S in the presence of a suitable biosensor, namely hsGFP, and its realization with optical devices. Simultaneous perception. Figure 4. Visualization of intracellular pyruvate, a byproduct of pH-control activity. (Left) Real-time 15 of the FRET biosensor Pyronic in response to 1 mM βCDA and 1 mM pyruvate. Traces. (Right) Rods upon application of extracellular pyruvate (n=5 / 34) and βCDA (n=5 / 34). This represents the maximum FRET response. A Student t-test was applied. Figure 5. Manipulating subcellular pH levels. a) Cytosol in response to 1 mM βCDA. (n=3 / 16), three different cellular structures including mitochondria (n=3 / 22) and the nucleus (n=3 / 33). a) Real-time SypHer biosensor traces in the region. b) Representative confocal images, pH 20 The sensitive biosensor SypHer3s (top panel) and pH-Control constructs (middle panel) are correctly identified. It shows the layout. The lower panels display fluorescence combined with bright field images. It shows images. Figure 6. Calibration of the pH biosensor SypHer3s. a) Scatter dot plot, at different pH values. Titration of extracellular imaging medium with levels of SypHer3s 25 These represent normalized ratio values. To make the cells permeable, 10 a) Titrated in the presence of 10 µM monensin and 10 µM nigerisin. b) As shown in the figure. (n=3 / 15) imaging media with different pH levels and monensin and nigericin Representative real-time image of HEK293T cells expressing the engineered SypHer3s biosensor. traces. 30 Figure 7. Overcorrection of intracellular pH levels following acute acidification. (left) In the presence of 10 µM nigerisin and 10 µM monensin, which permeabilize the plasma membrane (pink curve and (right) bars, n=3 / 15) and in its absence (green curve and (right) bars n=3 / 13) to 1 mM βCDA. 6 Real-time traces of HEK293T cells expressing SypHer and pH-Control in response. Student t-test was administered. Figure 8. pH control allows modulation of membrane potential in primary neurons. mM βCDA or high extracellular K+ levels (50 mM) and low Na+ levels (93 5 in primary DRG neurons that express pH-Control and ASAP2 together in response to (mM) Representative real-time traces of ASAP2 signaling. Individual cell responses from two different animals. It was taken from 4 independent experiments from the same group. Figure 9. Design of an untargeted dsRED-stDCyD plasmid. Figure 10. mito-DsRed-stDCyD plasmid design. Figure 11. DsRed-stDCyD-NES plasmid design 10 Figure 12. DsRed-stDCyD-NLS plasmid design. Figure 13. Design of an untargeted SypHer plasmid. Figure 14. mito-SypHer plasmid design. Figure 15. SypHer-NES plasmid design. Figure 16. SypHer-NLS plasmid design 15 Figure 17. Design of the dsRED-Y287FstDCyD plasmid. Detailed Description of the Invention This invention enables the simultaneous visualization of ultra-local acidification in living cells through genetic means. a novel chemogenetic approach combined with the biosensor SypHer3s, coded as 20 pH-Control is an abbreviation for Chemogenetic Operation of iNTracellular prOton Levels. It offers (Figure 1a). This combination involves the chemogenetic enzyme (sypHer biosensor). (by visualizing the incoming fluorescent signals) it was definitely possible to reduce the pH in the cells. This is important in terms of demonstrating it. This is what needs to be shown for the characterization of the approach. It was a critical thing. Substrate-based chemogenetic tools typically target an amino acid that is not naturally occurring. 25 They are recombinant proteins that remain silent until a biochemical stimulus is applied. Genetic These experimental systems, combined with biosensors that can be coded as such, have physiological and New research allows analysis of intracellular pathways that modulate pathological cellular responses. It has opened up areas. pH-Control, the novel chemogenetic tool described in this invention, is a red fluorescent protein 30 Salmonella variant called D-Cysteine ​​Desulfhydrase (stDCyD) It is a chimera of a Typhimurium derivative enzyme. In previous studies, stDCyD was found to be... it converts the unnatural amino acid β-chloro-D-alanine (βCDA) into the corresponding α-ketoaside. and in