A method for targeted triggering of mitophagy with recombinant HOK protein

Abstract

The invention relates to a method for triggering the mitophagy mechanism targeted at mitochondria through HOK (host killing) gene expression, intended for use in regenerative medicine, biological-medical research for the treatment of various diseases, gene therapy, and other fields. Specifically, the invention is applicable in research targeting neurodegenerative diseases, all cell biology, cell biochemistry, and cell molecular biology studies related to mitochondrial and mitophagy dynamics, as well as in the development of new drug therapies for diseases associated with mitophagy. It involves a method for triggering mitophagy using bacterial recombinant HOK protein targeted at the mitochondrial inner membrane via a mitochondrial targeting sequence (MTS), enabling the monitoring of mitophagy and subsequent cell death.

Description

[0001] A METHOD FOR TARGETED TRIGGERING OF MITOPHAGY WITH RECOMBINANT HOK PROTEIN

[0002] Technical Area

[0003] The invention relates to a method for triggering mitochondrial-targeted mitophagy mechanisms through HOK (host-killing) gene expression, intended for applications in regenerative medicine, biological-medical research for the treatment of various diseases, gene therapy, and similar fields.

[0004] In particular, the invention relates to a method for triggering mitophagy by a bacterial recombinant HOK protein targeted to the inner lining of mitochondria via the MTS (mitochondrial target sequence) signaling sequence and for monitoring mitophagy and associated cell death for use in research targeting neurodegenerative diseases in all cell biology cell biochemistry and cell molecular biology research dealing with mitochondria and mitophagy dynamics and in research for new drugs for mitophagy-induced diseases.

[0005] State of the Art

[0006] Mitochondria are essential organelles that regulate cellular energy homeostasis and cell death. The elimination or degradation of damaged mitochondria through a process called mitophagy, which enables their participation in cellular recycling, is crucial for maintaining cellular health and viability. Indeed, recent studies have shown that mitophagy plays a critical role in the terminal differentiation of red blood cells, paternal mitochondrial degradation, neurodegenerative diseases, and tissue damage due to ischemia or drug exposure. Mitophagy not only preserves the health of the mitochondrial network but also plays a significant role in maintaining the health of the cell and the organism as a whole.

[0007] Various chemicals are used to induce mitophagy in both in vitro and in vivo models. However, a primary issue with these models is that the chemicals employed affect not only the mitochondria but the entire cell. The chemicals, some of which are listed below, exhibit their effects particularly in in vitro cell models when added to the culture medium at specific concentrations. Chemicals and their mechanisms that trigger mitophaqy: Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP): CCCP disrupts proton flow by increasing mitochondrial membrane permeability, leading to a reduction in mitochondrial membrane potential. This triggers mitochondrial damage and depolarization, initiating mitophagy. Rotenone: Rotenone is an insecticide that inhibits mitochondrial complex I. Inhibition of complex I can damage the mitochondrial electron transport chain, thereby triggering mitophagy. Antimycin A: Known to inhibit mitochondrial ATP synthase complex V, Antimycin A can increase mitochondrial damage and initiate mitophagy. Rapamycin: Rapamycin is an inhibitor of mTOR (mammalian target of rapamycin). Inhibition of mTOR can stimulate autophagic pathways that initiate mitophagy.

[0008] These chemical agents contribute to the clearance of damaged mitochondria in cells by triggering mitophagy through various mechanisms. However, when such chemicals are used, they affect the entire cell rather than targeting only mitochondria. Although chemical agents induce mitophagy, the effects observed are not solely mitochondria-specific, as nearly the entire cell is impacted. Therefore, careful consideration is necessary when using these chemicals, and specific cellular models or genetic methods are recommended, as some chemicals have broad activity and can cause cytotoxic effects. Among these, CCCP is the most commonly used to induce mitophagy in vitro and to study mitochondrial dynamics. To induce mitophagy using CCCP, it is added to the cell culture medium, and mitochondrial network disruption is monitored through immunofluorescence microscopy. CCCP (carbonyl cyanide m-chlorophenyl hydrazone) and rotenone are chemicals that affect mitochondrial function. By inhibiting the mitochondrial electron transport chain, they disrupt mitochondrial activity and can initiate mitophagy. However, mitophagy induced by CCCP or rotenone is generally not considered a reliable cellular model. Some reasons for this limitation are provided below:

