These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by βIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An  They are found in eukaryotic cells where they form cytoskeletal architectures supporting a wide range of cellular functions, such as chromosome segregation, cell shape, intracellular transport and cellular movement ,2. The oAnti-microtubule drugs such as colchicine, nocodazole (NOC), benomyl and paclitaxel are commonly used to study the diverse functions of microtubules and to synchronize cells at mitosis. Drugs that both stabilize microtubules (e.g. taxanes such as paclitaxel) and destabilize microtubules  are also used as anti-cancer agents ,7. Most Schizosaccharomyces pombe, a single-cell eukaryote, that allows a rapid and economic workflow for screening and identifying the molecular targets of drugs. We executed a high-throughput phenotype-based screen for small molecules that inhibit fission yeast growth and then tested their effects on microtubules. Candidate molecules were then further tested for their ability both to disrupt microtubules in human cells and to specifically inhibit the growth of human breast cancer cell lines. These novel small molecules inhibit mitotic spindle assembly effectively in both fission yeast and human cells, and provide potential drug candidates for cancer therapies.In this paper, we aimed to identify new anti-microtubule drugs, with the objectives of generating new reagents for the physiological investigation of microtubular functions in cells and also for the identification of new lead drugs that could be used in cancer therapy. To search for new low-molecular-weight compounds with anti-microtubular activity, we used the fission yeast, . 2. 2.1a) and subsequently conducted a phenotypic screen of these to identify drugs that affect microtubule organization (figure 1b). After the MDR-sup cells were treated with 5 µM of each compound for 3 h, microtubules were monitored using GFP tagged α-tubulin Atb2 and live-cell fluorescence microscopy. In untreated interphase cells, microtubules form long cytoplasmic arrays that run parallel to the long cellular axis, and in mitotic cells, microtubules form a bipolar spindle in the nucleus and the cytoplasmic microtubules disappear (figure 1c(i), arrow). Tubulin is present in the nucleus in interphase cells but does not form detectable nuclear polymer structures. This screen identified 34 compounds that induced cytological phenotypes indicative of defects in microtubule organization or function. The observed defects could be grouped into three classes: (i) microtubules are undetectable, and α-tubulin accumulates in the nucleus (as seen with compound L1 in figure 1c(ii)); (ii) mitotic cells exhibit only short bipolar spindles (as seen with compound P2 in figure 1c(iii)); and (iii) tubulin is found as a single dot at the SPB without the formation of short or long microtubules (as seen with compound B8 in figure 1c(iv) and figure 1d).We have previously screened a collection of 10 371 small molecules to identify compounds that inhibit the growth of a multiple-drug-sensitive fission yeast strain (MDR-sup) ,13. We sfigure 1a). The control DMSO-treated cells progressed through mitosis with a mean mitotic duration of 77 ± 6 min. Among the 34 compounds we tested, nine caused a significant increase in mitotic duration in human cells (a), ranging from 122 ± 42 min (F15) to 720 ± 53 min (C16). Most of these compounds resulted in cells arresting in early mitosis, followed by abnormal asymmetric division and/or cell death (b). Cells treated with the four compounds K1, L1, J1 and B8 immediately induced cell death independent of the phase of the cell cycle, but at a lower concentration (0.5 µM), these compounds arrested cells initially in mitosis, followed by cell death . As described in the Discussion, we have not carried out a detailed analysis for B8 in this study. To determine whether these cell deaths were caused by apoptosis, caspase activity was assayed for 24 h using time-lapse microscopy in the presence of the 12 compounds. Caspase 3 and Caspase 7 activities were observed 12–24 h after the addition of the compounds . There were also other indications of apoptosis including the morphological chances reminiscing of membrane blebbing, chromatin condensation and cell explosion . Caspase-mediated apoptosis induction is similar to the effects of mitotic arrest caused by anti-microtubule drugs in HeLa cells [c). We conclude that these 12 compounds cause mitotic arrest followed by cell death through apoptotic activation.To determine whether these 34 compounds also induce mitotic abnormalities in human cells, a second screen of these compounds was conducted using HeLa cells. We used time-lapse microscopy to monitor mitotic progression in HeLa cells expressing mCherry-tagged histone H2B to mark chromatin, and EGFP-tagged α-tubulin to monitor microtubules over a 24 h time period in the presence of 2 µM of each of the 34 compounds. The duration of mitosis was determined by live-cell imaging, defined as the period from prophase to cytokinesis based on chromatin and microtubule morphologies a. The coan cells a, ranginll death b. Cells ll death a,b. As dLa cells ,15. At l. 2.2in vitro tubulin polymerization assays. Microtubule polymerization was induced with bovine brain tubulin (greater than 99% pure) in the presence of the compounds at 5 µM at 37°C (dotted lines in figure 3b) with NOC as a positive control. Tubulin polymerization was monitored every 1 min by measuring fluorescence absorbance at 340 nm for 1 h. As shown in the representative traces shown in figure 3a), and six compounds inhibited polymerization partially (figure 3b). The five most potent compounds inhibited in vitro tubulin polymerization in a concentration-dependent manner, with a similar dose dependency to NOC (figure 3c). While NOC had an IC50 of 0.56 µM, the five potent compounds had comparable IC50s of K1; 0.59 µM, L1; 0.47 µM, M2; 0.22 µM, A15: 0.50 µM, J3; 0.58 µM (figure 3c). These results established that these five compounds possess strong in vitro anti-tubulin polymerization activity, and that the other six compounds have partial inhibitory effects on tubulin polymerization. The 12th compound, F15 had no observable effects on polymerization at 5 µM and therefore appears to interfere with progression through mitosis by a mechanism not involving microtubule polymerization dynamics. F15 is also the least potent of the tested compounds for both mitotic arrest and cell death.To determine whether the 12 compounds affected mitotic spindle organization by directly interfering with tubulin polymerization, we performed . 2.3α/β-tubulin dimers, the effects of the most active inhibitors  on the stability of purified tubulins were assessed by differential scanning fluorimetry (DSF) analysis [−1) by gradually increasing the temperature from 25°C to 95°C in the presence of the excess Sypro orange fluorescent dye [figure 4a blue line, B blue bar). Upon addition of an increasing concentration of the inhibitors, the unfolding transition of tubulin was shifted to lower temperatures in a dose-dependent manner. This is because the inhibitors bind and destabilize the structure of the α/β-tubulin dimers at lower temperatures . By titrating the compound concentrations, we found that compounds L1, J3, A15 and M2 exhibited potent destabilizing effects with Tm ranging from 51°C to 55°C when 50 µM of the compounds was used, and from 53.5°C to 56.5°C when 10 µM was used. These effects were greater than either NOC or taxane (figure 4b).To determine if the compounds directly bound analysis . Becausecent dye –18. Fluoa. The compounds L1, K1, E2 and J1 all have structural similarities. L1, K1 and E2 have a common benzodioxole, which is also a part of polygamain (figure 5b), a known microtubule-depolymerizing lignan [50 of 0.47 µM, comparable to NOC with an IC50 of 0.56 µM (c). All microtubule destabilizing compounds screened in this study showed a similar phenotype in fission yeast: no interphase microtubules or mitotic spindles were detectable, and tubulin was found to be present in the nucleus . The same phenotype was also observed when fission yeast MDR-sup was treated with other known inhibitors of microtubule polymerization such as NOC and benomyl . The drug NOC that inhibits microtubule assembly [nda2-KM52 and the β-tubulin mutant nda3-KM311 were both hypersensitive to L1 at the permissive temperature of 30°C. No such drug hypersensitivity was observed in the cell cycle mutant cdc25-22, condensin mutant cut14-208 or actin mutant act1-48 . Altogether, our screen has successfully identified novel direct inhibitors of microtubule polymerization in both fission yeast and HeLa cells.The chemical structures of the most potent compounds L1, K1, J3, A15 and M2 are shown in figure 5g lignan , whereas 0.56 µM c. All mi nucleus c,b. The assembly  was hype. 2.4Since polygamain binds to tubulin within the colchicine-binding site, we