the presence of the cofactor pyridoxal 5' phosphate (PLP), hydrochloric acid (HCl), ammonium (NH4+) 7 and it has been shown to produce pyruvate byproducts. β-Chloro-D-alanine (βCDA) is a rootstock. It is an antibacterial substance and is metabolized by human cells and tissues. This is not possible. The StDCyD enzyme cannot be stimulated to produce hydrochloric acid from its substrate (βCDA). It can be differentially targeted to subcellular regions where the enzyme remains immobile. Theoretical calculations and experimental approaches show that the amount of by-products produced is negligible. It has been shown that it is at a manageable level (Table 1 and Figure 4). At the same time, in [H+] The change is a pH shift of one magnitude during the enzymatic activity of stDCyD. This is equivalent to a 900% increase (Table 1). Table 1. Treatment with βCDA when pH drops one order of magnitude (i.e., from 7 to 6). ions produced as (by)products of stDCyD enzyme activity after being retained and 10 Theoretical calculations of the percentage increase in the concentration of molecules. Initial concentrations, Roughly speaking, the ions / molecules in the human body (a single cell or tissue) taken from the literature. These are average levels. It is assumed that (by)products are produced as isomorphic and The average cell volume (HEK 293T) is assumed to be 5*10-12 L. First Name concentration (M) Last concentration (M) First mole Last mole Concentration changes percentage Hydronium Ion 10⁻⁷ 10⁻⁶ 5*10⁻¹⁹ 5*10⁻¹⁸ 900% Chloride ion 5*10-3 5.0009*10-3 2.5*10-14 2.50045*10-14 %0.018 Pyruvate 4*10-5 4.1*10-5 2*10-16 2.045*10-16 2.5% Ammonia (blood) 2*10-5 2.09*10-5 10-16 1.045*10-16 5% Overexpression of pH-Control with SypHer in cultured cells (HEK293T) results in cytosol, Even if it targets mitochondria or the cell nucleus differently (Figure 5), any visible No toxicity was observed (Figure 1b). High concentrations were applied to cells expressing pH-Control. βCDA application is potent, as documented by the pH-sensitive biosensor SypHer3s. It caused intracellular acidification. In contrast, wild-type cells, lacking the enzyme, underwent the same process for 20 minutes. It showed a marginal response (Figure 1c). A single molecule at position Y287F in the StDCyD enzyme mutation (a tyrosine residue at position 287 of the wild-type sequence of DCyD) (substitution with phenylalanine residue), upon provision of βCDA, cells This gave a dysfunctional control structure that could not acidify (Figure 1d). pH-Control expressing cells The constitutive activity of βCDA at different levels is concentration-dependent and completely reversible. 25 8 It exhibited a cyclical SypHer response (Figure 1e, left panel). This invention refers to pH-Control, a recombinant enzyme, lowers the pH in the presence of βCDA, and this decrease in pH, This can be visualized using a pH biosensor called SypHer. And the signal from SypHer, When it returns to the baseline after substrate removal, this is the reverse of the approach. This demonstrates its translatability. Also, only cells expressing SypHer 5 It remained unresponsive to the same treatment (Figure 1e, right panel). Selectivity tests showed that the enzyme was more resistant to D-alanine. remained agnostic and showed marginal responses to β-chloro-L-alanine (βCLA) compared to βCDA. This revealed that it showed (Figure 1f). Figure 1g shows that even low pH levels of the extracellular imaging medium can reduce the cytosolic pH. To acidify to the degree achieved by applying βCDA at concentrations (1 mM), 10 This indicates that it is insufficient. Predicting acidification levels using pH control. To do this, we calibrated the SypHer biosensor. The dynamic range of the pH biosensor is pH 7.5. We found it to be between 5.5 and 5.5 (Figure 6). Our results suggest that pH-control is a single measure of intracellular pH. During the process, manipulation of pH typically between 7.5 and 6.5 in the cytosol is allowed. This implies that it gave. Another critical observation is that after the withdrawal of βCDA, 15 The biosensor signal indicates cellular alkalization, consistent with a recent report. The problem was that it exceeded the baseline after the initial recovery (Figure 1c, 1g and Figure 5a). This problem was further addressed. To address this further, monensin and to resolve controlled proton transport across H+ channels... We visualized overcorrection in cells in the presence and absence of nigeris (Figure 7). Intracellular pH overcorrection was reduced when cells were permeabilized with monensin and nigericin. 