Graphene quantum dots harvest anti-trypanosomatid efficacy by disrupting antioxidant networks centered on trypanothione reductase

doi:10.21203/rs.3.rs-2457845/v1

Download PDF

Research Article

Graphene quantum dots harvest anti-trypanosomatid efficacy by disrupting antioxidant networks centered on trypanothione reductase

https://doi.org/10.21203/rs.3.rs-2457845/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Trypanosomiasis is a highly lethal infectious disease caused by trypanosome, leading to a severe social and economic burden worldwide. Due to the lack of mechanism research, application of the promising nanomaterials and nanotechnologies in treatment of trypanosomiasis is limited.

Results

Herein, the toxicological effects induced by graphene quantum dots (GQDs) on T. brucei and the underlying mechanism are investigated. First, the biological/cytotoxic effects are evaluated, including endotytosis, cell viability, apoptosis, ROS production and morphological defects of subcellular organelles. Considering the few experimentally-determined 3D structures of T. brucei proteins, next, a computed structure database of T. brucei genome-wide proteins is constructed from I-TASSER, AlphaFold2 and MD simulation. Then, the database is used for docking with GQDs, and two goups of potential target proteins with transporter activity and antioxidant activity are screened out. Last, TryR stands out as a vital target due to its high binding energy with GQDs at active site and its key role in the trypanothione-dependent antioxidant network of T. brucei, which is further verified by theoretical (MD simulation) and experimental (BLI, inhibition of enzyme activity) means.

Conclusions

Evidences from this study suggest that GQD-induced cytotoxicity on T. brucei results from interference of GQDs with the lineage-specific antioxidant network with TryR as a key target. These findings provide theoretical insights into the rational design of nanomedical materials for trypanosomiasis.

Trypanosoma brucei

Graphene quantum dots

Trypanothione reductase

Nano-protein interaction

Nanobiological effects

Trypanosomiasis imposes a considerable health burden on developing countries and areas with poor community living conditions, mainly involving African trypanosomiasis (sleeping sickness) and American trypanosomiasis (Chagas disease)[1]. African trypanosomiasis spreads in sub-Saharan Africa[2], caused by Trypanosoma brucei and transmitted by tsetse flies[3]. Two subspecies, Trypanosoma brucei gambiense[4] and Trypanosoma brucei rhodesiense[5], are responsible for chronic and acute forms of the disease respectively[6]. American trypanosomiasis occurs in Central and South America caused by Trypanosoma cruzi and transmitted by Tria bug[7], afflicting about 8 million people and leading to about 20,000 deaths each year (from CDC Website)[8]. The pathologic changes associated with trypanosomiasis severely impair the physical and mental health of infected patients and carry a high risk of stroke and sudden death if left untreated[9].

For the treatment of trypanosomiasis, hopes are high for chemotherapy compared with sluggish development of vaccines[10]. So far, however, only a few drugs, such as suramin, pentamidine[11], melarsoprol[12], eflornithine[13] and most recently fexinidazole[14], have been promoted as first-line agents, against a background of inefficient new drug development[15]. Unfortunately, most of these drugs are unsatisfactory due to limitations in efficacy, toxicity, administration route, or drug resistance[16]. Despite their shortcomings, these drugs are currently the preferred treatment for trypanosomiasis, reflecting the lack of more suitable alternatives. Considering the high costs and long cycles associated with new drug development, greater efforts must be made to develop new therapeutic strategies for optimal control of trypanosomiasis[17]. Recently, the application of nanotechnology offers new opportunities for the control of trypanosomiasis, involving drug delivery systems, therapeutic agents[18], diagnostic materials[19] and vaccine adjuvants[20], etc. Various nanomaterials, including polymers[21], lipids[22], and metallic/inorganic systems[18] have demonstrated their utility in the diagnosis and treatment of protozoan diseases including trypanosomiasis. Especially, ultra-small nanomaterials represented by graphene quantum dots[23] (GQDs) and noble metal nanoclusters[24] have shown excellent potential of anti-protozoal diseases, which rely on not only the universal advantages of nanomaterials, but also the antiprotozoal characteristics of materials themselves. More importantly, these smaller materials are better suited for endocytosis via a clathrin-dependent endocytic system at the flagellar pocket of trypanosomes, where internalized nanomaterials are limited in size (< 130 nm)[25].

Recently, GQDs have been an increasing interest for the fight against trypanosomiasis[26]. GQDs can be approximately defined as a zero-dimensional graphene material with atomically thin layer thickness (1–3 layers) and a plane size usually less than 10 nm[27]. The advantages of GQDs as a biomedical material for treatment of protozoosis are as follows: 1) unique planar structure gives GQDs a high specific surface area and strong adsorption capacity, allowing for higher drug load and more efficient drug delivery; 2) Oxygen-containing functional groups such as hydroxyl and carboxyl groups[28] at the edges endow GQDs with excellent water solubility[29] and easy surface-conjugation modification with targeting ligands or chemotherapeutic drugs[30]; 3) the peroxidase-like activity of recent concern renders GQDs highly toxic to pathogenic microorganisms (including protozoa) by inducing oxidative stress[31, 32]. In addition, the biomolecule-comparable size enables GQDs to interact with key protein molecules and affect their function, thereby triggering the toxicity of GQDs to protozoon pathogens[33]. Overall, the use of GQDs offers new opportunities for combating trypanosomiasis due to these excellent properties. However, given the lack of research in this field, greater efforts are needed.

Challenges in controlling trypanosomiasis arise primarily from the complex life cycle and diverse ontogeny of pathogenic parasites. For example, trypanosoma brucei brucei (T. brucei), a model organism for studying trypanosomiasis which is the causative agent of Nagana[34], includes the elongated bloodstream form (BSF) in mammalian host blood and the stubby procyclic form (PCF) in tsetse fly intestine. BSF and PCF are not only significantly different in morphology, but also in energy and substance metabolism[35]. Especially, the lineage-specific antioxidant system and involved regulatory enzyme molecules are often the therapeutic targets of interest[36]. Unlike humans, however, trypanosomes have evolved a unique thioredox system that relies on trypanothione, a condensation product of one spermidine and two glutathione molecules[37]. The conversion of trypanthione in its oxidized (TS₂) and reduced (T(SH)₂) forms is driven by the species-specific trypanthione reductase (TryR), a NADPH-dependent oxidoreductase[38]. Further, the Trx-like oxidoreductase tryparedoxin (TXN) catalyzes the transfer of electrons from T(SH)₂ to various target proteins, thereby forming a trypanothione-centered multi-enzyme-assisted antioxidant system. Overall, trapanothione metabolic homeostasis is critical for maintaining trypanosome infectivity and viability in host system[39], which has been a concerning focus of nanotoxicological research and nanodrug delivery design in recent years[26].