conducted a fluorescent-based colchicine displacement assay to evaluate the ability of L1 to inhibit colchicine binding to tubulin  figure . L1 comp. 2.550 5.6 nM) compared with the parental HeLa cell line (IC50 1.9 nM)  . These r. 2.6in vitro assay had specificity against human breast cancer cell lines. The sensitivity of the inhibitors L1, J3, A15 and M2 were evaluated in three breast cancer cell lines,  and a non-cancer cell line from the human mammary gland, MCF-10A , M2 (1.4–1.9 µM) and J3 (0.6–0.9 µM), were comparable between the breast cell lines and the non-cancer cell line. However, the IC50 of L1 was more than threefold higher in the non-cancer cell line MCF-10A cells (9.6 µM) compared with the three breast cancer cell lines (2.4 to 3.1 µM).We next investigated whether the four most potent microtubular inhibitors in the  MCF-10A . Drug sea,b). These results are consistent with a report that has shown a synergistic effect between tubulin and Plk1 inhibitors [figure 9c,d). As L1 displayed specific toxicity in a breast cancer cell line (figure 9e) and a non-cancer cell line (figure 9f), with various doses of L1 in the absence (black) or presence (red) of 0.1 nM BI2536. In the cancer cell line, the expected IC50 by the Bliss independence model is 2.1 µM, while the actual IC50 is 0.6 µΜ. In the non-cancer cell line, the expected IC50 is 7.7 µM and the actual IC50 is 6.7 µM. Thus, the synergy between L1 and Plk1 inhibitor is more effective in the breast cancer cell line CAL51 than in the non-cancer cell line MCF-10A.It has been reported that a microtubule-depolymerizing drug TH588 exhibits a synergistic effect with BI2536, an inhibitor against the mitotic kinase Plk1, on the growth of tissue culture cells. This effect is preferentially increased in cancer cells ,27. All figure 9ell line , we furtFinally, a clonogenic assay was used to further evaluate the synergy effects between the L1 and Plk1 inhibitors to inhibit colony formation of breast cancer CAL51 cells after a 12 h drug exposure. The percentages of colony formation of cells treated with 0.1 nM BI2536 alone and 0.5 µM L1 alone were 89% and 67%, respectively, while cells treated with both 0.1 nM BI2536 and 0.5 µM L1 together were 2%  indicati. 3Most microtubule inhibitors are natural products or their derivatives. We have explored here a diverse small-molecule chemical library to identify new microtubule inhibitors in a chemically unbiased manner. Unlike most previous small molecule screens, which identified tubulin inhibitors through their cytotoxic effects on human tissue culture cells, we first employed fission yeast for rapid and cost-effective primary screening followed by screening in human tissue culture cells. This approach was effective in the identification of small molecules which disrupt microtubules in both fission yeast and human cells.α/β-tubulin-RB3-tubulin tyrosine ligase (TTL) complex soaked with TUB092 showed that the benzodioxole group positions at the hydrophobic pocket of β-tubulin, a position which is similar to the site that the trimethoxyphenyl moiety (A-ring) of colchicine also occupies [α/β-tubulin-RB3-TTL complex with podophyllotoxin or its analogue, which have both trimethoxyphenyl and benzodioxole moieties, the position of the benzodioxole matches to the methoxytropolone C ring of colchicine [Among the 11 compounds that inhibit microtubule assembly, three of them  contained benzodioxole, which has been found in several known microtubule drugs that target colchicin-binding sites ,29, incloccupies . Moleculoccupies . Since Llchicine , not theet al. [in vitro IC50 or cellular IC50 of anti-tubulin effects . As B8 also exhibits mitotic defects in HeLa cells, B8 is a candidate for modulating microtubule dynamics in mitosis without directly binding to tubulins.Among the compounds that exhibited microtubule assembly defects in fission yeast, L8 and B8 did not show any detectable effect on This study also identified a panel of compounds that caused growth inhibition of drug-sensitive MDR-sup fission yeast cells, but did not exhibit obvious effects on mitosis in HeLa cells. Although further analyses are required to test if they inhibit pathogenic yeasts and fungi, drugs that selectively inhibit fungi but not human cell lines would be potential candidates for fungicides and antifungal drugs, possibly with potential for agriculture and therapeutic applications ,56.. 4. 4.1http://www.rockefeller.edu/htsrc/libraries). They were resuspended in DMSO (Sigma-Aldrich), and stock solutions were stored at −20°C.The screening libraries comprised 10 371 compounds from the Rockefeller University High-Throughput Screening Resource Center  [- and its derivative strains were used in the electronic supplementary material, figure S3: nda2-km52, nda3-km311 [cdc25-22 [cdc14-208 [act1-48 [The ::ura4+)  used fora3-km311 , cdc25-2cdc25-22 ,60, cdc1dc14-208  and act1[act1-48  spotted . 4.32 maintained in log phase growth and were routinely monitored for mycoplasma by PCR . HeLa cells stably expressing H2B-mcherry and mEGFP- α-tubulin (a gift of D. W. Gerlich) [βIII cell line (gifts from S. L. Mooberry) [−1, GIBCO).All cell lines were incubated at 37°C in a humidified incubator containing 5% COGerlich) , HeLa WTooberry) , MCF-10Aooberry)  were groFor live-cell imaging experiments, 200 000 cells were seeded onto glass-bottom culture dishes (MatTek P35GC-1.5-14-C). Cells were grown overnight before the addition of the compound, and the culture media was replaced with fresh media lacking phenol red to limit phototoxicity before imaging.. 4.4Live-cell imaging was performed in a LCV110 U VivaView FL incubator microscope (Olympus) equipped with an X-Cite-exacte illumination source (Excelitas Technologies) and Orca-R2 CCD camera (Hamamatsu Photonics). For screening, images were acquired with a 20 × objective every 15 min for 24–48 h in the differential interference contrast (DIC), m-cherry and GFP channels. For assessment of apoptosis, the cells were monitored every 15 min for 24 h. Individual cells were manually analysed using the CellCognition browser .. 4.5Assessment of apoptosis IncuCyte Caspase-3/7 Green apoptosis Reagent from Essen Bioscience (cat. no. 4440) was used to quantitatively evaluate apoptosis in HeLa cells expressing H2B-mcherry and EGFP-α-tubulin cells. Cells were seeded for 24 h onto glass-bottom culture dishes and apoptosis was assessed after each compound was added. The cells were monitored by live microscopy every 15 min for 24 h using DIC microscopy, m-cherry and GFP channels.. 4.6−1 of highly purified porcine brain tubulin heterodimer .The Tubulin Polymerization HTS Assay Kit  was used according to the manufacturer's instructions. All components were added into a 96-well microtiter plate , then the tubulin reaction mixture was quickly added to the wells, and tubulin polymerization was initiated and monitored every 1 min at 37°C for 1 h by recording fluorescence of excitation wavelength at 340 nm and emission at 450 nm. The tubulin reaction mixture was composed of 80 mM PIPES (pH 6.9), and 1 mM MgCl2, 1 mM EGTA, 1 mM GTP and 2 mg ml. 4.72 and 0.5 mM EGTA) was incubated and monitored in a RT-PCR instruments using the following program: an initial 2 min hold at 25°C, ramping up in increments of 1°C to a final temperature of 95°C. The Tm values were obtained from the midpoint of the transition.Thermal shift assays were carried out using an RT-PCR machine  and SYPRO orange  as a fluorescence probe. The 25 µl of reaction mixture containing 5 µM porcine brain tubulin  and 1 : 1000 diluted SYPRO orange dye in general tubulin buffer  in tubulin reaction mixture as above and displacement assays were performed with 2 µM colchicine or 2 µM colchicine with 0–50 µM L1 using a fluorescence spectrophotometer at 37°C for 2 h. Reaction mixtures were excited at 380 nm and the emission was measured at 438 nm ,64. Valu. 4.9Cells were plated in 384-well plates at a density of 800 cells per well in 22.5 µl of media. After 24 h, 2.5 µl drug was diluted in media and was added to each well. Following 5 days of incubation, viability was assessed using CellTiter-Glo (Promega) according to the manufacture's protocol. Luminescence in each well was measured using a Synergy Neo2 plate reader (BioTek).The Bliss independence algorithm was used to quantify the drug synergy in which drug combinations are compared to sensitivity to individual drugs at the same dose .. 4.10CAL51 cells were plated at a density of 200 cells per 60 mm tissue culture dish. After 24 h, they were treated with vehicle (DMSO), 0.1 nM BI2538, 0.5 µM L1 or the combination of 0.1 nM BI2538 and 0.5 µM L1, respectively, for 12 h. After an exposure, the cells were washed with warm sterile PBS, and incubated for additional 9 days in fresh media. After the incubation, cells were fixed and stained with a 20% MeOH: 0.01% crystal violet solution for 30 min. Colonies were counted using a stereomicroscope in three independent experiments.