20 Next, the pH-Control method was applied to mouse primary dorsal root ganglion (DRG) neurons. We tried to use it. Eight days after viral transduction, the cells showed both constructs, pH- Control and SypHer3 exhibited high expression levels (Figure 1h inset). Different βCDA Application of concentrations resulted in strong SypHer3s signals in DRG neurons (Figure 1h). Then, pH-Control mediated 25 to manipulate intact primary neurons. We tried to test whether acidification was sufficient. H+ production in the membrane targeting the outer cell membrane, assuming it will cause depolarization of its potential. We used the ASAP2 voltage sensor, a GFP-based biosensor (Figure 1i). βCDA, which is even more potent than extracellular potassium, provides depolarized primary neurons. (Figure 1j and Figure 8). Overall, our results show that the pH-Control method is effective for both cell 30 for cytosolic acidification, which is a functional consequence of both the streaks and primary cells. It shows that it is effective. 9 In conclusion, this involves the temporary and precise manipulation of intracellular pH levels. We developed pH-Control, a novel substrate-based chemogenetic method that enables neurons to... Even in complex cellular systems like these, pH control involves simultaneously managing intracellular acidification. It can be easily combined with any suitable biosensor for imaging. In the future, adding our new method to transgenic animal model systems will lead to the development of pathological 5 in addition to acidification and the ability to identify novel therapeutic targets involved in physiological pathways in turn, it is possible to dynamically change the pH balance in various cells and tissues. We expect it to happen. This discovery is unique because it synthesizes a toxic substance from a bacterium (Salmonella typhimurium). It uses an enzyme that is not present and is now recombinantly found in living cells. They can be produced using genetically encoded biosensors, namely SypHer for pH measurement. Or, if expressed together with hsGFP for H2S measurement, these parameters are ultra-sensitive space- It can be measured precisely with temporal resolution. In particular, intracellular pH and H2S levels in the cell. modulation on demand in six regions, unlike any traditional approach. This is not possible. Since stDCyD is genetically encoded, the internal pH (pHi) in the desired region must be 15. and allows precise manipulation of H2S levels in specific tissues and cells. It can be expressed in various cell types and subcellular regions. In addition, stDCyD exhibits fluorescence. Fusion with proteins allows for tracking the localization of the enzyme. Chemogenetics A unique element of these approaches is the absence of an activating substrate in the host body; this Therefore, a non-specific reaction is not expected, and the side effect of the enzymatic reaction is 20. The quantity of these products is negligible. Accordingly, in the case of stDCyD, βCDA or It remains silent until D-cysteine ​​is provided. In vivo, it reacts with other chemogenetic enzymes. As shown in our previous studies, substrates are given to animals via drinking water, injection, or It can be administered via eye drops. The enzyme's dose-dependent response is relevant to the biological question. According to this, it allows for the optimization of substrate concentration. 