In this work, we investigated the inhibitory effect of GQDs on T. brucei brucei and their underlying mechanism to evaluate whether GQDs are effective medical materials for the treatment of trypanosomiasis. After evaluating the effects of GQDs on T. brucei cell viability, apoptosis and ROS production, a computed structure database of T. brucei genome-wide proteins (reference genome Tb927) was constructed by utilizing I-TASSER and AlphaFold2 programs. Then, the interaction of GQDs and database proteins was performed by docking simulation (PatchDock program) and ranked according to the thermodynamic stability of GQD-protein complexes. Proteins in top ranked complexes were selected for the GO enrichment analysis, and more detailed interaction were analyzed with MD simulations using the GROMACS software package. For potential target protein candidates, recombinants were prepared by prokaryotic expression. Bio-layer interferometry (BLI) measurement, MD simulation and enzyme activity inhibition assay were performed to obtain the thermodynamic information on the interaction between GQDs and their target proteins. Base on the above analysis, the molecular mechanism by which GQDs interfere with signaling network and key regulatory protein molecules in T. brucei was finally determined.

Preparation of GQDs and their uptake by T. brucei

GQDs were synthesized as illustrated in Fig. 1a. According to TEM images of the prepared GQDs (Fig. 1b), the core sizes of GQDs were 2.15 ± 0.59 nm (n = 100) with a relatively narrow size distribution. Despite their promising quantum yield and bleach-resistant autofluorescence, GQDs alone cannot be clearly imaged in the complex biological environment of T.brucei. Given these inadequacies, fluorescein fsothiocyanate (FITC), a typical fluorescent dye, was modified onto the surface of GQDs by EDC/NHS to enhance the fluorescence intensity of GQDs (Fig. 1a). The fluorescence excitation and emission spectra of GQDs or GQDs-FITC were shown in Fig. 1c. Ex/Em of 488/550 nm were chosen for GQDs-FITC to investigate the endocytosis of GQDs by T. brucei. The endocytosis system of T. brucei has been identified as a clathrin-mediated endocytosis (CME). CME is the most ancient known as mechanism of internalization in many lineages, and the involved protein participants have been known[40]. In T. brucei, CME is AP-2 independent and involves a cohort of lineage-specific clathrin-associated interactomes that drives CME. Clathrin is localized in the collecting tubules of flagellar pocket, where clathrin and its partners are recruited and assembled to perform endocytosis in T.brucei. The flagellar pocket is a particular type of plasma membrane invagination that surrounds the base of the flagella, and all trypanosoma endocytosis and exocytosis are polarized to this site as an only gateway to and from the host-parasite interface.

Although CME pathway focusing on small molecules as the nutrients has been investigated well in T. brucei, few reports are provided on nanoscale materials. After 6h of incubation, the obtained images visualized the phase contrast image of T.brucei cells, involving the blue nucleus stained with DAPI and the green GQDs-FITC (200 µg/mL) distributed in the cytoplasm of T. brucei cells (Fig. 1d). Pitstop2 is a classical inhibitor of clathrin-mediated endocytosis that are widely used in mammalian cells. This inhibitor has been shown to work well in T. brucei cells[24]. As shown in the last line of images, the pre-treatment of T. brucei cells with Pitstop2 caused a significant reduction of internalized GQD, indicating a key role of CME in GQDs uptake in T. brucei. The internalization of GQDs indicated the two-dimensional nanomaterials represented by GQDs can be used as promising nanomedicine materials for the diagnosis and treatment of parasitic protozoa.

Cytotoxicity induced by GQDs on T. brucei

Firstly, the cytotoxicity induced by GQDs in T. brucei cells was evaluated by MTS assay. As shown in Fig. 2a, the GQDs inhibited significantly the cell viability of T. brucei by a dose-dependent pattern. Half-maximal inhibitory concentrations (IC₅₀) for GQDs after 24h incubation was 36.1 µg/mL, which was negligible toxic doses for mammalian cells[32]. In most cases, GQDS can cause significant toxicity in mammalian cells at doses between 150 and 300 µg/mL, suggesting GQDs can be applying as a potential anti-trypanosomic reagent or drug carrier in vivo. Next, the apoptosis/necrosis ratio of T. brucei cells treated with GQDs was assessed using the Annexin V-FITC/PI apoptosis assay. Flow cytometry (Fig. S1) and statistical analysis (Fig. 2b) demonstrated that GQDs mainly induced late apoptosis. In T. brucei, the apoptotic phenotypes have been concerned and described when the parasite was incubated with drugs[41]. However, unlike mammalian cells, there are no evidences that caspase-like activity on their respective substrates have been detected in pathogenic trypanosomatids. Up to now, the key factors contributing to this process including caspase genes, Bcl-2 family homologs and TNF-related receptor members have not been identified in T. brucei[42]. Instead, they have evolved other stress response mechanisms, such as prostaglandin D2(PGD2) treatment[43] and spliced leader RNA silencing (SLS) pathway.

Numerous reports have shown that apoptosis induced by both mitochondrial dysfunction and ER stress in trypanosoma are dependent on ROS, but independent of caspase-like proteases[44]. Oxidative stress is known to be the main pathway of apoptosis induced by nanomaterials, including GQDs. Thus, ROS production induced by GQDs was determined and results (Fig. 2c) showed that GQD treatment significantly increased the ROS level in T. brucei in a dose-dependent manner. The ultrastructural changes of subcellular organelles in T. brucei cell were significantly altered after exposure to GQDs, including a vesicular-like expanded endoplasmic reticulum (ER) and morphologically altered mitochondria (M) (Fig. 2d). In addition, a marked expansion of the flagellar pockets was observed. Usually, there is a significant increase in diameter and morphological defects following the occurrence of abnormal endocytosis or clathrin-associated protein depletion in flagellar pocket. In conclusion, T. brucei cells exposed to GQDs exhibited abnormal ultra-structural changes that were typical hallmarks of nano-induced toxicity.

Genome-wide target protein screening towards GQDs in T. brucei.

The ultra-small size, lamellar structure and oxygen-containing groups at the edges of GQDs enable them to interact specifically with protein molecules in T. brucei. Thus, identifying the key target proteins towards GQDs was a prerequisite for studying the cytotoxic mechanism induced by GQDs in T. brucei cells. A model of GQDs with a side length of 2.5 nm was constructed using Lerf-Klinowski model[45]. PDB files are provided in Supplementary Dataset S1. The 3D structures of 5190 proteins of T. brucei were obtained by three ways, including 283 proteins from PDB web, 3117 proteins using I-TASSER, and 1790 proteins using AlphaFold. The structures and thermodynamic parameters of the GQD-protein complexes were finally generated by molecular docking with the PatchDock program. PDB files of GQD-protein complexes were ranked according to binding strength which were provided in Supplementary Dataset S2. The top 200 proteins with high affinity to GQDs (Supplementary Dataset S3) were categorized into different groups based upon subcellular localization (Fig. S2), molecular function (Fig. S3), or biological process (Fig. S4). Results showed that a large proportion of GQD-binding proteins were located in mitochondria, endoplasmic reticulum, and membranes' integral components (Fig. 3b). The molecular functions of top 200 proteins could be mainly divided into two parts: transporter activity and oxidoreductase/ antioxidant activity (Fig. 3c).