Tendinopathy is a common musculoskeletal condition causing pain and dysfunction. Conventional treatment and surgical procedures for tendinopathy are insufficient; accordingly, recent research has focused on tendon-healing regenerative approaches. Tendon injuries usually occur in the hypoxic critical zone, characterized by increased oxidative stress and mitochondrial dysfunction; thus, exogenous intact mitochondria may be therapeutic. We aimed to assess whether mitochondrial transplantation could induce anti-inflammatory activity and modulate the metabolic state of a tendinopathy model. Exogenous mitochondria were successfully delivered into damaged tenocytes by centrifugation. Levels of Tenomodulin and Collagen I in damaged tenocytes were restored with reductions in nuclear factor-κB and matrix metalloproteinase 1. The dysregulation of oxidative stress and mitochondrial membrane potential was attenuated by mitochondrial transplantation. Activated mitochondrial fission markers, such as fission 1 and dynamin-related protein 1, were dose-dependently downregulated. Apoptosis signaling pathway proteins were restored to the pre-damage levels. Similar changes were observed in a collagenase injection-induced rat model of tendinopathy. Exogenous mitochondria incorporated into the Achilles tendon reduced inflammatory and fission marker levels. Notably, collagen production was restored. Our results demonstrate the therapeutic effects of direct mitochondrial transplantation in tendinopathy. These effects may be explained by alterations in anti-inflammatory and apoptotic processes via changes in mitochondrial dynamics.  Tendinopathies are common musculoskeletal problems associated with aging and sports . RotatorMitochondrial dysfunction has been observed in a broad spectrum of diseases, and studies of therapies using or targeting mitochondria have increased exponentially ,20. Clin2. Human tenocytes  and L6 myoblasts  were purchased. Human tenocytes and rat cells (L6 myoblasts) were cultured in Dulbecco’s Modified Eagle Medium (HyClone) supplemented with 10% FBS (HyClone) and 1% P/S (HyClone) at 37 °C in a humidified atmosphere containing 5% CO2. Tenocytes were cultured on pre-coated flasks/plates with collagen type 1  for 24 h and rinsed with 1× Dulbecco’s phosphate-buffered saline  prior to seeding cells. UC-MSCs (passages 5–7), tenocytes (passages 3 or 4), and L6 myoblasts (passages 5 or 6) were used. Those cells were subcultured when they reached approximately 80–90% confluence.This in vitro study was approved by the CHA University Institutional Review Board (IRB number: NON2018-003). Umbilical cord-mesenchymal stem cells (UC-MSCs) were obtained from the Wharton’s jelly as in our previous study . BrieflyRecombinant human tumor necrosis factor-α (TNF-α) was purchased from PeproTech . TNF-α was dissolved with pure ethanol to prepare a 20 µg/mL stock stored at 4 °C. The test concentrations used were 10, 50, 100, and 500 ng/mL in a growth medium. Tenocytes were finally treated with TNF-α (10 ng/mL) for 24 h to establish an in vitro tendinopathy model .7-cell suspension, cells were mechanically disrupted using a 1 mL syringe (100 strokes) . To remove unbroken cells and nuclei, centrifugation was performed at 1100× g for 3 min at 4 °C. The supernatant was collected and centrifuged at 12,000× g for 15 min. To wash the crude separated mitochondria, the pellet was suspended in 1.5 mL of SHE buffer and then centrifuged at 20,000× g for 10 min. After removing the supernatant, the final mitochondrial pellet was suspended in 1.5 mL of DPBS (Welgene) and centrifuged at 20,000× g for 5 min. After removing the supernatant, the mitochondrial pellet was suspended in 200 μL of DPBS (Welgene); 10 μL of the mitochondrial solution was obtained, and the concentration was determined using a bicinchoninic acid assay  , 2 mM EGTA, 0.1% defatted bovine serum albumin] to a 2 × 10IL, USA) .5 tenocytes. Isolated mitochondria were mixed slowly with tenocytes stored in PBS. The mixture was centrifuged at 1500× g for 5 min and washed with PBS two times. Then, cells were seeded into a plate according to the experimental design. This process allowed the transfer of mitochondria to tenocytes. All subsequent analyses were performed 24 h after mitochondrial transfer. Mitochondria from UC-MSCs were transferred into tenocytes pretreated with TNF-α by centrifugation . In brie5 cells/well) into a 6-well plate, and UC-MSCs were seeded (1 × 105 cells/flask) into a T-175 culture flask. Tenocytes were treated with TNF-α at 10 ng/mL for 24 h. After 24 h, for visualization of mitochondria incorporation, damaged tenocytes and donor cells were stained with MitoTracker Green or CMXRos red, respectively, according to the manufacturer’s protocols. The cells were washed with Hank’s balanced salt solution (HyClone) and cultured with 300 nM MitoTracker Green and CMXRos red at 37 °C and 5% CO2 for 30 min. Then, mitochondria were isolated from donor cells, and extracted mitochondria were transferred into tenocytes by centrifugation. After adding 4′6-diamidino-2-phenylindole  to tenocytes, slides were prepared  and MitoTracker CMXRos red (CMXRos red) (Thermo Fisher) were used to confirm the mitochondrial transfer to tenocytes by fluorescence microscopy. First, tenocytes were seeded (1 × 10prepared . A 100× n = 3), positive control (n = 6), low mitochondrial concentration (n = 6), and high mitochondrial concentration (n = 6) groups. The Achilles tendons of both hind limbs were injected with 0.6 mg/40 μL collagenase . After 2 weeks, isolated mitochondria from L6 cells were injected into the tendon by local injection. Bodyweight was the weight on an electronic scale, and limb thickness was measured using a caliper every week. Animals in each group were sacrificed using carbon dioxide (CO2) after 2 weeks to evaluate the efficacy of mitochondrial transplantation.This animal study proceeded after approval from the Institutional Animal Care and Use Committee at CHA University (IACUC190102). Twenty-one male Sprague–Dawley rats  were randomly distributed into the following four groups: negative control .Tendon tissues were examined to visualize delivered mitochondria after local mitochondrial injection. Stained mitochondria from L6 cells with CMXRos red were injected locally into the Achilles tendon. Tissue samples were used for cryosectioning. Right after harvesting, the tendon tissue was fixed in 4% paraformaldehyde  with 4 °C overnight. After rinsing the tissue with 1× PBS, the fixed material was shaken with 10% and 20% sucrose for 3 h at 4 °C and overnight with 30% sucrose at 4 °C, then we waited until the tissue subsides in a 50 mL conical tube. After making a square mold with foil, the OCT compound  was added appropriately, and then the tissue was embedded and frozen rapidly on nitrogen without bubbles. After that, the compound should be stored at −70 °C. The tissues were cut to 5 μm thick by the cryostat  at −20 °C. The temperature of the cutting chamber was occasionally adjusted ±2 °C. After attaching sections to a slide, 300 nM MitoTracker Green dye was added to the sections for 30 min at 37 °C and 5% CO4 cells per well and then cultured in a growth medium for 24 h at 37 °C and 5% CO2. When cells reached about 80–90% confluence, they were treated with TNF-α, and mitochondria were delivered. After 24 h, cells were washed once with PBS and incubated for 30 min with 10 μM DCF-DA. Fluorescence intensity was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a Synergy Mix Multi-Mode Reader .Intracellular ROS was measured using the 2′,7′-dichlorofluorescein diacetate (DCF-DA) probe . Cells were seeded in a 48-well plate at a concentration of 1 × 104 cells/well and then cultured in a growth medium for 24 h at 37 °C and 5% CO2. When cells reached about 80–90% confluence, they were treated with TNF-α and mitochondria were delivered. After 24 h, cells were washed with once with PBS and incubated for 30 min with 1 μM MitoSOX Red at 37 °C and 5% CO2. Fluorescence intensity was measured at an excitation wavelength of 510 nm and an emission wavelength of 528 nm using a Synergy Mix Multi-Mode Reader (BioTek Inc.). Moreover, images of mROS level at 200× magnification were obtained using a fluorescence microscope (Leica Microsystems).mROS was measured using MitoSOX Red, a mitochondrial superoxide indicator . Cells were seeded in a 48-well plate at a concentration of 1 × 10ATP was measured using the CellTiter-Glo Luminescence Kit . Growth medium (100 μL) and CellTiter-Glo Luminescence Test Solution (100 μL) were added to cells treated with TNF-α and mitochondria, and the treated cells were left first for 10 min and then for 2 min with shaking at room temperature (22 °C–23 °C). Emission signals were measured using a Luminescence microplate reader .A JC-1 assay was performed. When JC-1 dye accumulates in healthy mitochondria, emission shifts from green (528 nm) to red (590 nm). The JC-1 dye was stored at 7.7 mM after dilution in dimethyl sulfoxide. The growth medium was supplemented with 2 µM JC-1 immediately before the experiment. Cells were incubated for 20 min at 37 °C and then washed two times with DPBS (Welgene). Then, cells in DPBS (100 µL) (Welgene) were analyzed using a microplate reader .Tnmd), Collagen 1 (CoL1), and matrix metalloproteinase 1 (MMP1) were analyzed by qPCR  with protease inhibitor. After centrifugation at 13,572× g for 15 min at 4 °C, the supernatant was collected. The protein contents were determined by the BCA assay using an ELISA microplate reader at 562 nm. The proteins contents were normalized so that all samples had the same concentration and volume for further analysis. Each sample was heated with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer  at 100 °C for 3 min. Proteins were separated by 12% SDS–PAGE and transferred to a PVDF membrane at 0.35 mA for 3 h. PVDF was blocked in pure methanol before use. The membrane was blocked in 1× TBS containing 3% bovine serum albumin  and 0.1% Tween-20 for 1 h at room temperature. Membranes were incubated overnight with the following primary antibodies diluted at a 1:1000 ratio in 3% BSA: anti-BID , anti-Bax , anti-Bcl2 , anti-Cytochrome C , anti-Tenomodulin (TNMD) , anti-Tenascin C (TNC) , anti-Collagen 1 (COL1) , anti-matrix metalloproteinase 1 (MMP1) , p-NF-kB p65 (Ser536), NF-kB p65 , anti-Fis1 , anti-Drp1 , anti-Mitofusin 2 (MFN2) , total OXPHOS rodent , and anti-β-actin  antibodies. After the membranes were washed three times for 10 min with TBS-T, they were reacted with a secondary antibody conjugated with horseradish peroxidase (HRP), goat anti-mouse antibody  or mouse anti-rabbit antibody , at a dilution of 1:5000 in 3% BSA in TBS-T for 1 h at room temperature. After washing membranes three times with TBS-T for 10 min, signals were visualized by enhanced chemiluminescence , and detected using a LAS-4000 . Uncropped raw images of all blots were included in After treatment with TNF-α and the transplantation of mitochondria, cells were cultured at 37 °C under 5% COg for 5 min. The supernatant was harvested and analyzed. IL-6 and IL-1β were analyzed according to the manufacturer’s instructions provided with the ELISA kit . Next, IL-6 and IL-1β standard proteins, analysis buffer, and biotin-binding detection antibody were added to the cells, and the cells were cultured for 24 h. After incubation, each plate was washed with PBS and incubated with streptavidin-HRP for 1 h. After the color reaction with a TMB solution, absorbance was measured at 450 nm using an Epoch spectrometer/microplate reader (BioTek Inc.).To analyze IL-6 and IL-1β activities, the culture medium was collected 24 h after stimulation of TNF-α and transfer of mitochondria. Cells were centrifuged at 212× 5 cells per well in a 6-well plate and treated with TNF-α (10 ng/mL) for 24 h. Then, the isolated mitochondria were transferred, and RNA was extracted after 24 h using the Easy-spin Total RNA Extraction Kit (iNtRON Bio) and a NanoDrop spectrophotometer (Thermo Fisher) (260:280 > 1.9 and 260:230 > 1.9). The mitochondrial concentration (yield) was determined to be 1 μg, and the purity was measured. Microarray analysis was performed using Affymetrix HuGene 2.0ST Chips . A heatmap clustering analysis was performed using Morpheus.Tenocytes were seeded at 1 × 10g for 5 min. After collecting the supernatant, an oxidation mixture was added and reacted at room temperature for 20 min. The developer and DMAB concentrate were added at 50 μL per vial and reacted at 65 °C for 45 min. Measurements were obtained at 560 nm using a Synergy Mix Multi-Mode Reader (BioTek).A hydroxyproline assay was performed to confirm the total tendon collagen content. The sacrificed tissue was rapidly frozen in liquid nitrogen and stored at −80 °C. Tissue disruption and experimental methods were performed following the manufacturer’s instructions for a hydroxyproline colorimetric assay . After breaking the tissue, proteins were treated with 10 N NaOH and HCl and then centrifuged at 10,000× The inhibitor Mdivi-1 was processed to confirm the impact of mitochondria fission. Mdivi-1  was treated on tenocytes in DMEM (HyClone) supplemented with 10% FBS (HyClone) for 24 h with TNF-α treatment. After 24 h of mitochondrial transplantation, western blot analysis was performed.p < 0.05. All statistical analyses were performed using SigmaPlot 11.0 . The significance of differences between groups was evaluated by one-way analysis of variance, with post hoc tests for multiple comparisons. Quantitative results are shown as mean ± SEM values, and statistical significance was defined at The data that support the findings of this study are available from the corresponding authors upon reasonable request. Isolated mitochondria from UC-MSCs were then evaluated using mitochondrial markers  . IsolateTNMD, the most specific tenocyte marker reported to date [p < 0.05, versus without TNF-α exposure) and recovered at 24 h after mitochondrial transfer in a dose-dependent manner    and increased in the mitochondrial transfer groups  , and declined after mitochondrial transplantation. All three doses  of mitochondria reduced the expression of MMP1 to the level observed before TNF-α exposure  (We investigated tenocyte-related markers (TNMD and COL1) and MMP1 after the mitochondrial transfer ,28. The  to date , decreasransfer) B. COL1 eransfer) C. The legulation , was eleransfer) D.p < 0.05, versus without TNF-α exposure) and recovered to the pre-damage state upon mitochondrial transfer     E. The exransfer) F. The MMransfer) G. We alsp < 0.05, versus TNF-α exposure without mitochondrial transfer)  and mitochondrial (MitoSOX) ROS levels induced by TNF-α declined upon mitochondrial transplantation in a concentration-dependent manner as follows: for cellular ROS, 19.8%, 25.0%, and 32.6% and for mitochondrial ROS, 26.0%, 33.6%, and 32.5%  A. The hioduction .p < 0.05, versus without TNF-α exposure). However, the membrane potential increased to that in the pre-damage state in the mitochondrial transfer group    B. Additip < 0.05). The amount of intracellular ATP was mostly restored by exogenous mitochondria in a dose-dependent manner  (We further found that ATP content was 33% lower in the TNF-α-treated group for 24 h than in the pre-damage state (ransfer) C.The mitochondrial membrane complex consists of the electron transport chain (ETC) (complexes I to V) and is indispensable for oxidative phosphorylation (OXPHOS) and ATP production ,32. Protp < 0.05, versus without TNF-α exposure)   level decreased by about 39% (xposure) A–C. Compransfer) A. After ransfer) B. As a fransfer) C. The leransfer) .p < 0.05, versus TNF-α exposure without mitochondrial transfer)    (ransfer) D. Mitochransfer) E,F. Moreransfer) G. BID anransfer) .p < 0.05). Mitochondrial transfer suppressed IL-1β    expression  the increased phosphorylation of NF-κB (approximately 1.5-fold upon TNF-α exposure), a key pathway in the inflammatory cascade (p < 0.05) in each group and created heatmaps to illustrate the expression patterns for TNF-α exposure and TNF-α exposure with mitochondrial transfer (Levels of the pro-inflammatory cytokines IL-1β and IL-6 in tenocytes increased in response to TNF-α (by about 2-fold) compared with those in the control group not subjected to TNF-α (ransfer) H and IL-pression I. Mitoch cascade J. We nextransfer . In thisp < 0.05). Swelling subsided at 1 and 2 weeks after local injection of 10 and 50 μg mitochondria into Achilles tendons  , positive control injured by collagenase , low mitochondrial concentration 10 μg , and higher concentration 50 μg  (A rat model of tendinopathy was prepared by collagenase injection into the Achilles tendon . The tenber = 6) .We evaluated whether injected exogenous mitochondria were incorporated into Achilles tendon tissues. Mitochondria stained with CMXRos were administered to the Achilles tendon. After 2 weeks, the tendon tissues were harvested, and CMXRos signals were detected by confocal microscopy B.p < 0.05, versus collagenase-injected tissue without mitochondrial transfer)    and MMP1 were measured. TNC is involved in the tendon tissue repair process, which modulates MMP expression and whose level increases under pathological conditions. TNC level increased approximately 2.6 folds in the injured group  and decreased by 82.3 ± 2.5% and 76.8 ± 4.3% in the groups treated with 10 and 50 μg of mitochondria, respectively (ransfer) C. The leransfer) D. Collagransfer) E.p < 0.05). After the delivery of 50 µg mitochondria by local injection, Fis1 level was reduced  (p < 0.05)   A. Drp1 l < 0.05) B. MFN2 lransfer) C.p < 0.05, versus collagenase-injected tissue without mitochondrial transfer)  returned to the control level upon 10 and 50 μg mitochondrial transplantation   were assessed at 2 weeks after mitochondrial transplantation. Collagenase I-induced increases in BID (by approximately 1.9-fold) and Bax (approximately 3.5-fold) levels declined upon mitochondrial transplantation  (ransfer) D,E. The ransfer) F.p < 0.05, versus collagenase-injected tissue without mitochondrial transfer)     G. IL-1β ransfer) H. Besideransfer) I. Furtheransfer) J.This study showed that intact exogenous mitochondrial transplantation could have protective effects against TNF-α-induced damage in tenocytes and a collagenase-induced animal model of tendinopathy. Inflammation is the primary pathogenic mechanism of tendinopathy ,34, and Tendinopathy is characterized as “failed healing” which refers to the incomplete recovery of mechanical strength and inferior tissue quality mainly due to tendon tissue avascularity ,36. SuchMitochondria were successfully incorporated into tenocytes using centrifugation and revitalized damaged tenocytes. TNMD, COL1, MMP1, and TNC levels were altered upon mitochondrial transplantation, along with reductions in pro-inflammatory marker levels. TNMD is a specific tenocyte-related marker . The knoExogenous mitochondria might contribute to or replace the activity of endogenous mitochondria. Mitochondrial function can be measured by changes in ROS and ATP levels and mitochondria-membrane potential . In partWhen mitochondria fail to function properly, the equilibrium between mitochondrial fusion and fission shifts , causingMitochondria are a major source of ROS under physiological conditions; ROS mediate redox signaling . ExcessiThe delicate balance between fission and fusion determines mitochondrial network morphology and affects mitochondrial functions . MitochoThese findings were validated by microarray data related to TNF signaling, apoptosis, calcium signaling, inflammation-mediator regulation, and other processes. Significantly affected signaling pathways in this analysis correspond to those reportedly affected in previous cell and animal studies.5 tenocytes) and outcome variables. This might be attributed to the ceiling effect of higher concentrations of mitochondria. Various molecules released from mitochondria  can induce pro-inflammatory properties and have been implicated in pathologic conditions [This study had several limitations. First, we could not obtain consistency between the dose-dependent effects of delivered mitochondrial doses (1, 5, and 25 µg per 1 × 10nditions . Higher nditions . Centrifnditions . Third, nditions . Fourth,nditions . Newer pThis study provides the first evidence for the therapeutic effects of mitochondrial transfer in tendinopathy. Exogenous mitochondria were successfully delivered into damaged tenocytes and tendons. They modulated inflammation and apoptosis and recovered the collagen component of the extracellular matrix. Exogenous mitochondrial transfer may become a novel approach for the treatment of tendinopathy.