25 The figure below illustrates the basic principle of the chemogenetic approach developed in this invention. This is shown (Figure 1a). The StDCyD enzyme converts the unnatural amino acid beta-chloro-D-alanine. It can convert to hydrochloric acid. A genetically encoded biosensor. In its presence, this event can be measured as an optical readout. Accurate localization of the enzyme. To visualize this, a red 30 was used to create a new recombinant chimera (Figure 1b). It is fused to a fluorescent protein. Living cells subjected to the enzyme substrate exhibit cellular activity. It was able to acidify the regions. In the absence of the enzyme, only marginal pH changes were detected, and The pH sensor was documented by SypHer3 (Figure 1c). Another critical feature of this approach is that it creates a concentration-dependent and reversible effect in cells. This allows the formation of HCl (Figure 1e). The enzyme has a biochemical stimulus. As long as the biochemical stimulus (substrate application) is present, HCl can be produced. If removed, the pH returns to normal levels within minutes. As shown in Figure 4A, only beta-chloro-D-alanine (βCDA) but not D-alanine in cell 5 It can acidify the cell compartments. Additionally, it can affect intracellular pH levels in relation to the substrate. The manipulation is selective. Since D-cysteine ​​is another substrate of stDCyD, it is a weak acid. We believe that the pH was increased by applying 10 mM D-Cysteine, which is caused by H2S formation. A marginal decrease was observed. More importantly, with a related biosensor (SypHer). When combined, this allows for the precise determination of the actual pH levels in the relevant cellular region. By allowing this, pH levels can be calibrated (Figure 9). In the literature, the Y287F mutation inactivates stDCyD with respect to all substrates. It is known that this leads to the mutant enzyme being inactive in the presence of βDCA in living cells. To demonstrate that it remained, a mutant version of the enzyme was developed and expressed in cells. (Figure 2). 15 The stDCyD enzyme, utilized as a novel chemogenetic approach, is multifunctional and relevant. If the substrate is supplied to the cells, it allows H2S formation. A suitable biosensor, Therefore, in the presence of hsGFP, H2S production can be selectively detected in real time by optical devices. This can be done (Figure 3). Methods Creating Clones Primers and template plasmids are listed in Table 2. All constructs were converted to pLenti-MP2. (Addgene plasmid #36097) has been cloned. 25 targeting sequences (mito, NES, and NLS) have been added. Cloning fragments are cloned using Taq DNA polymerase (NEB, M0273) or Q5 high-accuracy DNA. The material was amplified by PCR with polymerase (NEB, M0491S). PCR products and cloning vectors were then processed in the respective databases. Double-digested with restriction enzymes (all purchased from NEB), from agarose gel. purified and ligated using T4 DNA ligase (NEB, M0202T). The ligation products were further They were then transformed into competent stbl3 E. coli (NEB) cells. The Y287F stDcyD mutant construct was used for 30 days. This was done via overlapping PCR. First, overlapping fragments were separated into primer sets using SalI- using stDcyD-For / stDcyD-Y287F-Rev and ApaI-stop-stDcyD-Rev / stDcyD-Y287F- They were amplified by PCR. The PCR products were then processed using SalI-stDcyD-For / Apa1-Stop-stDCyD-Rev. The primers were used as common templates for the next round of amplification. 11 The final product was cloned in pLenti-MP2 as previously described. All constructs were identical to the entire plasmid. This was confirmed by the arrangement. Tampons and Chemicals Dulbecco's modified Eagle medium (DMEM) is a phenol-free DMEM containing penicillin and 5. streptomycin, trypsin, and fetal bovine serum (FBS) were purchased from Pan Biotech (Aidenbach, Germany). Transfection reagent Polyjet was purchased from Signagen (Maryland, USA). β- Chloro-D-alanine was purchased from Biosynth Ltd (Compton, United Kingdom). β-Chloro-L- Alanin was purchased from Medchem (Istanbul, Türkiye). Unless otherwise stated, all The chemicals were purchased from NeoFroxx (Einhausen, Germany). CO2 incubation chamber 10 The cells outside the container were treated with 2 mM CaCl2, 5 mM KCl, 138 mM NaCl, 1 mM MgCl2, and 1 mM HEPES. (Pan-Biotech, Aidenbach, Germany), 0.44 mM KH2PO4, 2.6 mM NaHCO3, 0.34 mM NaH2PO4, 10 mM D-Glucose, 0.1% MEM Vitamins (Pan-Biotech, Aidenbach, Germany) 0.2% essential amino acids (Pan-Biotech, Aidenbach, Germany), 