Of the top 200 proteins (Supplementary Dataset S3), about one fifth had transporter activity (Fig. 3c), which involved important roles in a wide range of cellular activities such as nutrient uptake, metabolite release, and signal transduction. Representative target proteins with high affinity included: (1) glucose transporters[46] (Tb927.10.8440, Tb927.10.8490, Tb927.10.8530 and Tb927.10.8510) response to the maintenance of energy metabolism in T. brucei; (2) vacuolar ATPases (Tb927.10.200, Tb927.10.3760, Tb927.8.2100 and Tb927.11.7480) mainly distribute in the lysosome and acidocalcisomes of T. brucei, contributing to macromolecular degradation, nutrients storage and ion homeostasis[47]; (3) aquaglyceroporins (AQP1, Tb927.6.1520; AQP3, Tb927.10.14160) regulate osmosis and uptake of low molecular weight solutes such as glycerol and urea[48]; (4) mitochondrial carrier family (MCF, Tb927.10.14840, Tb927.9.4310) localize in the mitochondrial inner membrane, mediating the transport of various metabolic intermediates or maintain the mitochondrial ADP/ATP ratio in T. brucei[49, 50]. Thus, by interacting with transporters, GQDs affected the uptake, storage and degradation of nutrients, osmoregulation, ion homeostasis, energy metabolism and mitochondrial phosphorylation in T. brucei. As a result, GQDs could indirectly induce ROS production, and thus trigger trypanosoma apoptosis.

Strikingly, another large percentage of potential targets towards GQDs with high affinity were a group of enzymes associated with redox regulation activity (Fig. 3c). Trypanosomes are early-diverging eukaryotes that are susceptible to ROS, but lack the major disulfide reductases, such as glutathione reductase and thioredoxin reductase which control thiol-redox homeostasis in most living organisms. Trypanosoma have evolved a unique thiol-redox system based on trypanothione[51], which is condensation compound of GSH and spermidine synthesized by trypanothione synthetase (TS). The regeneration of reduced/oxidized trypanothione (T(SH)₂/TS)₂) are regulated by trypanothione reductase (TryR) using NADPH as co-factor[52]. During the thiol redox process, oxidized glutathione (GSSG), dehydroascorbate (DHA), and the species-specific dithiol protein tryparedoxin (TXN) can accept the reducing equivalents as intermediary molecules, and then (in their reduced state) transfer electrons to the antioxidant enzymes including peroxidases, reductases, glyoxalases, and transferases[53]. Docking simulation results (Supplementary Dataset S3) showed that up to 20 proteins related to the T(SH)₂-TXN-NADPH system had high affinity with GQDS, suggesting that GQDs could directly disrupt the anti-redox system of T. brucei and thus initiate apoptosis.

Identification of TryR as vital target for GQDs in T. brucei.

Based on the T(SH)₂-TXN-NADPH system, the species-specific antioxidant network consisting of enzymatic and nonenzymatic redox-active molecules, is responsible for the detoxification of ROS/RNS (H₂O₂, O₂^•−, ^•NO, ONOO⁻) in T. brucei. These redox-active enzymes located in distinct subcellular compartments in T. brucei have high susceptibility to oxidative stress, which are considered attractive candidates for drug targeting[54]. Results (Fig. S5) showed that GQDs could induce the ROS increase in a short time with a dose- and time-dependent relationship. Combined with the previous docking results (Fig. 3c), in which most antioxidant enzymes involved in T(SH)₂-TXN-NADPH system had high affinity with GQDs, it was speculated that GQDs could interact directly with antioxidant enzyme molecules to trigger ROS-mediated apoptosis in T. brucei. To identify the crucial target proteins, MD simulation was performed using GROMACS software package to find out the details of interfacial interaction between GQDs and antioxidant enzymes (Fig. 4).

Twenty redox-active enzymes with high affinity towards GQDs were selected as potential targets to be investigated by MD simulations. Representative snapshots of nano-protein complex structures from MD simulations (Fig. 4a) provided the interfacial morphology between GQDs and enzymes. Bonding strength between GQDs and enzymes was measured using Gibbs free energy change (ΔG) obtained by averaging frames over 80–100 ns (Fig. 4b). Bonding strength consists of “ineffective bonding” and “effective bonding”. Ineffective bonding can be defined as a bonding that has little or no effect on enzyme activity. Effective bonding can be defined as a bonding that affects enzyme activity, and the bonding site is usually located at or near the enzyme activity center. Usually, an effective bonding event is the interaction with high bonding strength that occurs in the enzyme active center. In addition to bonding strength (Fig. 4b), the centroid distance between GQDs and the active site of the target protein was employed to identify effective bonding events (Fig. 4c). The active sites of enzymes were marked with the highlighted amino acids. Overall, TryR exhibited high interaction energy and the shortest centroid distance when binding to GQDs. TryR plays an extremely critical role in the trypanothione-dependent antioxidant network of T. brucei, by reducing TS₂ to T(SH)₂ with reducing equivalents provided by NADPH. In conclusion, TryR was identified as the most important target protein towards GQDs in T. brucei.

Validation of GQDs targeting TryR

To explore the inhibitory mechanism of the GQDs on TryR, the ultrastructures of the contact interface between GQDs and TryR were simulated by MD. According to the results (Fig. 5a), the strong interactions resulted from the hydrophobic interactions (VAL443, ILE339, PRO336, TRP21), hydrogen bonds (GLU18, ASN22, ASP116, SER109, THR335), and ionic bonds (ARG496, LYS108, LYS61) between GQDs and TryR amino acid residues. Among them, CYS52 and CYS57 residues are the components of TryR active site, meaning that GQDs can inhibit the activity of TryR by directly interacting with the amino acid residues of active sites in TryR. Considering that TryR functions as a dimer under physiological conditions, MD simulation method was further used to simulate the interaction between GQDs and TryR dimer. Results (Fig. 5b) showed that GQDs were inserted into the contact sites of TryR dimer to form a tight complex, where the activity center of TryR was completely blocked. The interaction energies between GQDs and TryR were calculated based on MD simulations (Fig. 5c), and the K_D of 1.6×10^− 8 M indicated a strong interaction between GQDs and TryR.

To validate the targeting effect of GQDs on TryR, biolayer interferometry (BLI) analysis was employed to quantify GQD-TryR interaction in vitro. His-tagged TryR recombinant (HIS-TryR) was expressed using a prokaryotic expression system and analyzed by SDS-PAGE (Fig. 5d). During BLI sensing, the HIS-TryR was first immobilized on the Ni-NTA biosensor, then immersed in the GQDs solution to capture the GQDs and subsequently dissociated in the dissociation solution (Fig. 5e). Dynamic characteristics of the GQD-TryR interaction based on BLI analysis were provided in Fig. 5f. The K_D value (2.4×10^− 8 M) obtained by BLI was comparable to that (1.6×10^− 8 M) in MD (Fig. 5c), which also validated the accuracy of the previous theoretical simulation. To further verify whether GQDs affect TryR function, an enzyme activity evaluation system was developed by employing TS₂ as catalytic substrate with the reducing equivalent provided by NADPH. The effect of GQDs on TryR enzymatic activity was evaluated by determining the absorbance decrease of NADPH at 340 nm. In the presence of TryR, NADPH (the maximum absorbance at 340 nm) will be converted to NADP⁺, which has no absorption at 340 nm. Considering the interference from GQDs absorbance themselves, a 3000-D ultrafiltration tube filter device was employed to remove the GQDs after interacting with TryR. Schematic diagram of the developed method and operation process was shown in Fig. 5g. The results (Fig. 5h) showed that GQDs exhibited a significant inhibitory effect on TryR activity. The inhibition rate reached 83.45% at 40 µg/mL of GQDs and 95.17% at 120 µg/mL of GQDs. In conclusion, GQDs could significantly inhibit TryR activity through strong interaction with TryR active site.