[0009] ■ Non-Specific Effects: Agents like CCCP and rotenone affect various points in the mitochondrial electron transport chain, meaning they act on multiple targets rather than a single specific one. This can lead to unintended effects when attempting to study a specific mitophagy mechanism.

[0010] Cellular Stress and Toxicity: Compounds such as CCCP or rotenone can induce cellular stress and cause toxic effects, potentially leading to cellular responses that differ from the normal regulation of the mitophagy process. ■ Confounding Effects of Mitochondrial Damage: Because CCCP and rotenone cause mitochondrial damage, assessing the outcomes of this damage can be complex. When used to evaluate mitophagy, this may reflect a generalized damage response rather than an accurate assessment of the overall mitochondrial status within the cell.

[0011] ■ Challenges of In Vitro Models Representing In Vivo Conditions: Mitophagy induced by chemicals like CCCP or rotenone represents a state occurring in cell culture conditions, which may differ from mitophagy mechanisms that occur in a living organism. For these reasons, cellular models based on more specific and natural stimuli are often preferred to understand mitophagy mechanisms. For example, more targeted approaches, such as natural stimuli affecting cellular energy levels or genetic manipulations, may enable a more precise investigation of mitophagy mechanisms.

[0012] The Hok / Sok system is a post-segregational killing mechanism employed by the R1 plasmid in Escherichia coli. The Hok / Sok system consists of the following three genes:

[0013] 1. HOK - host-killing

[0014] 2. SOK - suppressor of killing

[0015] 3. MOK - modulator of killing

[0016] Within this system, the native Hok gene encodes a 52-amino acid toxin protein capable of penetrating the cell membrane, leading to loss of electrochemical potential and cell death.

[0017] In the field, significant progress has been made in identifying the molecular components of mitophagy through extensive in vitro and characterization studies; however, in-depth research is still required to fully understand all components of mitophagy. The unknown molecular details and mechanisms of mitophagy are being investigated using cellular models. Nevertheless, current mitophagy models use chemical agents such as CCCP, 6-OH dopamine, and rotenone to induce mitophagy, which affect not only mitochondria but the entire cellular system. The additional cellular effects of these agents, which are not representative of a natural model, are often overlooked. Consequently, it is challenging to assess the accuracy of current cellular mitophagy models in replicating natural mitophagy processes and to determine how closely the data obtained from these models reflect in vivo mitophagy. Therefore, in the current state of the art, there is a need for a method that selectively targets mitochondria to induce mitophagy using the HOK protein without the use of chemical agents. An example of the known state of the art in the literature search is patent application number WO2020124236A1 . The application relates to a screening method for identifying Parkin- mediated mitophagy activating agents. The document states that a mitophagy-inducing agent is used, selected from a proton ionophore, an iron chelator, or a mitochondrial toxin. The proton ionophore is specified as carbonyl cyanide m-chlorophenylhydrazone (CCCP). However, the document does not describe a model or method involving the induction of mitophagy by a mitochondrially targeted HOK protein.

[0018] In conclusion, due to the drawbacks mentioned above and the inadequacy of current solutions, further advancements in this technical field have become necessary.

[0019] Purpose of the Invention

[0020] The invention is inspired by existing situations and aims to solve the above-mentioned problems.