100 µg / mL Penicillin (Pan- Biotech, Aidenbach, Germany) and 100 U / mL Streptomycin (Pan- Biotech, Aidenbach, 15 It was stored in a storage buffer containing (Germany). The pH was adjusted using 1 M NaOH. Set to 7.43. Cell storage buffer, 0.45 µm ambient filter (Isolab, Germany) It was sterilized using [method]. For live cell imaging experiments, 2 mM CaCl2, 5 mM KCl, 138 mM NaCl, 1 mM A HEPES-buffered physiological saline solution consisting of MgCl2, 10 mM HEPES, and 10 mM D-glucose. Nigerisin (N7143, Merck) was used, and the pH was adjusted to 7.43 using 1 M NaOH. For experiments with monensin (M5273, Merck), 5 mM KCl, 138 mM NaCl, 1 mM Imaging involving MgCl2, 20 mM HEPES, 10 mM D-glucose, 0.2 mM EGTA, and pH. The buffer was adjusted to the desired value using 1 M NaOH or 1 M HCl. β-Chloro-D- To prepare alanine solutions, the powder was dissolved in imaging buffer and the pH was adjusted to 7.43. Reset. Cell culture Characterization studies were carried out in a humidified incubator (37°C, 5% CO2) with 10% FBS and A full 30-day high-glucose (4.5 g / L) solution containing 100 μg / ml streptomycin and 100 U / ml penicillin. cultured human embryonic kidney cells (HEK293) grown in medium This was performed. 24 hours prior to transfection, the cells were placed on a 30 mm glass coverslip No. 1. (Glaswarenfabrik Karl Knecht Sondheim, Germany) was seeded onto (~3 x per cavity) (10⁵ cells). Cells at ~70-80% confluence, transfected via PolyJet according to manufacturer's instructions. 12 Differential targeting of SypHer3 or DsRed-stDCyD enzymes using the reagent transfected with CMV-guided mammalian expression vectors (pLenti-MP2) All imaging experiments were performed 24 hours after transfection. HEK293T The cells were cultured up to passage 30. Primary Neuron Culture: WT C57BL / 6 mice were euthanized via cervical dislocation. 5 Bilateral DRGs were dissected from all segments and then subjected to an ice-cold RPMI. Placed in medium 1640 (R0883, Gibco). For enzymatic dissociation, ganglia, Neural Basal 2% B27 (17504-044, Gibco), 2 mM in media (NBA, 10888-022, Gibco) at 37C, 5% CO2 Glutamax-I (35050-61, Gibco), 100 U penicillin and 100 mg streptomycin (15140-122, Gibco) Incubated with 100 U / mL collagenases (C9407, Sigma) in a neural environment containing 100 U / mL collagenases. 40 minutes After an incubation period, the collagenase-containing medium was removed and the ganglia were examined by Hank. The ganglia were washed using buffered saline solution (H9269, Sigma). The ganglia were also examined for neural involvement. The experiment was conducted using 1 mg / mL trypsin (25300-054, Gibco) at 37 °C, 5% CO2 for 15 minutes. was enzymatically broken down over a period of time. At the end of the incubation, the free DNA fragments To inhibit trypsin, 50 mg / mL DNase (D4513, Sigma) 15 was added to the cell suspension. The tissues were added and powdered by pipetting to obtain single cells. 37°C, After 30 minutes of incubation at 5% CO2, the cell suspension was 120 g for 3 minutes. It was rotated and 10% fetal calf serum and 700 mg / mL were added to inhibit enzymatic activity. The neural environment was resuspended after supplementing with a trypsin inhibitor (T6522, Sigma). To purify DRG neurons from satellite cells and cellular debris, cell 20 The suspension was carefully fitted to 10%, 35%, and 60% percoll gradients (P4937, Sigma). and rotated at 300 g for 20 minutes. Total sensory neurons were approximately 35% percoll. It was collected from the layer and the cell suspension was spun at 120 g for 3 minutes. The pellet, The virus was resuspended in an antibiotic-free neural environment for transfection. Following this procedure, the cells were placed in a petri dish coated with 10 mg / ml laminin (L2020, Sigma) 25 They were seeded into containers. One day after incubation, the cells were treated with lentivirus as described above. It was transformed. High titer virus purification HEK293T cells were transferred at a 1:4 plasmid:PEI ratio when 70% confluence was reached. Along with plasmids (pLenti-MP2-SypHer3s or pLenti-MP2-DsRed-stDCyD). MDL, RSV-rev, VSVG (gifts from Didier Trono, Addgene plasmids #12251, #12253 and A gift from Arthur Nienhuis & Patrick Salmon, transfected with Addgene plasmid #35616. Lentivirus-containing medium was collected 72 hours after transfection, then divided into 3000 g portions. 