In T. brucei, cargo uptake is though a highly divergent clathrin-mediated endocytosis (CME) pathway located at polarized flagellar pocket[55], lacking AP2 complex but with an unusually high endocytosis rate that facilitates immune escape[56]. Significantly, this species-specific endocytosis system only works for cargos smaller than ~ 130 nm[25]. The ultra-small size of GQDs is comparable to that of nutrients and smaller than most proteins, allowing GQDs to be rapidly endocytized by T. brucei. After the internalization of GQDs, T. brucei exhibited classic toxicity-related pathomorphological changes, including abnormal mitochondrial morphology, swellen RE, and dilated flagellar pocket. The characteristic cytotoxic hallmarks induced by GQDs included ROS production and the resulted apoptosis. The doses of GQDs that could cause significant apoptotic symptoms in T. brucei are safe for mammalian cells, suggesting the potential of GQDs as a therapeutic agent for trypanosomiasis. Given the high affinity of GQDs to most of the key enzymes involved in the antioxidant network in T. brucei, the cytotoxic effect of GQDs on T. brucei was speculated to be due to the ROS increase caused by the interaction of GQDs with these enzymes as potential targets.

The lineage-specific T(SH)₂-TXN-NADPH system governs the cellular redox homoeostasis in T. brucei[57]. GQDs could disturb the T(SH)₂-TXN-NADPH network through high affinity interaction with a variety of enzymes that have redox activity/antioxidant activity in the system. Representative enzymes with high affinity towards GQDs and their functions were summarized in Fig. 6. TryR, can reduce TS₂ to T(SH)₂ with the coenzyme NADPH. TryS, performs T(SH)₂ biosynthesis in an ATP-dependent manner by condensating two GSH molecules to spermidine. TryR and TryS maintain homeostasis in the central T(SH)₂/TS₂ cycle and establish an intracellular reduction environment. The T(SH)₂/TS₂ cycle utilizes GSH, ASC or tryparedoxin (TXN) as the primary physiological redox rheostats to transfer electron to the whole antioxidant network. ERV1, Grx and TryP catalyze the redox state transition of DHA/ASC, GSSG/GSH and TXN I_ox/TXN I_red, respectively. APxCcP, performs the H₂O₂ metabolism in ER, mitochondria, and plasma membrane with ASC or Cyt C as the electron donors. GPx-II, catalyzes the reduction of lipid or Organic hydroperoxides (LOOH or ROOH) by GSH/GSSG at cytosol. MPX (mitochondrial) or CPX (cytoplasmic), decomposes H₂O₂ and ONOO⁻ with TXN as the reducing substrate. MsrA, as a methionine sulfur oxide reductase, reduces Met-SO to Met-SH via TXN. As TXN-dependent peroxide dismutase, Fe-SODA (mitochondria) or Fe-SODB (glycosome) is mainly used for detoxification of O₂•⁻ and inhibition of ONOO⁻ formation. Other proteins directly or indirectly related to Trypanosoma redox systems, including PTR1, PDH, and desaturase (FAD, Δ6FAD, ENR2, and SL4D), were also affected by GQDs.

GQDs are a class of nanometer-size graphene pieces with single-atom thickness that have unique optical and electronic properties with many promising applications in nanotechnology[58]. GQDs have a wide range of potential target proteins in organisms resulting from their unique structural morphology and physicochemical properties. (1) The ultra-small size (2–3 nm, comparable to biomolecules) and large surface to volume ratio enable GQDs to interact with target proteins. (2) The lamellar structure facilitates the insertion of GQDs into specific domains of target proteins. The best example in this study is the tight insertion of GQDs into the contact interface of TryR dimer. (3) The oxygen-containing groups at the edges and the hydrophobic environment in the lamellar middle endow GQDs the forces required for interaction with proteins, including hydrophobic interaction, hydrogen bonding, salt bridge, and van der Waals force. TryR was identified as the most critical target protein towards GQDs in T. brucei mainly based on the key role of TyrR in the T(SH)₂-TXN-NADPH system, as well as the strong affinity between TryR active site and GQDs. For other antioxidant enzymes that GQDs do not directly interact with their active sites, GQDs still affect their activity by impairing the protein structures or forming steric hindrance, when GQDs interact with them. Therefore, the actual situation should be that GQDs trigger the oxidative stress-mediated cytotoxicity by inhibiting most enzymes in the antioxidant network of T. brucei to varying degrees.

Moreover, the interactions between GQDs and proteins with transporter activity were also non-negligible factors in the GQD-induced cytotoxicity. Transporters play important roles in a wide range of cellular activities such as nutrient uptake, metabolite release, and signal transduction. Although transporters are not directly involved in the antioxidant regulation, the interference of GQDs on the transporter functions undoubtedly also increased ROS levels with an indirect pathway in T. brucei. However, the cytotoxic mechanism induced by GQDs may go far beyond these. The peroxidase activity of GQDs has been the focus of recent studies on nanobiological effects of GQDs[59], which should be considered when GQDs are applied to T. brucei. Results (Fig S5) showed that the prepared GQDs had peroxidase activity. In conclusion, the cytotoxicity caused by GQDs in T. brucei was a complex mechanism, involving the dominated inhibition to antioxidant enzymes, the secondary interference with the transporters, and their own minor peroxidase activity, due to their unique structural morphology and physicochemical properties.

In summary, this study found that ultra-small GQDs can be rapidly internalized by T. brucei and cause significant cytotoxicity, including morphological defects, decreased viability, increased ROS and apoptosis. The unique physicochemical properties of GQDs enabled them to have high affinity for multiple target proteins with antioxidant enzyme activity or transporter activity after internalization, leading to their complex toxicological mechanism against T. brucei. Further, a computed structure database of T. brucei genome-wide proteins was constructed, based on which TryR was screen out as a key target protein. Prominently, GQDs could significantly inhibit TryR activity through strong interaction with the active site of TryR. Considering the critical role of TryR in the antioxidant network of T. bruci, the toxicological mechanism of GQD-induced oxidative stress with TryR as the target was finally determined after experimental verification. These findings enhance our understanding of the nanotoxicology in trypanosomal parasites and provide theoretical reference for the application of nanomaterials in trypanosomiasis.