[0021] The primary objective of the invention is to establish a controllable mitophagy model that can be induced (triggered) without the use of chemical agents. The invention aims to provide a method that enables the induction of mitophagy, monitoring of mitophagy progression, and associated cell death by targeting a recombinant bacterial HOK protein to mitochondria through an MTS (mitochondrial targeting sequence) signal sequence. With this invention, HOK protein creates pores in the inner mitochondrial membrane, disrupting the mitochondrial membrane potential and thereby triggering mitophagy.

[0022] One aim of the invention is to selectively target mitochondria to trigger mitophagy, allowing the temporal tracking of mitochondrial disruption as regulated by HOK expression. This approach ensures that mitophagy and related cell death occur solely by targeting mitochondria. The pores formed by the HOK (Host Killing) protein in the mitochondrial inner membrane trigger mitophagy and associated cell death by exclusively affecting mitochondria. Furthermore, since HOK expression can be regulated by tetracycline in cells, the invention aims to offer a mitophagy and cell death model that can be controlled precisely from initiation to completion.

[0023] Another aim of the invention is to provide a model for mitophagy that is reversible, restartable, and stoppable as needed. Through this invention, HOK gene expression in eukaryotic cells can be initiated on demand and halted when necessary.

[0024] To achieve the aforementioned objectives, the invention is a method for triggering mitophagy specifically targeting mitochondria without the use of chemical agents, comprises: i. Applying codon optimization to the bacterial HOK gene sequence, ii. Following optimization, adding a mitochondrial targeting sequence (MTS) to the N- terminal end of the HOK gene sequence and a green fluorescent protein (GFP) sequence to the N-terminus to obtain the recombinant HOK construct (MTS-Hok- GFP), iii. Expressing the HOK recombinant construct into a eukaryotic expression vector to obtain the HOK construct, iv. Transferring the pTagRFPMito vector into the HEK 293T TetR+ cell line to enable mitochondrial labeling, v. Transferring the HOK construct into the HEK 293T TetR+joTagRFPMito cell line, vi. Induction of tetracycline into the cells created in process step V to synthesize HOK protein and target it to mitochondria and trigger mitophagy by disrupting the mitochondrial netw

[0025] The structural and characteristic features of the invention, along with all its advantages, will be more clearly understood through the detailed explanation provided below, and therefore, the assessment should be conducted considering these shapes and detailed descriptions.

[0026] Figures to Help Understand the Invention

[0027] Figure 1 shows the map of the pTagRFP-mito vector (Evrogen).

[0028] Figure 2 presents an immunofluorescence microscopy image of pTagRFP-positive HEK293T cells using a 100x 1 .3 objective.

[0029] Figure 3 illustrates the establishment of cell lines: A) Transfection of the pTagRFPMito vector into the HEK293T TetR+ cell line resulting in the creation of the HEK293T TetR+_PTagRFPMito cell line; B) Transfection of the MTS-GFP-HOK construct into the HEK293T TetR+_PTagRFPMito cell line leading to the generation of the HEK293T TetR+_PTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK cell line.

[0030] Figure 4 demonstrates the localization of the HOK protein in mitochondria through colocalization: A) DAPI nuclear (core) staining, B) MTS-GFP-HOK expression, C) RFPMito expression, and D) Overlay of the three images (100X objective).

[0031] Figure 5 shows the characteristic network structure observed in mitochondria after 48 hours of HOK expression induction, resulting in a punctate distribution (A: HOK expression not induced; B: HOK expression induced). Figure 6 shows the increase in HOK expression and time-dependent degradation of the mitochondrial network in HEK293T TetR+joTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK cells after tetracycline induction, as evidenced by confocal microscopy.

[0032] Figure 7 shows the increase in HOK expression and time-dependent degradation of the mitochondrial network in HEK293T TetR+_pTagRFPMito_pcDNA4 / TO-MTS-HOK-GFP cells after tetracycline induction, as evidenced by confocal microscopy.

[0033] Figure 8 presents ACT graph indicating the increase in HOK mRNA following tetracycline induction (Act: Actin internal control, HOK: Host Killing).