To the Editor: Pemphigus is a group of chronic autoimmune blistering diseases affecting skin and mucosa with a huge impact on the quality of life (QOL).P < .001 and P = .001, respectively). For both, the decrease was most pronounced between M0 and M6  and between M0 and M12 . The number of patients in the DLQI category “no/minimal impairment” increased from 13 to 24 (+84.6%) between M0 and M6 and from 11 to 24 (+118.2%) between M0 and M12. The number of patients in the category “severe impairment” decreased from 10 to 0 (−100%) between M0 and M6 and 11 to 1 (−90.9%) between M0 and M12. The number of patients in the TABQOL category “low impact” increased from 10 to 16 (+60.0%) between M0 and M6 and from 10 to 17 (+70.0%) between M0 and M12. The number of patients in the category “high impact” decreased from 8 to 4 (+50.0%) between M0 and M6 and 8 to 2 (−75.0%) between M0 and M12. Hospital Anxiety and Depression Scale anxiety decreased among M0, M0.5, M6, and M12 , specifically between M0 and M12 . Hospital Anxiety and Depression Scale depression showed no significant decrease during the rituximab treatment . Visual analog scale pain decreased among M0, M0.5, M6, and M12 , in particular between M6 and M12  and between M0 and M12 . In this study, we showed the positive effect of rituximab on patients’ well-being, QOL, and treatment-specific QOL during rituximab treatment. The improvement in DLQI and TABQOL was observed after 6 and 12 months, proving the short-term and long-term effects of the first 2 infusions. The observed decline in the anxiety scores during treatment may contribute to the positive effect of rituximab. Contrarily, no improvement in depression was observed, possibly because it is a result of a combination of long-term non–disease-related factors and disease burden. A limitation of this study is the absence of the assessment of patient-reported outcome measurements in patients with pemphigus treated with other systemic therapies.,Dr Horváth reports fees from Janssen-Cilag , AbbVie , Novartis Pharma , UCB Pharma (Advisory Boards and Consultations), Leo Pharma (Consultations), Solenne B.V. (Investigator Initiative Studies), Celgene (Consultations and Investigator Initiative Studies), Akari therapeutics (Consultations and Investigator Initiative Studies), Philips (Consultation), Roche (Consultation), Regeneron (Consultation), and Sanofi (Consultation). Drs Rashid, Poelhekken, Meijer, and Bolling have no conflicts of interest to declare.


Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP+ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP+ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PDdef). We first solved the crystal structure for G6PDK403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP+ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PDdef), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PDK403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PDdef and provide a foundation for future drug discovery efforts.Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme that generates NADPH. The NADPH produced by G6PD provides the cell with reductive power and is essential for the regeneration of oxidized glutathione—reduced NADPH levels cause redox imbalance . Due to def) is a genetic trait present in ∼400 million people worldwide , one for an NADPassembly , 11, 12.nterface , 14.+ and G6P, both of which alter the oligomeric state of G6PD  of G6PD, which improves the activity and stability of several Class II and III G6PD clinical variants he dimer , 19. The+ (A), and studies by Wang et al. , forms an important contact with a phosphate group of the structural NADP+ A, and stg et al.  revealedWT was selected: 500 nM G6PD, 80 mM sodium phosphate (NaP) (pH 8.0). The enzymes used in subsequent assays were expressed and purified as indicated in the Experimental procedures section. In the presence of 80 mM NaP (pH 8.0), G6PDWT consisted of approximately 25% tetramer, 40% dimer, and 22% monomer, with each species clearly defined . Under the same condition, no G6PDK403Q tetramer was observed . Instead, 74% of G6PDK403Q existed as a dimer, with no change in the percentage of monomer. Interestingly, an intermediate species was detected between the dimer and monomer peaks of G6PDWT; however, the intermediate was not detected for G6PDK403Q. This intermediate is likely the result of a dynamic dimer–monomer equilibrium, with intermediate SEC species reported for G6PD  was used, and a buffer condition where monomeric, dimeric, and tetrameric species could be observed for G6PDfor G6PD .WT with 10 μM NADP+, NADP+ did not alter the relative percentage of tetramer; however, it slightly increased the dimer (∼8%), resulting in a subsequent reduction of the monomer (C and S1C). Additionally, the monomer migrated through the column faster, as indicated by a slight shift in the monomer elution volume, from 10.5 ml to 10.25 ml. In contrast, NADP+ had no effect on G6PDK403Q oligomerization (D and S1D); there were no changes in the distribution of species and no changes in location of peaks, which is consistent with the inability of G6PDK403Q to bind structural NADP+.When incubating G6PD monomer C and S1Crization D and S1DWT and G6PDK403Q in 50 mM Tris (pH 7.4), 150 mM NaCl, and 10 mM G6P, conditions that should dissociate the dimer . In contrast, G6PDK403Q had minor changes in the relative distribution of oligomers (E and S1F). Since differences in G6P binding may explain why G6PDWT oligomerization was severely impacted but not G6PDK403Q, we removed G6P. In the absence of G6P, we still observed major differences in the G6PDWT oligomeric distribution; a 16% reduction in the tetramer, a 15% reduction in dimer, and a 34% increase in monomer (G). Therefore, changes in the buffer composition alone affect the quaternary structure for G6PDWT, but not for G6PDK403Q. The fact that G6PDWT had an increase in the relative percentage of monomers but G6PDK403Q did not suggests that the G6PDK403Q dimer has a higher binding affinity than G6PDWT in this condition, and loss of structural NADP+ binding favors the dimeric state.When incubating G6PDncreased E and S1Eligomers E and S1F monomer G. TherefK403Q dimer less prone to dissociation, it should have a higher propensity to form a tetramer; however, lack of tetramer formation suggests that although the dimer is present, loss of structural NADP+ binding may induce allosteric changes that prevent the tetramer from forming. Using partial native PAGE (P-Native PAGE), described in previous work . However, unlike SEC, three bands were observed near the expected molecular weight (MW) of the G6PD dimer (116 kDa), and major differences between G6PDWT and G6PDK403Q were observed; the G6PDK403Q dimer had a higher apparent MW than the G6PDWT dimer.With the G6PDous work , differeric G6PD , F and GWT and G6PDK403Q were subjected to SDS-PAGE, and no differences between the monomer MW were detected, confirming that MW differences were due to differences in mobility (H). Interestingly, although G6PDK403Q forms a dimer that is less prone to dissociation, it is inactive and thermally unstable (I and J). Loss of activity can be explained by loss of catalytic NADP+ and/or G6P binding as the Michalis–Menten equation could not reproducibly fit our data (Km > 1 mM) with maximum substrate concentrations reaching 1 mM G6P and NADP+. Given that activity, thermostability, and amino acid location correlate with the clinical severity of G6PD variants : G6PDP396L, G6PDR393H, and G6PDK238R, with G6PDK238 and G6PDR393H forming direct contacts with the structural NADP+. Like G6PDK403Q, Class I variants at the structural NADP+-binding site have impaired NADP+ binding (B); the monomer peak of the SEC chromatogram displayed a well-defined peak at 10.5 ml in the presence of 10 μM NADP+. Additionally, the dimer was less prone to dissociation, formed a dimer, which migrates slower on P-Native PAGE, could not form a tetramer, and had reduced catalytic activity (C and D).Like G6PDexamined A: G6PDP3 binding B; the moactivity , C and DK238R, although the activity is compromised, it maintained 34% activity, whereas G6PDP396L, G6PDR393H, and G6PDK403Q were catalytically inactive. Additionally, the G6PDK238R dimer dissociated to a greater extent and formed less of the top dimer species. G6PDK238R may retain activity due to the nature of the amino acid substitution; lysine and arginine have a similar size and positive charge. This may reduce, but not eliminate, structural NADP+ binding.Interestingly, for G6PD+-binding site, exhibit the same features, three additional Class I mutations, G6PDF216L, G6PDE274K, G6PDK275N, distant from the structural NADP+-binding site were generated (E). Like mutations located at the structural NADP+-binding site, G6PDF216L, G6PDE274K, G6PDK275N displayed properties like G6PDK403Q (F–H). This suggests that mutations at the dimer and/or tetramer interface, with G6PDF216L connecting to the dimer interface while G6PDE274K and G6PDK275N are located at the tetramer interface, can induce allosteric changes, which cause loss