Materials

Pitch carbon fibers (CF) were obtained from Aladdin (Shanghai, China). Sodium acetate (CH₃COONa), 3,3',5,5'-Tetramethylbenzidine (TMB), 4% paraformaldehyde, Fluorescein-5-isothiocyanate (FITC), kanamycin were purchased from Macklin (Shanghai, China). NADPH tetrasodium salt and glycine were provided by Solarbio (Beijing, China). Sodium bicarbonate (NaHCO₃), acetic acid (CH₃COOH), sodium hydroxide (NaOH), nitric acid (HNO₃), hydrogen peroxide (H₂O₂) and sulfuric acid (H₂SO₄) were purchased from Merck (CA, USA). All chemicals were commercially available and used without further purification. Amicon® Ultra-0.5 Centrifugal Filter Devices were obtained from Millipore (Massachusetts, USA). CellTiter 96® AQueous One Solution Cell Proliferation Assay was purchased from Promega (Wisconsin, USA). The Annexin V-FITC Apoptosis Detection Kit and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from the Beyotime Institute of Biotechnology (Haimen, China). Fetal bovine serum (FBS) was obtained from Biological Industries. All other chemicals, unless specified, were obtained from Sigma or sources mentioned previously. The T. brucei strain STIB 920 was presented as a gift by Professor Dehua Lai from Sun Yat-sen University.

GQDs preparation and characterization

GQDs was prepared according to the literature with slight modifications[60]. In brief, 0.50 g Pitch carbon fibers were added into a mixture of concentrated 30 mL H₂SO₄ and 10 mL HNO₃. The black solution was sonicated for 2 h and stirred for one day at 150°C. The mixture was cooled and diluted with 200 mL deionized (DI) water. The pH was adjusted to 8 with NaHCO₃. The final product solution was further dialyzed in a dialysis bag (3500 Da) for 3 days and stored at 4°C. The fluorescence spectra were performed using a Hitachi (Japan) F-7100 fluorescence spectrophotometer. The TEM (transmission electronic microscopy) characterization was obtained on Tecnai G2 T20 U-Twin electron microscope.

GQDs-FITC preparation and Endocytosis of GQDs-FITC by T. brucei

For FITC modification, the EDC (20 mg)/NHS (20 mg) in 1 mL deionized water were added into 5 mL GQDs (5 mg/mL) incubating for 8 h at room temperature with gentle stirring. The resulting NHS-activated particles were reacted with 1 mL EDA for 4h. Then, 200 µL FITC (in DMSO, 50 mg/mL) was covalently linked to the resulting particles. The final product solution was further dialyzed in a dialysis bag (3500 Da) for one day and stored at 4°C[61]. For imaging, 1x10⁶ T. brucei cells were plated in 24-well plate. Then, 200 µg/mL GQDs-FITC was added to the cells. After 6 h of incubation, the medium concluding GQDs-FITC was removed from the cells and washed twice with cold PBS. The treated cells were fixed using 4% paraformaldehyde and stained with DAPI (Vector Labs). Endocytosis of GQDs-FITC by T. brucei were observed using a confocal laser scanning microscope (olympus).

Pitstop2™, a clathrin inhibitor[62, 63], was used to confirm the clathrin-mediated endocytosis pathway. First, T. brucei cells that grow logarithmically were pre-incubated with 10 µM Pitstop 2™ for 30 min at 37°C. Next, GQD-FITC at the desired concentrations were added and incubated another 6 hours in the presence of 0.5 µM Pitstop 2™. Finally, T. brucei cells were harvested and performed above imaging operation.

T. brucei viability and apoptosis with treatment of GQDs

T. brucei cells were grown in HIM-9 medium containing 10% FBS and 1% Penicillin-Streptomycin solution at 37°C in 5% CO₂ atmosphere. The viability of T. brucei cells was measured with MTS Cell Proliferation and Cytotoxicity Assay Kit. T. brucei cells grown on 96-well plates (1 × 10⁶ cells/well), and incubated with different concentrations of GQDs, involving 300 µg/mL, 150 µg/mL, 80 µg/mL, 40 µg/mL, 20 µg/mL and 10 µg/mL for 24 h. Then, 20 µL MTS was added to per well and incubated for another 4 h. Assay plates were read with absorbance of solubilized formazan salts at 490 nm on a VICTOR Nivo Multimode Plate reader (PerkinElmer, USA).

The cell apoptosis assay was performed by propidium iodide and annexin V-FITC staining using BD FACSAria III flow cytometer (BD Biosciences, USA). Briefly, T. brucei cells seeded in 24-well plates were treated with GQDs (50, 100, 200, 350 and 500 µg/mL) at 37°C in 5% CO₂ atmosphere for 24 h. Then, T. brucei cells were washed twice with cold PBS and stained with a cell apoptosis kit (125 µL binding buffer, 1.25 µL annexin V-FITC, and 1.25 µL propidium iodide/well), followed by incubation for 30 min at room temperature in the dark and immediately analyzed by flow cytometry.

Assessment of ROS levels after treatment with GQDs

The intracellular ROS levels were measured with Reactive Oxygen Species Assay Kit (DCFH-DA). T. brucei cells were seeded on 96-well plates (1 × 10⁶ cells/well) and then treated with different concentrations of GQDs (50, 150 and 300 µg/mL) for 24 h. The cell suspension was added to a V-bottom 96-well plates and centrifuged at 1500 rpm. After that, the cultural media was carefully removed and washed with cold 1×PBS, followed by added 10 mmol/L DCFH-DA to each well for 30 min incubation at 37 ^oC in the dark. Last, T. brucei cells washed with PBS twice, and immediately analyzed by flow cytometry.

Structural model of GQDs

According to the TEM measurements (Fig. 1b), a square trilayer graphene sheet with a side length of 2.5 nm (about 2 nm in thickness) was constructed based on the Lerf-Klinowski graphene oxide (GO) model[45]. Oxidized groups were modified on the exposed upper, lower surfaces and sides of GQD model, including epoxy, hydroxyl, and carboxyl groups. The force field parameters describing GO were adapted from the work of Zhou et al[64].

Construction of T. brucei protein structure library

The information of T. brucei, including “Gene ID”, “sequence”, “structure”, “localization”, and “GO Terms”, were downloaded from tritrypdb.org. The total number of T. brucei proteins recorded in this webserver is 11,886 (reference genome Tb927). The 3D structures of proteins were obtained from three ways, including (i) about 283 T. brucei protein structures were available in the Protein Data Bank (PDB); (ii) The protein structure with less than 1500 amino acids was predicted using I-TASSER and the predicted number was 3117; (iii) the rest structures were predicted by AlphaFold2 programs and the predicted number was 1790. Thus, the total number of protein structures constructed in the T. brucei protein structure library was 5190. Proteins with no predicted structure were mainly due to excessive protein molecular weight or unpredictable multiple transmembrane structures involving complex hydrophobic domains.