13 It was centrifuged for 1 minute and filtered through a 0.45 µm PES filter (SLHP033RS, Millipore). Lentiviral particles were incubated at 10,000 g on a 20% sucrose cushion for 4 hours at 4°C. Purified by centrifugation and then centrifuged again in ice-cold 1x PBS. It was further concentrated with its parameters. 70% conjugation of AAV-HEK293 cells, 1:4 plasmid:PEI ratio with pAAV-hSyn-ASAP2s, 5 pAdDeltaF6 and pAAV2 / 1 (a gift from François St-Pierre and a from James M. Wilson) Gift, plasmids (purchased from Addgene plasmids #112867, #112867, #101276) They were transfected. The cells were collected with a cell scraper 72 hours after transfection and The cells were pelleted by centrifugation at 300 g for 5 minutes. The cell pellets were then immersed in lysis buffer (150 mM). NaCl, 20 mM Tris, 1 mM MgCl2, pH: 8) was resuspended and subjected to 3 freeze-thaw cycles. cycle and subsequent sonication and 45 minutes of treatment with Benzonase (E8263, Sigma) at 37°C. It was then broken down. The cellular debris was removed by centrifugation at 4°C for 20 minutes at 300 g. The supernatant containing AAV was subjected to a 2-hour gradient of Iodixanol (D1556, Sigma) for further purification. Ultracentrifuged at 220,000 g via (60%, 40%, 25%, 17%). AAV particles, 40%- Collected from the interface between 60% gradients. Iodixanol was removed and AAV particles, 15 3x centrifugation at 4oC for 30 minutes with 300g and 100K column (UFC910024, Millipore) Concentrate in a storage buffer (1x PBS, 5% D-sorbitol, 200 mM NaCl) using It was done. Live cell imaging 20 Wide field imaging experiments were performed using the Colibri 7 LED light source (423 / 44 nm, 469 / 38 nm, 555 / 30), Plan-Apochromat 20× / 0.8 dry objective, Plan-Apochromat 40× / 1.4 oil immersion objective The lens is a Zeiss Axio Observer Z1 / 7 equipped with a monochrome CCD camera, the Axiocam 503. This was carried out at Carl Zeiss AG, Oberkochen, Germany. The substrates are a metal. Apply to cells placed in a perfusion chamber (NGFI, Graz, Austria) and return 25 A custom-made pump-driven perfusion system was used to draw up the SypHer signals, beam with separators (FT455 (for SypHer low, F420) and FT495 (for SypHer high, F490)) Alternating excited cells using a pair of filter wheels equipped with motors. Emissions were viewed by a band-pass filter (BP 525 / 50). DsRed-stDyCD emissions were collected using FT570 (BS) filter combinations and 30 Data was collected using a 605 / 70 emission filter. Data collection during live viewing, Developed using Zen Blue 3.1 Pro software (Carl Zeiss AG, Oberkochen, Germany). Confocal imaging with Plan-Apochromat 40x / 1.3 DIC (UV) VIS-IS oil immersion objective. using an equipped laser scanning confocal microscope LSM 800 (Zeiss, Germany) 14 This was done. SypHer biosensors were excited with 488 nm and 405 nm lasers and a 509 nm filter system. Emissions were collected using SypHer signals, a Multialkali-PMT detector. This was achieved using a GaAsP-PMT detector and a 400–565 nm filter. DsRed- stDCyD structures were excited using a 561 nm laser, and the emission wavelength was between 616 and 700 nm. It was caught between them. Digital detector gain was set to 1 for all channels; detector gain was 5 It was applied between 500 and 1000 V. Laser intensities were between 0.90% and 0.95%, needle The hole size was adjusted between 29 and 32 μm depending on the expression level of the fluorescent proteins. Bright field mode was visualized using a photodiode detector. Areas of interest... Zen Blue 3.1 software (Zeiss, Germany) was used in its determination. Statistical analysis Image analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). This was done using [method]. All experiments were repeated in at least three copies, and the complete experiments were performed. The number is given as 'N', and the total number of cells displayed is indicated by 'n.' in the figure descriptions. This is expressed as follows. For example: 3 / 32, where N = 3 (triple