of structural NADP+ binding and formation of a dysfunctional dimer. It is likely all variants that exhibit these allosteric changes result in the most severe G6PDdef.To understand if other Class I mutations, where the mutation is distant from the structural NADPenerated E. Like m6PDK403Q , F–H. ThK403Q were the result of changes in protein structure, we solved the crystal structure of G6PDK403Q (PDB 7SEI) at 3.65-Å resolution and observed significant structural changes , which caused major changes in the globular structure of the dimer ; though, loss of G6P binding inhibits its conversion.To confirm whether the altered biochemical and biophysical properties of G6PD changes  and S2. +-binding site share a similar structural defect and that a dysfunctional dimer may be a means to regulate G6PDWT activity. It should be noted that the resolution of G6PDK403Q is relatively low, 3.65-Å, with the crystallographic structure having high B-factors. However, we have previously characterized five other Class I and Class I-like dimer structures of G6PD, all having mid to poor resolution with high B-factors, with 6VAQ having the highest resolution—2.95-Å. All of these structures share the same dimer and structural features, which are distinct from the G6PDWT dimer.These findings are consistent with recently solved structures of several G6PD Class I variants , suggestK403Q tetramer was generated by reflecting a biological G6PDK403Q dimer along the axis of symmetry, and the G6PDK403Q tetramer was compared with the G6PDWT tetramer  salt bridges between K290 of one monomer and E287 of another monomer stabilize opposing αj-helices across the tetramer interface (E). (2) K275, a residue extending from the αj-helix, forms a salt-bridge with E347, which is located on a ß-sheet loop extending across the tetramer interface on an opposing monomer (G). However, in G6PDK403Q, the αj-helix is shifted, preventing K290/E287 and E347/K275 salt bridges from forming, explaining loss of tetramerization  K2 monomer G. HoweveK403Q and Class I variants could not form a tetramer, we aimed to understand how the tetramer contributes to enzymatic activity and stability. To do this, we made synthetic mutants favoring distinct oligomeric states. Nonnative cysteines were introduced at either the dimer or tetramer interface to cross-link G6PD oligomers together. For generation of the dimer-locked (dL) synthetic mutant, a cysteine was added to the C-terminal end; the C-terminal tail of monomer-1 faces the C-terminal tail of monomer-2 at the dimer interface (A). In theory, a disulfide bond would bridge monomer-1 to monomer-2. Indeed, this was the case as the G6PDWTdL monomer was not present on P-Native PAGE following cross-linking and did not dissociate to a monomer on SEC in monomer conditions (B and C).Since G6PDnterface A. In thenditions , B and Cet al. (A277C)—to form a disulfide bond across the tetramer interface, and we disrupted a salt bridge between E347 and K275 across the tetramer interface (G6PDE347A)—to prevent tetramerization (D). These mutations are effective due to each disulfide bond pair being multiplied by 4 in the tetramer interface. Consistent with findings by Ranzani et al., the G6PDA277C mutant forms a stable tetramer that does not dissociate when subjected to monomer conditions (E and F). Additionally, the G6PDE347A mutant does not form a tetramer when placed in conditions favoring tetramer formation; low pH, ionic strength, with addition of NADP+ and MgCl2 (E and G). These mutations did not modify the biophysical properties of the dimer, as G6PDE347A and G6PDA277C behaved similarly to G6PDWT; P-Native PAGE results show that ∼70% of G6PDWT and G6PDA277C was in monomeric form (H). Interestingly, G6PDE347A had a slight increase in dimer dissociation, ∼80% in the monomeric form, suggesting that the tetramer may aid in preventing monomer–monomer dissociation.To generate synthetic mutants stabilized in or inhibited from being in the tetrameric state, we followed work done by Ranzani et al. ; we mutarization D. These nditions , E and Fnd MgCl2 , E and Gric form H. IntereE347A, reduced enzymatic activity by ∼40% (I). Consistent with these findings, stabilization of the tetramer, G6PDA277C, increased the catalytic activity by ∼80%, reinforcing the importance of tetrameric G6PD for catalysis. When comparing the dimeric form, G6PDE347A, against the tetrameric form, G6PDA277C, the tetramer had ∼300% increase in catalytic activity. The Km for NADP+ and G6P for G6PDWT, G6PDE347A, and G6PDA277C supports increased activity as the tetramer binds G6P more readily; a KM of 115 versus 93 μM for G6PDE347A and G6PDA277C, respectively (J). Additionally, the tetramer is more thermally stable than dimeric G6PD (K). Together, these findings suggest that the tetrameric state provides maximum enzymatic activity and stability of G6PD.When examining the enzyme kinetics of the generated mutants, we found that impairment of the tetramer, G6PD by ∼40% I. Consisectively J. Additiric G6PD K. TogethK403Q has been established as a good model for Class I variants, we sought to determine if stabilizing the dimer or tetramer could correct activity or stability for G6PDK403Q. Using the same mutagenic strategy described above, we made double and triple mutants, the G6PDK403Q mutation combined with either dL, or both dL and tetramer-locked (tL), to determine if inducing oligomerization could recover activity and/or stability for G6PDK403Q.Previous work in the lab found that stabilizing the dimeric state with a small molecule increases the catalytic activity of Class II and III variants , 19. As K403Q forms a stable dimer, locking G6PDK403Q in the dimeric states offered little improvement in activity (A). In contrast, locking G6PDK403Q in the tetrameric state improved activity significantly, ∼200-fold. Similar trends were obtained at substrate concentrations recommended by the WHO (200 μM NADP+ and 600 μM G6P) and when comparing with G6PDWT, tetramer stabilization activated a catalytically dead enzyme to 16.5% of G6PDWT activity (B).Consistent with our findings that G6PDactivity A. In conactivity B.Figure K403Q series (C). The Km reveals that G6PDK403QdL improved both G6P and NADP+ binding, as we were able to generate a reliable fit to the Michalis– Menten equation, whereas G6PDK403Q could not (previously discussed). However, although dimer locking improved the Km for G6P and NADP+, the Km exceeded 800 μM, binding substrates poorly. Consistent with the fact that tetramer locking was able to activate G6PDK403Q, G6PDK403QdLtL improved G6P and NADP+ binding significantly, as the Km changed from 1382 to 102 μM for G6P and from 802 to 59 μM for NADP+. The Km for G6PDWT appears different (J and 5C) because different protein concentrations were used; ∼3 nM was used for Figure 4J, whereas ∼70 nM was used for Figure 5C—higher concentrations were needed to accurately measure G6PDK403Q kinetics.To better understand the relationship between substrates and activity, the Michalis–Menten kinetics were measured for the G6PDQ series C. The Kmifferent J and 5C + in erythrocytes has been reported at 25 to 40 μM NADP+ . These findings are consistent with those obtained with the impaired tetramer mutant, G6PDE347A, and tL mutant, G6PDA277C; the tetramer is more stable than the dimer.The physiological concentration of NADPμM NADP+ , 22, witto 60 μM , 24. Thef G6PDWT D. These K403QdLtL indeed forms a tetramer. When running G6PDK403Q and G6PDK403QdLtL on SEC in monomer conditions, up to 80% of G6PDK403QdLtL was a tetramer, confirming that the G6PDK403QdLtL mutant can form a tetramer (E and S3A). To further characterize the G6PDK403QdLtL structure in solution, we used SEC-SAXS, with the G6PDK403QdLtL dimer and tetramer fractions having an equal distribution (A and S5A).To further confirm the biochemical and thermostability measurements, we used analytical SEC and SEC coupled with small-angle X-ray scattering (SEC-SAXS) to see if the G6PDtetramer E and S3Aribution A and S5AK403QdLtL dimer and tetramer fractions, as shown in the I(q) versus q as a log-linear plot and the Kratky plot (F and G). Additionally, Guinier analysis, P(r) analysis, and Porod volume estimates for dimer and tetramer fractions yielded different Rg, Dmax, and MW values, corresponding to dimers and tetramers . This indicates that the G6PDK403QdLtL dimer has a greater population of flexible regions. Therefore, by forming the tetramer, the structural flexibility of the dimeric enzyme is reduced; this is consistent with and likely explains increased stability by tetramer stabilization.Analysis of SEC-SAXS data reveals two distinct signatures for the G6PDtky plot , F and GtL dimer G. This iK403QdLtL tetramer; unfortunately, we were only successful at solving the structure of G6PDK403QdLtL (PDB 7SEH) in the dimeric form at 2.8 Å resolution . Additionally, the globular shape of the dimer is modified, with a hinge-like shift in one of the monomers relative to the other monomer in the dimer . These observations confirm that the dimer-locked and tetramer-locked mutations induce