Genome-wide target protein screening for GQDs in T. brucei

Docking simulation of T. brucei protein structure library with GQDs was performed using the PatchDock program. The proteins were treated as receptors and GQDs as ligand. Twenty complex structures were provided by each docking process. The optimized complex structures were then filtered by a scoring function defined as the binding energy per contact area. The top-ranked conformer was selected for each protein complex. Finally, all complexes (5154 complexes) were ranked according to the score given by the scoring function.

Identification of redox-associated proteins targeting GQDs

Top 200 complexes based on the scoring function were selected for the “GO Enrichment Analysis Advanced” using OmicShare 6.4.5. The P-value was obtained by converting the scores of each complex. The top 30 annotations were used for functional analysis of target proteins. Ultimately, the most suspected targets with "oxidoreductase activity" were identified, consisting of 20 target proteins, which were listed in Table S1.

Molecular dynamics (MD) simulations

MD simulations were performed to explore the binding mode of GQDS with the 20 redox-related target proteins using the GROMACS software package (version 2020). Proteins and GQDs with oxidation groups were described using CHARMM27 force field[65] solvated with the TIP3P water model [66]. The temperature was modulated at 300 K by using a v-rescale thermostat[67] with a damping coefficient of 1 ps^− 1. The pressure was controlled by Parrinello − Rahman algorithm[68] in the semi-isotropic (X + Y, Z) directions of the system, and the target pressure was 1 atm. The cut-off distance of short-range electrostatic interaction was set to 12.0 Å, and the particle grid Ewald (PME) method was employed to describe the long-range electrostatic interaction[69]. Standard periodic boundary conditions were applied throughout all the directions. Each system was firstly minimized to eliminate unfavorable geometric clashes and then equilibrated for 2 ns in the NVT ensemble, which was used in production runs with a 2-ps time step, and the coordinates were saved every 100 ps. In total, 100-ns length simulation was performed for each system, and all simulation snapshots were rendered using the program VMD. The interaction energy of GQD-protein complex was calculated from the sum of electrostatic energy and VDW energy. The centroid distance between the GQDs and the protein active site was calculated from their last 20ns trajectories.

Preparation and characterization of recombinant TryR

The crystal structure of TryR was from Protein Data Bank (PDB) data (PDB: 2WOI). The full-length gene sequence of TryR was obtained from PlasmoDB (https://plasmodb.org/plasmo/app), and synthetized by Sangon Biotech (Shanghai, China). The TryR gene was cloned into the pET-28a(+) expression vectors through restriction enzymes and expressed in E. coli BL21 cells. The transformed E. coli cells were grown in LB medium with 100 mg/L kanamycin at 37℃ until OD₆₀₀ = 0.6, and induced for protein expression by adding 0.5 mM IPTG at 25℃ for overnight. After sonication, lysate was centrifuged at 12000 rpm for 15 min and keep the supernatant. TryR was purified by His GraviTrapTM 147 affinity columns (GE Healthcare), and identified by SDS-PAGE.

Inhibition of TryR by GQDs.

Experiments to calculate the IC₅₀ of GQDs for inhibition of TryR activity were performed by varying GQDs concentrations. Briefly, 10 µL TryR (40 nM) and 10 µL GQDs (from 2.5 to 120 µg/mL) were pre-incubated at 25°C for 2 h. The mixture of TryR and GQDs was then added to the substrate solution consisting of 40 mM HEPES (pH 7.5), 150 mM NaCl, 100 µM NADPH and 50 µM TS₂, and incubated for another 2 h at room temperature. The chromogenic solution was separated by centrifugation at 14,000 × g for 20 min in a 0.5 mL ultrafiltration tube to remove interference from GQD absorption. Finally, place the chromogenic solution (100 µL) on a 96-well plate and measure the absorbance at 340 nm.

Biolayer interferometry (BLI) Assays

The affinity of GQDs to TryR was measured using BLI (ForteBio, Menlo Park, CA). Briefly, Ni-NTA biosensors used in BLI were equilibrated in 1× Kinetics Buffer; the prepared recombinant TryR was diluted using BLI kinetic buffer at the desired concentrations and immobilized onto the Ni-NTA biosensor by 10 min incubation. The TryR-adsorbed Ni-NTA biosensors were first combined with GQDs with a series of concentrations dispersed in BLI kinetic buffer at 37°C for 10 minutes; then, immersed in the Kinetic Buffer for another 10 min to dissociate the analytes. The equilibrium dissociation constant (Kd) was calculated using Octet data analysis software by globally fitting the data to a 1:1 binding model.

Peroxidase-like activity of GQDs

The peroxidase-like activity assays[70] of GQDs were performed as follows: 20 µL TMB (in DMSO, 10 mg/mL) + 15 µL H₂O₂ (10 M) + 10 µL various concentrations of GQDs (from 0.4 µg/mL to 24 µg/mL) + 1 mL acetate buffer (pH 4.5). The absorbance of the ox-TMB (652 nm) represented the peroxidase-like activity was recorded at certain reaction times via the SpectraMax® Plus 384 microplate spectrophotometers (Molecular Devices, USA).

Acknowledgements

The authors gratefully acknowledge Professor Dehua Lai of Sun Yat-sen University in China for the gift of trypanosoma bruci strain.

Author contributions

DZ, RJ and LML contributed equally to this work. DZ, RJ and LML performed the experiments and analyzed the experimental data. LLY, QWJ, RL and NW performed computer simulation and analyzed the simulation data. NW and XFH assisted with most of the research. XFH and XYW supervised this study. DZ, RJ and XYW wrote this manuscript. XYW conceived the study and finalized the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Subject Construction Project of Dalian University (5901050), the Department of Science & Technology of Liaoning Province (2019-MS-270), the Research Foundation of Educational Department of Liaoning Province (LSNZD201901), and the natural Science Foundation of Hebei Province (H2022206507).

Availability of data and materials

All data and materials of this study can be obtained from the corresponding author under reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agree to be published.

Competing interests

The authors declare that they have no competing interests.

Author details

¹ Key Laboratory of Glycolipid Metabolism of Liaoning Province, School of Life and Health, Dalian University, Dalian, Liaoning, 116622, China; ² College of Sciences, Shenyang Agricultural University, Shenyang, Liaoning, 110866, China; ³ State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, Hubei, 430070, China; ⁴ Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100176, China; ⁵ Pharmaceutical College, Hebei Medical University, Shijiazhuang, Hebei, 050017, China.