[0034] Figure 9 illustrates the cell death assay associated with HOK expression (*p<0.005, **p<0.001).

[0035] Figure 10 displays transmission electron microscopy (TEM) images of mitochondria in HEK293T TetR+_pTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK cells following HOK expression (Hok+ cells).

[0036] Detailed Description of the Invention

[0037] This detailed description outlines the preferred constructs of the method that is the subject of the invention, aimed solely at enhancing the understanding of the topic.

[0038] The invention relates to a method for triggering mitophagy in a mitochondria-targeted manner without the use of chemical agents. The method involves the following steps: i. Application of codon optimization to the bacterial HOK gene sequence, ii. Following optimization, the addition of a mitochondrial targeting sequence (MTS) at the N- terminal end and a sequence corresponding to green fluorescent protein (GFP) at the C- terminal end to obtain the recombinant HOK construct (MTS-Hok-GFP), iii. Expression of the HOK recombinant construct within an eukaryotic expression vector to obtain the HOK construct, iv. Transfer of the pTagRFPMito vector into the HEK293T TetR+ cell line to mark the mitochondria, v. Transfer of the HOK construct into the HEK293T TetR+joTagRFPMito cell line, vi. Synthesis of the HOK protein in the cells generated in step v through tetracycline induction, enabling targeting to the mitochondria and triggering mitophagy due to the disruption of the mitochondrial network. In the method of the invention, codon optimization is necessary for the expression of the HOK protein in vitro in eukaryotic cell systems (conversion to protein). The ability of the expressed recombinant HOK protein to trigger mitophagy is essential for monitoring associated mitochondrial dynamics and cell death.

[0039] In the method of the invention, the transfer of the generated MTS-GFP-HOK construct to pTagRFP-Mito-positive (carrier) HEK293T TetR+ cells is necessary for the expression of the HOK protein and for tracking mitophagy.

[0040] The synthesis of the HOK protein, which forms the basis of the invention, within the cells is crucial for its localization to the inner mitochondrial membrane, and that all these are important in terms of both tracking the HOK protein with GFP and monitoring mitochondrial changes with RFPMito.

[0041] The applications and analyses performed using the method of the invention are outlined below.

[0042] 1 . Codon Optimization

[0043] The process of codon optimization is a necessary procedure for expressing a bacterial protein in a eukaryotic system, taking into account the differences in the genetic code. This optimization allows for more efficient translation of the bacterial gene in the target cells by mimicking the genetic code of eukaryotic cells. During this process, the codons of the bacterial gene are modified to favor those that are commonly used in the target eukaryotic cells, thereby enhancing expression efficiency. It is important to select codons based on their usage frequency. Since HOK is a gene of bacterial origin, codon optimization is essential for its expression in eukaryotic cells. For this purpose, codon optimization and GC content balancing were performed using GENEius software (Eurofins Genomics). After this optimization, a mitochondrial targeting sequence (MTS) was added to the N-terminal end, and a sequence corresponding to green fluorescent protein (GFP) was added to the C- terminal end. The recombinant gene containing the complete HOK gene (NC_004998.1), which we optimized, was synthesized by Eurofins and cloned into the pEX vector, and subsequently sent to us.

[0044] The non-optimized gene sequence of HOK is as follows. The HOK optimized gene sequence is as follows

[0045] 2. Design of Recombinant Plasmid Vectors

[0046] From the plasmid construct that arrived in our laboratory, the MTS-GFP-HOK segment was extracted and subcloned into pcDNA4 / TO (Life Tech, USA), resulting in the construct pcDNA4 / TO-MTS-GFP-HOK. The pcDNA4 / TO vector is a eukaryotic expression vector that allows for the expression of the gene of interest upon tetracycline (Tet) induction in eukaryotic cells. The verification of the presence of the MTS-GFP-HOK gene in all colonies was confirmed by running the digested products with EcoRI and Hindlll on a 2% EtBr gel ( gel containing Ethidium Bromide) . The subcloned recombinant vector was transformed into E. coli DH10B cells in our laboratory, from which glycerol stocks were prepared, and endofree plasmid preparations were made for transfection studies.