cultures) and n = 32 (15 in this experiment). (number of cells displayed) is shown. Statistical comparison of the two groups, two The results were evaluated using a Student's t-test with tails. Homeostasis of pHi and pHe is involved in neuronal burst firing, silencing, and in neurotransmission, and therefore in the physiology and pathophysiology of the central nervous system They play critical roles. The correlation between pHi and neuronal excitability, for example, 20 chemosensitive neurons, hippocampal neurons that exhibit milder responses compared, they will show higher levels of excitatory responses to intracellular acidosis. It appears that pHi depends on the type of neurons. Changes in pHi are seen in the locus coeruleus. To evaluate how it affects the chemosensitive response of neurons, Filosa et al., hypercapnic acidosis, isocapnic acidosis, isohydric hypercapnia, acidified Hepes buffered 25 They applied medium or HCO3- buffer containing propionate and then changed the pH and this They measured the neuronal firing rate related to the change. In this way, they showed that acidic pH loading most that it is responsible, at least in part, for the increased firing rate in locus coeruleus neurons They demonstrated it. However, the precise sequential mechanisms and pathways remained unclear. This challenge, Basically, managing intracellular pH levels in a reliable, controlled, and physiological manner. 30 This stems from the lack of a robust and universally applicable approach. On the other hand, more than 60 genes, directly or indirectly, influence pH via the cell membrane. It codes for genes that are directly responsible for regulation. Therefore, in cells Regulating pH by manipulating the endogenous proton regulation mechanism is plausible. It will not happen and in most cases it seems likely to backfire. More importantly, the proton Its level is not independent of other ions and molecules in the intracellular matrix, and pH Changing pH by manipulating regulatory elements affects different molecules and ions. (e.g., Na+, K+ and Cl-) bioavailability, i.e., perturbation of counterions. It affects and all the following data provided in this way will be suspect. 5 In contrast, the chemogenetic tool described in this invention addresses this issue with the highest accuracy and It can effectively process the StDCyD enzyme. The StDCyD enzyme is produced exogenously and can be processed without any... It remains dormant until the substrate, which can be stopped in time, is complete. Therefore, a It adds a layer of specificity and improves the spatio-temporal resolution of the chemogenetic approach. It increases considerably. 10 pH changes affect cells in general and at the macro level, such as in cell migration and proliferation. In addition to unraveling how pH affects protein structure and behavior in a physiological context, Its effect on function is important. Some proteins harbor hypersensitive switches and Dramatic conformational and functional changes occur with a slight change in in vitro pH. This indicates that, however, the only way to reliably track this phenomenon is at pH 15. It is the highly controlled intracellular regulation of the pH level; otherwise, pH The changes can be subtle or pronounced and may not provide physiologically relevant data. In in vitro traditional methods attempting to elucidate the role of pH level in cellular signaling In contrast to previous studies, this innovative chemogenetic tool has high spatiotemporal resolution. Promising research into downstream pH-dependent signaling pathways in a cellular environment. 