Barrett MP, Burchmore RJS, Stich A, Lazzari JO, Frasch AC, Cazzulo JJ, Krishna S. The trypanosomiases. Lancet. 2003; 362:1469-1480.
Kennedy PGE. Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 2013; 12:186-194.
Dewar CE, Casas-Sanchez A, Dieme C, Crouzols A, Haines LR, Acosta-Serrano A, Rotureau B, Schnaufer A. Oxidative Phosphorylation Is Required for Powering Motility and Development of the Sleeping Sickness Parasite Trypanosoma brucei in the Tsetse Fly Vector. Mbio. 2022; 13:e02357-21.
Jamonneau V, Truc P, Grebaut P, Herder S, Ravel S, Solano P, De Meeus T. Trypanosoma brucei gambiense Group 2: The Unusual Suspect. Trends Parasitol. 2019; 35:983-995.
Steverding D, Tyler KM. Decolonising Parasitology: The Case of Trypanosoma brucei rhodesiense. Trends Parasitol. 2021; 37:461-464.
Kasozi KI, MacLeod ET, Ntulume I, Welburn SC. An Update on African Trypanocide Pharmaceutics and Resistance. Front Vet Sci. 2022; 9:828111.
Moretti NS, Mortara RA, Schenkman S. Trypanosoma cruzi. Trends Parasitol. 2020; 36:404-405.
Moncayo A, Yanine MIO. An update on Chagas disease (human American trypanosomiasis). Ann Trop Med Parasit. 2006; 100:663-677.
Buscher P, Cecchi G, Jamonneau V, Priotto G. Human African trypanosomiasis. Lancet. 2017; 390:2397-2409.
Bottieau E, Clerinx J. Human African Trypanosomiasis Progress and Stagnation. Infect Dis Clin N Am. 2019; 33:61-77.
Baker N, de Koning HP, Maeser P, Horn D. Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 2013; 29:110-118.
Kennedy PGE. An alternative form of melarsoprol in sleeping sickness. Trends Parasitol. 2012; 28:307-310.
Sjoerdsma A, Schechter PJ. Eflornithine for African sleeping sickness. Lancet. 1999; 354:254-254.
Deeks ED. Fexinidazole: First Global Approval. Drugs. 2019; 79:215-220.
Maxmen A. Pill treats sleeping sickness. Nature. 2017; 550:441-441.
Altamura F, Rajesh R, Catta-Preta CMC, Moretti NS, Cestari I. The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets. Drug Develop Res. 2022; 83:225-252.
Sabia Junior EF, Santos Menezes LF, Silva de Araujo IF, Schwartz EF. Natural Occurrence in Venomous Arthropods of Antimicrobial Peptides Active against Protozoan Parasites. Toxins. 2019; 11:e563.
Baiocco P, Ilari A, Ceci P, Orsini S, Gramiccia M, Di Muccio T, Colotti G. Inhibitory Effect of Silver Nanoparticles on Trypanothione Reductase Activity and Leishmania infantum Proliferation. Acs Med Chem Lett. 2011; 2:230-233.
Lima D, Hacke ACM, Ulmer B, Kuss S. Electrochemical sensing of trypanosome- and flavivirus-related neglected tropical diseases. Curr Opin Electroche. 2021; 30:100838.
Gu W, Bobrini VA, Chen S-PR, Wang Z, Schoning JP, Gu Y, Chen W, Chen M, Jia Z, Monteiro MJ. Biodistribution of PNIPAM-Coated Nanostructures Synthesized by the TDMT Method. Biomacromolecules. 2019; 20:625-634.
Kannan S, Harel Y, Levy E, Dolitzky A, Sagiv AE, Aryal S, Suleman L, Lellouche J-P, Michaeli S. Nano-Leish-IL: A novel iron oxide-based nanocomposite drug platform for effective treatment of cutaneous leishmaniasis. J Control Release. 2021; 335:203-215.
Oliveira CB, Rigo LA, Dalla Rosa L, Gressler LT, Prestes Zimmermann CE, Ourique AF, Da Silva AS, Miletti LC, Ruver Beck RC, Monteiro SG. Liposomes produced by reverse phase evaporation: in vitro and in vivo efficacy of diminazene aceturate against Trypanosoma evansi. Parasitology. 2014; 141:761-769.
Murugan K, Nataraj D, Jaganathan A, Dinesh D, Jayashanthini S, Samidoss CM, Paulpandi M, Panneerselvam C, Subramaniam J, Aziz AT, et al. Nanofabrication of Graphene Quantum Dots with High Toxicity Against Malaria Mosquitoes, Plasmodium falciparum and MCF-7 Cancer Cells: Impact on Predation of Non-target Tadpoles, Odonate Nymphs and Mosquito Fishes. J Clust Sci. 2017; 28:393-411.
Wang X, Zhang D, Jiang N, Wang X, Zhang N, Zhang K, Sang X, Feng Y, Chen R, Yang N, Chen Q. Induction of apoptosis in Trypanosoma brucei following endocytosis of ultra-small noble metal nanoclusters. Nano Today. 2021; 38:101122.
Grunfelder CG, Engstler M, Weise F, Schwarz H, Stierhof YD, Morgan GW, Field MC, Overath P. Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers. Mol Biol Cell. 2003; 14:2029-2040.
Xie Y, Liang H, Jiang N, Liu D, Zhang N, Li Q, Zhang K, Sang X, Feng Y, Chen R, et al. Graphene quantum dots induce cascadic apoptosis via interaction with proteins associated with anti-oxidation after endocytosis by Trypanosoma brucei. Front Immunol. 2022; 13:1022050.
Yan Y, Gong J, Chen J, Zeng Z, Huang W, Pu K, Liu J, Chen P. Recent Advances on Graphene Quantum Dots: From Chemistry and Physics to Applications. Adv Mater. 2019; 31:e1808283.
Jang M-H, Yang H, Chang YH, Park H-C, Park H, Cho HH, Kim BJ, Kim Y-H, Cho Y-H. Selective engineering of oxygen-containing functional groups using the alkyl ligand oleylamine for revealing the luminescence mechanism of graphene oxide quantum dots. Nanoscale. 2017; 9:18635-18643.
Ma L, Sun S, Wang Y, Jiang K, Zhu J, Li J, Lin H. A graphene quantum dot-based fluorescent nanoprobe for hypochlorite detection in water and in living cells. Microchim Acta. 2017; 184:3833-3840.
Biswas MC, Islam MT, Nandy PK, Hossain MM. Graphene Quantum Dots (GQDs) for Bioimaging and Drug Delivery Applications: A Review. Acs Mater Lett. 2021; 3:889-911.
Garg B, Bisht T, Ling Y-C. Graphene-Based Nanomaterials as Efficient Peroxidase Mimetic Catalysts for Biosensing Applications: An Overview. Molecules. 2015; 20:14155-14190.
Wang X-y, Lei R, Huang H-d, Wang N, Yuan L, Xiao R-y, Bai L-d, Li X, Li L-m, Yang X-d. The permeability and transport mechanism of graphene quantum dots (GQDs) across the biological barrier. Nanoscale. 2015; 7:2034-2041.
Tu Z, Guday G, Adeli M, Haag R. Multivalent Interactions between 2D Nanomaterials and Biointerfaces. Adv Mater. 2018; 30:e1706709.
Rijo-Ferreira F, Carvalho T, Afonso C, Sanches-Vaz M, Costa RM, Figueiredo LM, Takahashi JS. Sleeping sickness is a circadian disorder. Nat Commun. 2018; 9:62.
Bily T, Sheikh S, Mallet A, Bastin P, Perez-Morga D, Lukes J, Hashimi H. Ultrastructural Changes of the Mitochondrion During the Life Cycle of Trypanosoma brucei. J Eukaryot Microbiol. 2021; 68:e12846.