structural changes in the G6PDK403QdLtL dimer, though the structural modifications that occur in the G6PDK403QdLtL tetramer remain to be determined.We next attempted to solve the crystal structure of G6PDsolution . Interes6PDK403Q A. Additiy 20.4 Å B. These WT tetramer (PDB 6E08) and G6PDK403QdLtL dimer (PDB 7SEH) structures with missing flexible regions (C–F), and we used these models to generate theoretical I(q) versus q as a log-linear plot and Kratky plots (A and B). The theoretical G6PDWT tetramer and G6PDK403QdLtL dimer plots fit well to the experimental SAXS data for the tetramer (A and B) and dimer fraction, respectively (A and B). This confirms that the G6PDK403QdLtL dimer crystallographic structure matches the structure in solution. Additionally, the experimental values derived from SEC-SAXS analysis agree with Rg (Å), Dmax (Å), and MW estimations generated from CORAL modeling (H), further confirming that the G6PDK403QdLtL mutant forms tetramer in solution.We used CORAL  to model regions , C–F, anky plots , A and Btetramer , A and Bectively , A and Bmodeling H, furtheK403Q and Class I variants were unique to these mutants and whether clinically relevant mutants could be activated or stabilized via the induction of oligomers. To answer this, several clinical variants falling into different clinical classes and located throughout different regions of the protein structure were examined, as shown by the color coding of variants on the G6PD tetramer (A). G6PDCanton, G6PDMediterranean (MED), G6PDKaiping, and G6PDA-, four of the most common G6PD variants, were included in our study.We next questioned whether biophysical and biochemical properties observed for G6PDtetramer A. G6PDCaWT, in contrast to that observed for G6PDK403Q and Class I variants (B), confirming that a dysfunctional dimer is unique to Class I variants and the Class-I like variant, G6PDK403Q. Since G6PDK403Q had improved catalytic activity following induction of the tetrameric state, we hypothesized that inducing the tetramer would also improve biochemical parameters for Class I mutants and that this activation would not occur with induction of the dimeric state. Additionally, we hypothesized that induction of the dimeric state and/or tetrameric state may improve catalytic activity and/or stability of other clinical variants. To test our hypotheses, we made double and/or triple mutants for each clinical variant depicted in Figure 6A and measured the catalytic activity of each mutant series (A–M).Using P-Native PAGE, we found that most Class II, III, IV variants dissociated to a monomer, like G6PDvariants B, confirt series , A–M.C and D). Interestingly, dimer locking did not improve activity for Class I, II, and III variants tested with one exception; G6PDA-, a Class III double variant, had ∼60% activation (A–M). In contrast, Class I variants had significant improvements in catalytic activity when locked in the tetrameric state (A–G). In fact, activation via tetramer locking was specific for Class I variants, as tetramer locking did not improve the catalytic activity of most Class II and III variants; only G6PDMED, a Class II variant, exhibited a twofold increase in activity (H–M). Consistent with our findings that Class I mutations at the structural NADP+-binding site benefit from tetramer stabilization, G6PDW509A, a nonclinical variant located on the C-terminal tail, which sandwiches the structural NADP+-binding site (D).The change in catalytic activity from the nonlocked (nL) enzyme, the normal mutant enzyme, as a function of the position of the mutation is depicted onto the three-dimensional structure for both the dimer-locked and tetramer-locked variants , C and Dtivation , A–M. Inic state , A–G. Inactivity , H–M. Coing site , also exing site D.N). This is consistent with our data about the lack of benefit of increased dimerization on Class I variants and lack of structural NADP+ binding. Interestingly, select variants were inhibited by tetramer locking; G6PDE274K and G6PDR227W/Q. It is possible that tetramer locking with a disulfide bond causes steric clashing at the tetramer interface, suggesting that linker length may be an important determinant of activation.AG1, a small-molecule activator of Class II, III, and WT that stabilizes the dimer state  did not Fig. S9E and F). The effect on thermostability for various classes of clinical variants varied. For example, G6PDK275N, a Class I variant, had a 2.2 °C increase when dimer locking (G6PDK275NdL), and an additional 3.1 °C increase when tetramer locking (G6PDK275NdLtL), bringing G6PDK275NdLtL T1/2 to only 0.7 °C below that of G6PDWT (E). Interestingly, G6PDCanton was stabilized by dimer and tetramer locking, despite lack of catalytic activation. G6PDCanton, a Class II variant, had a 4.0 °C increase in T1/2 when dimer locking (G6PDCantondL), with tetramer locking (G6PDCantondLtL) adding an additional 3.6 °C to the T1/2, bringing the G6PDCantondLTL T1/2 to 5.2 °C above that of G6PDWT (F). Stabilizing the dimer/tetramer may be a therapeutic strategy to improve stability for catalytically competent mutants, such as Class II or III, with tetramer stabilization being beneficial to Class I variants by improving both catalytic activity and stability.Although dimer locking did not affect catalytic activity for most mutants, it stabilized Class I and Class II variants , E and Ff G6PDWT E. Interef G6PDWT F. StabilK403Q and Class I mutations at and distant from the structural NADP+ are impaired in their ability to bind structural NADP+, resulting in a dysfunctional dimer with modified biophysical properties; variants with this dimer have low activity and stability, are less prone to dissociation, and are unable to form a tetramer. Although these observations were striking, the structural mechanism remained unclear. Therefore, we solved the protein structure for G6PDK403Q and confirmed that major structural alterations occur in the G6PDK403Q dimer: the structural NADP+ is not bound, the catalytic G6P-binding site is occluded, and there are major changes in the globular structure of the dimer. These observations explain our biochemical data; G6P occlusion hinders G6PD activity, and dimer distortion prevents salt-bridge interactions at the tetramer interface, and these findings are consistent with previous structures solved for Class I variants  suggest that P-Native PAGE can distinguish between G6PD, which can or cannot bind structural NADP+. Furthermore, our findings using G6PDK403Q are physiologically relevant, since the G6PDK403Q mutation mimics in vivo acetylation of G6PDWT , whereas G6PDK275N had <10% of G6PDWT activity and formed a dysfunctional dimer (G and H). It is likely that when structural NADP+ is bound, NADP+ binding is sufficient to stabilize the ß-sheet, making loss of tetramerization less severe. However, if structural NADP+ is not bound, loss of this salt-bridge interaction may allow for ß-sheet shifting.Combining our findings with the structural insight from Horikoshi utations . As A277al dimer , H and Ial dimer , G and Hdef, as RBCs lack nuclei, and therefore, proteins must maintain proper stability for the entire life span of the RBCs (60–90 days). Consequently, as RBCs age, their residual G6PD activity is largely dependent on protein levels. We therefore suggest that improving the stability of any mutant with lower than WT catalytic activity, or in the case of Class I, a significant improvement in stability combined with a small increase in activity, may be sufficient to improve the clinical phenotype; clinical observations suggest that a shift from Class I to Class II-like state will result in a less severe phenotype , NADPH (Millipore Sigma – 10107824001), Glucose-6-phosphate , resazurin (ACROS Organics – AC189900050). Reagents: TALON Superflow resin (cytiva – 28957502), High Purity Bovine Thrombin (BioPharm Laboratories – 91-030-006), protease inhibitor cocktail (Millipore Sigma – p8340), Diaphorase . Antibodies purchased from Everest Biotechnology, G6PD (aa 308–320) goat (Everest Biotech – EB07841). Analytical SEC column: Superdex 75 10/300 Gl . Purification SEC column: Superdex 200 HiLoad 16/600 . SEC-SAXS column: Superdex 200 Increase PC 3.2/300 column (Cytiva).Chemicals: NADPNde I and Sal I restriction sites, with a stop codon added at the C-terminal end before the C-terminal his-tag. For mutants, site-directed mutagenesis was performed using the Agilent QuikChange II kit.G6PD was inserted into a pET-28a (+) vector, between E. coli cells. Cells were grown in TB medium, and protein expression was induced with IPTG at 28 °C for 24 h. Cell lysate was incubated with TALON Superflow resin for 1 h at 4 °C. Following resin washing, protein was eluted via overnight Thrombin cleavage at 4 °C to remove the N-terminal his-tag. For X-ray crystallography and SEC-SAXS experiments, protein was subjected to further purification on a Superdex 200 HiLoad 16/600  column in 50 mM Tris (pH7.4), 150 mM NaCl, 10 mM G6P.G6PD-pET-28a (+) was transformed into OverExpress C43 (DE3) chemically competent WT: 3 nM recombinant G6PD was incubated