20 The windows are opening. The primary sequence of the instrument referred to in the invention is shown above. The boldly highlighted sequence is 25 red represents the primary sequence of the fluorescent protein DsRed, and black is highlighted. The sequence is the primary enzyme derived from Salmonella Typhimurium, called stDCyD. The sequence is as follows: A single mutation results in a non-functional enzyme (as shown underlined). MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAKLQVTKGGPLPFAWDILSPQFQYGSKA YVKHPADIPDYMKLSFPEGFTWERSMNFEDGGVVEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTA GWEPSTEKLYPQDGVLKGEISHALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQ YEHAEARHSGSQVDATMPLHHLTRFPRLEFIGAPTPLEYLPRLSDYLGREIYIKRDDVTPIAMGGNKLRKLEFLVA DALREGADTLITAGAIQSNHVRQTAAVAAKLGLHCVALLENPIGTTAENYLTNGNRLLLDLFNTQIEMCDALTDP DAQLQTLATRIEAQGFRPYVIPVGGSSALGAMGYVESALEIAQQCEEVVGLSSVVVASGSAGTHAGLAVGLEHL MPDVELIGVTVSRSVAEQKPKVIALQQAIAGQLALTATADIHLWDDYFAPGYGVPNDAGMEAVKLLASLEGVL LDPVFTGKAMAGLIDGISQKRFNDDGPILFIHTGGAPALFAYHPHVDIIDIITG* 16 It has been brought to position 287 as indicated. Primers and template plasmids are shown in Table 2, and these engineered plasmids... The intended uses are listed below; Untargeted dsRED-stDCyD: altering pH throughout the cell without specific targeting. for, 5 mito-DsRed-stDCyD: To change pH only in the mitochondrial matrix, DsRed-stDCyD-NES: used to alter pH only in the cytosol. DsRed-stDCyD-NLS: for changing pH only in the core, Untargeted SypHer: pH changes in all cells without specific targeting. To visualize, 10 mito-SypHer: for visualizing pH changes only in mitochondria, SypHer-NES: for visualizing pH changes in the cytosol, SypHer-NLS: for visualizing pH changes in the nucleus, dsRED-Y287F stDCyD: observed pH changes are due solely to enzyme function. It is caused by and if the enzyme is rendered dysfunctional by the Y287F mutation, the pH will no longer be 15 To show that it cannot be changed. 17 Table 2. Primers and template plasmids Plasmite Name Forward Primer Reverse Primer Untargeted dsRED-stDCyD BamHI-DsRed-stD-FWD ApaI-stop-AgeI-stD-dsRed-REV ATAATAGGATCCTCTAGAGCCACCATGGAC AACACCG TATTATGGGCCCTTAACCGGTTATGATATCT ATGATATCAACGTGGG myth-DsRed- stDCyD Myth-stD-BamHI-FWD Myth-stD-XbaI-REV TATGGATCCGAATTCGCCACCATGTCTGTTC ATATCTAGACTTGGCCCTGGGGACTG DsRed-stDCyD- NES AgeI-stD-NES-FWD stD-NES-ApaI-REV FATHERCCGGTCTGCCCCCCCTGGAG FATHERGGCCCGTGGCAACTTCCAG DsRed-stDCyD- NLS stD-AgeI-NLS-FWD stD-NLS-ApaI-REV CAAGTTCTGCTTCTGACCGAGGGC ATAGGGCCCTTCTAGAGGCTCGAG Hedeflenmemiş SypHer BamHI-UN.SypHer-pLenti-FWD XbaI-UN.SypHer-pLenti-REV TATGGATCCACTAGTGCCACCATGTCCGGA C GATATCTAGATACGCGTCTCGAGAACCG CCTGTTTTAAAAC myth-SypHer XbaI-SypHer-myth-pLenti-FWD ApaI-SypHer-myth-pLenti-REV TATTCTAGAGCCACCATGTCCGGACCGCTG GCAATAGGGCCCTTAAACCGCCTGTTTTAA AACTTTATCG SypHer-NES MluI-SypHer-NES-pLenti-FWD XbaI-SypHer-NES-pLenti-REV TATACGCGTCTGCCCCCCCTGGAG ATATCTAGAGTGGCAACTTCCAGGGCCAG SypHer-NLS MluI-SypHer-NLS-pLenti-FWD stD-NLS-ApaI-REV GTATACGCGTACCGGTGATCCAAAAAAG ATAGGGCCCTTCTAGAGGCTCGAG dsRED-Y287F stDCyD SalI-stDcyD-For ApaI-stop-stDcyD-Rev CAGGTCGACGCCACCATG TATCTGTACAGGGCCCTATGAATTC stDcyD-Y287F-For stDcyD-Y287F-Rev CGATCCAGTTTTCACTGGGAAGG GCCTTCCCAGTGAAAACTGGATCG

Claims

18 REQUESTS 1. As a chemogenetic tool for manipulating intracellular (internal) pH (pHi). In vitro use of recombinant D-cysteine ​​desulfhydrase (DCyD) enzyme.

2. pH-dependent production of recombinant D-Cysteine ​​Desulfhydrase (DCyD) enzyme according to Claim 1. In vitro use in signaling and / or metabolic pathways.

3. The DCyD enzyme is derived from Salmonella Typhimurium, in accordance with Claims 1-2. In vitro use of recombinant D-cysteine ​​desulfhydrase (DCyD) enzyme. 10 4. It is a chemogenetic method for modulating internal pH, and its characteristic feature is; i. Provision of the D-Cysteine ​​Desulfhydrase (DCyD) enzyme sequence, ii. Cloning the aforementioned enzyme sequence into the cloning vector, 15 iii. Transferring the cloning vector into the living cells whose pH is to be altered. iv. Application of enzyme substrates to cells in which recombinant enzyme is produced. It includes the steps.

5. A chemogenetic method that complies with claim 4, characterized by the addition of at least one to the cloning vector. This involves the cloning step of a pH-sensitive fluorescent protein sequence.

6. A chemogenetic method that complies with claim 4, and is characterized by having at least one pH biosensor. This involves the step of transferring the vector containing the sequence into living cells. 25 7. This is a chemogenetic method conforming to claim 4, characterized by the use of β-chloro- enzyme substrates. It can be either D-alanine (BCDA) or D-cysteine.

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