Scotti L, Mendonca FJB, Jr., da Silva MS, Scotti MT. Enzymatic Targets in Trypanosoma brucei. Curr Protein Pept Sc. 2016; 17:243-259.
Manta B, Comini M, Medeiros A, Hugo M, Trujillo M, Radi R. Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids. Bba-Gen Subjects. 2013; 1830:3199-3216.
Lu J, Vodnala SK, Gustavsson A-L, Gustafsson TN, Sjoberg B, Johansson HA, Kumar S, Tjernberg A, Engman L, Rottenberg ME, Holmgren A. Ebsulfur Is a Benzisothiazolone Cytocidal Inhibitor Targeting the Trypanothione Reductase of Trypanosoma brucei. J Biol Chem. 2013; 288:27456-27468.
Krauth-Siegel RL, Comini MA. Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Bba-Gen Subjects. 2008; 1780:1236-1248.
Adung'a VO, Gadelha C, Field MC. Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis. Traffic. 2013; 14:440-457.
Dofuor AK, Djameh GI, Ayertey F, Bolah P, Amoa-Bosompem M, Kyeremeh K, Okine LK, Gwira TM, Ohashi M. Antitrypanosomal Effects of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler Extracts on African Trypanosomes. Evid-Based Compl Alt. 2019; 2019:1730452.
Welburn SC, Barcinski MA, Williams GT. Programmed cell death in trypanosomatids. Parasitol today. 1997; 13:22-26.
Sadok Menna-Barreto RF. Cell death pathways in pathogenic trypanosomatids: lessons of (over)kill. Cell Death Dis. 2019; 10:93.
Helms MJ, Ambit A, Appleton P, Tetley L, Coombs GH, Mottram JC. Bloodstream form Trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes. J Cell Sci. 2006; 119:1105-1117.
Lerf A, He HY, Forster M, Klinowski J. Structure of graphite oxide revisited. J Phys Chem B. 1998; 102:4477-4482.
Schmidt RS, Macedo JP, Steinmann ME, Salgado AG, Butikofer P, Sigel E, Rentsch D, Maser P. Transporters of Trypanosoma bruceiphylogeny, physiology, pharmacology. Febs Journal. 2018; 285:1012-1023.
Baker N, Hamilton G, Wilkes JM, Hutchinson S, Barrett MP, Horn D. Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes. P Natl Acad Sci Usa. 2015; 112:9112-9117.
Quintana JF, Field MC. Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins. Parasitology. 2021; 148:1137-1142.
Colasante C, Zheng F, Kemp C, Voncken F. A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei. Mol Biochem Parasit. 2018; 221:36-51.
Colasante C, Diaz PP, Clayton C, Voncken F. Mitochondrial carrier family inventory of Trypanosoma brucei brucei: Identification, expression and subcellular localisation. Mol Biochem Parasit. 2009; 167:104-117.
Ebersoll S, Bogacz M, Guenter LM, Dick TP, Krauth-Siegel RL. A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes. Elife. 2020; 9:e53227.
De Gasparo R, Halgas O, Harangozo D, Kaiser M, Pai EF, Krauth-Siegel RL, Diederich F. Targeting a Large Active Site: Structure-Based Design of Nanomolar Inhibitors of Trypanosoma brucei Trypanothione Reductase. Chem-Eur J. 2019; 25:11416-11421.
Reckenfelderbaumer N, Krauth-Siegel RL. Catalytic properties, thiol pK value, and redox potential of Trypanosoma brucei tryparedoxin. J Biol Chem. 2002; 277:17548-17555.
Saccoliti F, Di Santo R, Costi R. Recent Advancement in the Search of Innovative Antiprotozoal Agents Targeting Trypanothione Metabolism. Chemmedchem. 2020; 15:2420-2435.
Field MC, Carrington M. The trypanosome flagellar pocket. Nat Rev Microbiol. 2009; 7:775-786.
Manna PT, Obado SO, Boehm C, Gadelha C, Sali A, Chait BT, Rout MP, Field MC. Lineage-specific proteins essential for endocytosis in trypanosomes. J Cell Sci. 2017; 130:1379-1392.
Krieger S, Schwarz W, Ariyanayagam MR, Fairlamb AH, Krauth-Siegel RL, Clayton C. Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Mol Microbiol. 2000; 35:542-552.
Lu H, Li W, Dong H, Wei M. Graphene Quantum Dots for Optical Bioimaging. Small. 2019; 15:e1902136.
Deng S, Fu A, Junaid M, Wang Y, Yin Q, Fu C, Liu L, Su D-S, Bian W-P, Pei D-S. Nitrogen-doped graphene quantum dots (N-GQDs) perturb redox-sensitive system via the selective inhibition of antioxidant enzyme activities in zebrafish. Biomaterials. 2019; 206:61-72.
Peng J, Gao W, Gupta BK, Liu Z, Romero-Aburto R, Ge L, Song L, Alemany LB, Zhan X, Gao G, et al. Graphene Quantum Dots Derived from Carbon Fibers. Nano Lett. 2012; 12:844-849.
Farokhzad OC, Jon SY, Khademhosseini A, Tran TNT, LaVan DA, Langer R. Nanopartide-aptamer bioconjugates: A new approach for targeting prostate cancer cells. Cancer Res. 2004; 64:7668-7672.
Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan GB. HIV Enters Cells via Endocytosis and Dynamin-Dependent Fusion with Endosomes. Cell. 2009; 137:433-444.
Eichel K, Jullie D, von Zastrow M. beta-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol. 2016; 18:303-310.
Tu Y, Lv M, Xiu P, Huynh T, Zhang M, Castelli M, Liu Z, Huang Q, Fan C, Fang H, Zhou R. Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nat Nanotechnol. 2013; 8:594-601.
MacKerell JAD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B. 1998; 102:3586-3616.
Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J Chem Phys. 1983; 79:926-935.
Bussi G, Donadio D, Parrinello M. Canonical Sampling through Velocity Rescaling. J Chem Phys. 2007; 126:014101.
Parrinello M, Rahman A. Polymorphic Transitions in Single-Crystals - a New Molecular-Dynamics Method. J Appl Phys. 1981; 52:7182-7190.
Darden T, York D, Pedersen L. Particle Mesh Ewald - an N.Log(N) method for Ewald sums in large systems. J Chem Phys. 1993; 98:10089-10092.
Chen Q, Liu Y, Liu J, Liu J. Liposome-Boosted Peroxidase-Mimicking Nanozymes Breaking the pH Limit. Chem-Eur J. 2020; 26:16659-16665.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Graphene quantum dots harvest anti-trypanosomatid efficacy by disrupting antioxidant networks centered on trypanothione reductase

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Results

Validation of GQDs targeting TryR

Discussion

Conclusions

Materials And Methods

Materials

GQDs preparation and characterization

Assessment of ROS levels after treatment with GQDs

Structural model of GQDs

Identification of redox-associated proteins targeting GQDs

Molecular dynamics (MD) simulations

Preparation and characterization of recombinant TryR

Biolayer interferometry (BLI) Assays

Peroxidase-like activity of GQDs

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1