in buffer containing 50 mM Tris (pH 7.4), 10 mM MgCl2, 200 μM NADP+, 600 μM G6P, and 0.005% Tween20. G6P was added to start the reaction and NADPH absorbance at 340 nm was measured for 5 min at 23 °C. The slope (ΔAbs/ΔTime) was used to compare enzyme activity for mutants, with data normalized to G6PDWT activity. (2) Michaelis–Menten kinetics for G6PDWT, G6PDE347A, G6PDA277C: Activity assay 1 conditions were used, however, to prevent NADP+ depletion, 1 U/ml diaphorase and 0.1 mM resazurin fluorescence were used (NADPH conversion to NADP+ is coupled to resorufin production). The excitation/emission at 565 nm/590 nm respectively was measure and converted to NADPH/sec with an NADPH standard curve. Data were fit to the Michaelis–Menten equation in GraphPad Prism. (3) Activity assay for G6PDK403Q locked in dimer and/or tetrameric state: Activity assay 1 conditions were used; however, enzyme concentration was increased to 70 nM so relative difference in activity could be accurately assessed. For the condition with low NADP+, NADP+ was reduced to 10 μM. (4) Michaelis–Menten Kinetics for G6PDK403Q series: Activity assay 2 conditions were used; however, enzyme concentration was increased to 70 nM so differences in activity could be accurately assessed. (5) Activity assay pathogenic variants locked in dimer and/or tetrameric state: Activity assay 3 conditions were used at 10 μM NADP+; however, 70 nM of Class I variants protein was used, while 6 nM of less severe variant protein was used. Activity assay for AG1: 3 nM recombinant enzyme was incubated in buffer containing, 50 mM Tris (7.4), 3.3 mM MgCl2, 10 μM NADP+, 100 μM G6P, 0.010% Tween20, 0.5 mM EDTA, 1 U/ml diaphorase, and 0.1 mM resazurin. In total, 100 μM AG1 was incubated with mixture for 5 min at 23 °C. G6P was added to start reaction, and fluorescence was measured and slopes used to compare activity between series. These conditions were established by Hwang et al. ((1) Activity assay for biochemical characterization of clinical mutants relative to G6PDg et al.  for AG1.+ for 20 min at various temperatures. Enzyme activity, after temperature incubation, was measured according to conditions described in activity assay 1. The reaction rate was normalized to the maximum activity for a given temperature interval, and the normalized activity was plotted and fit to the Boltzmann sigmoidal equation in GraphPad Prism. The temperature at which the enzyme maintained half activity (T1/2), or the melting temperature, was generated for each mutant. Unique temperature intervals were used for independent experiments, in triplicates, and temperature intervals were combined to generate three Boltzmann sigmoidal fits, with each fit representing one triplicate.In total, 333 nM recombinant enzyme was incubated in 50 mM Tris (pH7.4), 150 mM NaCl, and 5 μM NADPK403Q and Class I variants versus G6PDWT: 500 nM recombinant G6PD was incubated in 80 mM NaP (pH 8.0) buffer with and without 10 μM NADP+ for 30 min at 23 °C. The protein was run on FPLC using 80 mM NaP (pH 8.0). (2) Monomer condition: 1 μM recombinant G6PD was incubated in 50 mM Tris (pH7.4), 150 mM NaCl, and 10 mM G6P for 30 min and ran on FPLC using 50 mM Tris (pH 7.4), 150 mM NaCl. (3) Tetramer condition: 1 μM recombinant G6PD was added to buffer containing 80 mM NaP pH 6.0, 20 mM MgCl2, 10 μM NADP+, 20 μM EDTA, 0.005% Tween20. For SEC chromatograms, data were normalized to total peak area, followed by normalization to the data maxima, so that chromatograms have a maximum absorbance of ∼1.All analytical SEC experiments were performed on a Superdex 75 10/300 Gl column and ran at a flow rate of 0.4 ml/min. (1) SEC for G6PDE, 270 nM G6PD was incubated in tetramer conditions , 150 mM NaCl.Laemmli sample buffer devoid of SDS and BME was added to samples. The sample was not boiled and instead immediately ran on SDS-PAGE. (1) P-Native PAGE: 850 nM protein was incubated in 50 mM Tris (pH 7.4), 150 mM NaCl, at 23 °C for 30 min followed by P-Native PAGE and western blotting. P-Native blots were quantified using Fiji (2) Cross-linking followed by P-Native PAGE: For Figure 4K403Q, 1 μl of 1 mM AG1, 2 mM NADP+, 8 mg/ml G6PDK403Q were mixed and incubated with 1 μl 100 mM MES pH 6.5, 13% PEG8000, 250 mM MgCl2, 1.4% 1,6-hexanediol and incubated at 20 °C using the sitting drop vapor diffusion method. For G6PDK403QdLtL dimer, 2 μl of 1 mM AG1, 2 mM NADP+, 5 mg/ml G6PDK403QdLtL dimer were mixed and incubated with 2 μl 100 mM MES pH 6.5, 12% PEG4000, 60 mM MgCl2, and incubated at 20 °C using the hanging drop vapor diffusion method.For G6PDK403Q and G6PDK403QdLtL were collected at 100˚K at the BL12-2 of the Stanford Synchrotron Light Source using Pilatus 6M detectors. Crystals for the G6PDK403Q were cryo cooled using well solution supplemented with 20% glycerol. Crystals of the G6PDK403QdLtL dimer were cryo cooled using well solution supplemented with 30% ethylene glycol. We collected 360 degrees of data with 0.2 degree per image. Diffraction data were processed with XDS suite of programs (K403Q was solved by molecular replacement with Phaser (6VA8) as the search model. The structure of G6PDK403QdLtL dimer was solved by molecular replacement with AMoRE (6VA7) as the search model. Iterative rounds of model building and refinements were performed using Coot  using 50 mM Tris pH 7.4, 150 mM NaCl, 10 mM G6P running buffer. In total, 500 images were collected with 1-s exposure every 5 s at a 0.05 ml/min flow rate. After the 100th image (blank data collections), the X-ray shutter was closed until just before the sample was eluted to keep the sample cell clean from debris formed by X-ray radiation. An additional sample cell wash was executed during the shutter closure. Data reduction and initial analyses were performed using the BL4-2 automated SEC-SAXS data processing and analysis pipelines, SECPipe (https://www-ssrl.slac.stanford.edu/smb-saxs/node/1860). It implements the program SASTOOL (https://www-ssrl.slac.stanford.edu/smb-saxs/node/1914) and ATSAS AUTORG /λ, 2θ is the scattering angle, and λ is the wavelength of the X-ray. After careful manual inspection, a total of ten images were selected to generate the averaged profile for further analysis. The program GNOM was used for the indirect Fourier transform to estimate the distance distribution function P(r)  was used an input model, and disordered N-terminal portion of 31 residues was modeled with P22 symmetry. For G6PDK403QdLtL dimer, a subunit of the crystal structure (PDB 7SEH) was split into three rigid bodies and three disordered regions in the subunit  were reconstructed during the modeling with P2 symmetry. Twenty independent runs were performed, and the model with the lowest χ2 value was selected as the best model. All models showed that the disordered portions were located virtually at the same position (C and D). The SAXS parameters of the best CORAL models were computed using the program CRYSOL (SEC-SAXS experiments were performed at SSRL beamline 4-2 as the previous report with some modifications . G6PDK40S AUTORG . The dation P(r) . The SAXion P(r) . For theposition , C and Dm CRYSOL . MW was m CRYSOL . ExperimI and J, G6PDK403Q was compared with G6PDWT, and statistical differences were calculated by a two-tailed unpaired t test. In, Figure 1G, G6PDK403Q was compared with G6PDWT, and statistical differences were calculated for each group of oligomeric species (dependent variables) independently, using four two-tailed unpaired t tests. In C and G, 4H, and 6B, mutants were compared with G6PDWT, and statistical differences were calculated for each group of oligomeric species (dependent variables) independently by four one-way ANOVAs, Dunnett’s multiple comparisons test. In D and H, mutants were compared with G6PDWT, and statistical differences were calculated by ordinary one-way ANOVA, Dunnett’s multiple comparisons test. In I and K, A, B and D and E and F, and A–M, statistical differences between mutants were calculated by ordinary one-way ANOVA, Tukey’s multiple comparisons test. For Fig. S9N, statistical differences between the no treatment group and AG1-treated group were calculated via a two-way ANOVA, Šidák’s multiple comparisons test. For all plots with bars displayed and Km tables, error bars represent mean ± standard deviation. Asterisks symbolize p values: n.s p > 0.05, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, and n represents a biological replicate, where each n was performed independently. For thermostability assays, measurements were derived from multiple independent experiments, performed in triplicates. Independent measurements were combined to generate a Boltzmann sigmoidal fit, with n representing triplicates and error representing standard deviation among the triplicate fit values.In K403Q, G6PDK403QdLtL dimer, respectively. All other data are included in the manuscript.Protein structures have been deposited in the Protein Data Bank (PDB) with accession codes 7SEI, 7SEH for G6PDThis article contains The authors declare that they have no conflicts of interest with the contents of this article.