[0047] The design of this plasmid vector is essential for the transfer of the optimized HOK gene and the associated mitochondrial inner membrane signal sequences and GFP protein sequences into eukaryotic cell systems.

[0048] The digestion of the pcDNA4 / TO-MTS-GFP-HOK construct with EcoRI and Hindlll and the subsequent analysis on a 2% Ethidium Bromide gel is important to demonstrate that the resulting vector contains the MTS-HOK-GFP construct.

[0049] 3. Transfection of pTagRFPMito Vector into Hek293T TetR+ Cells

[0050] The pTagRFPMito (Evrogen) vector was used to label mitochondria in Hek 293T TetR+ cells. This enabled the tracking of mitochondria under fluorescent microscopy. After the transfer of the pTagRFPMito vector into Hek293T TetR+ cells, the selection of cells expressing pTagRFPMito was carried out using the antibiotic Geneticin (G418). Following three weeks of selection with 600 pg / ml Geneticin, distinct colonies formed on 10 cm culture plates. These colonies were individually harvested using cylinders and transferred into 24-well culture plates. Each of the colonies transferred to the 24-well culture plates was examined under a fluorescent microscope, and the colonies exhibiting strong RFP signal indicating mitochondria were selected for further studies (Figure 2).

[0051] 4. Transfection of pcDNA4 / TO-MTS-GFP-HOK Plasmid into Hek293T TetR+joTagRFPMito Cells

[0052] The pcDNA4 / TO-MTS-GFP-HOK construct was transferred to the Hek293T TetR+joTagRFPMito cell line (Figure 3-B). The cells that received the constructs were selected in the presence of the antibiotic zeocin, and the resulting colonies were identified. Stable cell lines were established from the colonies selected in the presence of zeocin. Among the established cell lines, those exhibiting the strongest MTS-GFP-HOK expression were examined under fluorescent microscopy for GFP fluorescence after induction with tetracycline (10 pM / ml). The colony with the highest MTS-GFP-HOK expression was identified and designated as Hek293T TetR+_pTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK. Co-localization studies were conducted in stable Hek293T-TetR+ cells expressing both MTS- GFP-HOK and RFPMito proteins (Figure 4).

[0053] Additionally, the effect of HOK expression on mitochondria and the mitochondrial network was investigated through controlled experiments in Hek293T TetR+_pTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK cells. HOK protein expression was induced with tetracycline for 24 hours. The mitochondrial network was monitored via the RFP protein both before and after HOK protein induction. Fluorescent microscopy imaging revealed that mitochondria marked in red with RFP maintained their normal network structure prior to HOK induction, while it was observed that the expression of HOK protein disrupted the mitochondrial network following tetracycline induction (Figure 5).

[0054] Changes in the mitochondrial network following the synthesis and targeting of HOK protein to mitochondria were demonstrated over time using both MTS-GFP-HOK fluorescence microscopy (Figure 6) and the simultaneous tracking of RFPMito and MTS-GFP-HOK (Figure 7). These time-dependent studies indicate that the disruption of the mitochondrial network caused by HOK protein, which triggers mitophagy, began after the fourth hour. 5. Demonstration of HOK Expression via RT-PCR

[0055] Due to the absence of a commercial antibody against HOK protein, it is not possible to monitor the changes in HOK protein expression before and after tetracycline induction using Western blot analysis. Therefore, to demonstrate how HOK mRNA expression changes after 24 hours of tetracycline induction, RT-PCR primers were designed by us and obtained from lontek. The results of the RT-PCR analysis showed an increase in HOK mRNA following tetracycline induction (Figure 8).

[0056] 6. Demonstration of Cell Death Following HOK Expression

[0057] To reveal the effect of HOK expression on cell viability as a consequence of the disruption it causes in the mitochondrial network during mitophagy, a cell viability assay was conducted. In this study, Hek293T TetR+_pTagRFPMito cells were used as the control group, while Hek293T TetR+_pTagRFPMito_pcDNA4 / TO-MTS-GFP-HOK cells capable of HOK expression were used as the experimental group. Cells were cultured under two different conditions using low glucose (1 g / l) and high glucose (4.5 g / l) culture media. Following a 24- hour period of HOK protein expression, a Trypan blue exclusion assay was performed to determine the number of viable cells, comparing cells expressing HOK protein with those that did not. The results indicated that HOK protein expression significantly reduced cell viability in both low and high glucose conditions due to mitophagy (Figure 9).

[0058] 7. Demonstration of Changes in Mitochondria of Cells Expressing HOK Protein via Electron Microscopy Studies

[0059] The changes in mitochondrial morphology before and after HOK expression were directly monitored using Transmission Electron Microscopy (TEM). When comparing HOK- expressing cells (Hok+ cells) with cells that do not express HOK, it was observed that the structure of the mitochondria was compromised, with some being engulfed by autophagosomes (Figure 10).

[0060] The activity of mitophagy contributes to the regulation of mitochondrial morphology and function by mediating the processes of fusion and fission between mitochondria. Chemical agents such as CCCP and rotenone can trigger mitophagy activity by affecting mitochondrial functions. CCCP is a chemical that induces mitochondrial damage by reducing the mitochondrial membrane potential, while rotenone creates mitochondrial stress by inhibiting the mitochondrial electron transport chain. These chemical agents influence mitochondrial fusion and fission processes by triggering mitophagy activity. However, agents like CCCP and rotenone can be harmful to cells, which limits their use in biological systems.

[0061] Advantages of Mitophagy Triggered by Bacterial HOK Protein Using the Method of the Invention: 1 . Only mitochondria are targeted. There is no interference with other physiological activities of the cell.

[0062] 2. HOK expression is controlled by tetracycline, so allowing for the triggering of mitophagy at the desired level, the facilitation of a gradual progression, or the stopping of the process at a specific point. This enables the controlled investigation of mitophagy or associated cell death.

[0063] 3. After HOK transfection, cells expressing the HOK protein will be subjected to selection, resulting in a cell line that is monoclonal for HOK protein expression. Therefore, when HOK expression is induced, a uniform effect will be observed across all cells.

Claims

CLAIMS1. A method for the mitochondria-targeted triggering of mitophagy without the use of chemical agents, characterized by comprising; the following steps i. The application of codon optimization to the bacterial HOK gene sequence, ii. Following optimization, the addition of a mitochondrial targeting sequence (MTS) at the N-terminal end and a sequence from the green fluorescent protein (GFP) at the N-terminus to obtain the HOK recombinant construct (MTS-Hok-GFP), iii. The expression of the HOK recombinant construct within a eukaryotic expression vector to obtain the HOK construct, iv. The transfer of the pTagRFPMito vector into the Hek 293T TetR+ cell line for mitochondrial labeling, v. The transfer of the HOK construct into the Hek293T TetR+_pTagRFPMito cell line, vi. By Induction of tetracycline into the cells generated in step v. synthesis of the HOK protein, facilitating its targeting to mitochondria and triggering mitophagy via the disruption of the mitochondrial network.

2. The method in accordance with Claim 1 , characterized int hat: the obtaining of the following HOK recombinant sequence in step (ii)3. The method in accordance with Claim 1 , characterized in that: in the process step (iii), the HOK construct (pcDNA4 / TO- MTS-GFP-HOK) was obtained by tetracycline (Tet) induction of expression of the HOK recombinant gene into a pcDNA4 / TO- eukaryotic expression

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