Augmenting Vacuolar H+-ATPase Function Prevents Cardiomyocytes from Lipid-Overload Induced Dysfunction.

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H+-ATPase (v-ATPase). Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis.

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

Toosendanin, a novel potent vacuolar-type H+-translocating ATPase inhibitor, sensitizes cancer cells to chemotherapy by blocking protective autophagy.

Macroautophagy/autophagy is the process of self-digestion through the lysosomes; it disassembles unnecessary or dysfunctional long-lived proteins and damaged organelles for the recycling of biomacromolecules. Unfortunately, cancer cells can hijack this mechanism to survive under metabolic stress or develop drug resistance during chemotherapy. Increasing evidence indicates that the combination of autophagy inhibition and chemotherapy is a promising cancer treatment strategy. However, effective autophagy inhibitors with satisfied potency, bioavailability, and clearly-defined drug targets are still rare. Here, we report the identification of a potent autophagy inhibitor toosendanin which can effectively block autophagosome maturation, causing the accumulation of autophagy substrates in multiple cancer cells. Toosendanin did not inhibit the fusion process between autophagosome and lysosome but elevated lysosomal pH and impaired lysosomal enzymes activity. Using rat liver lysosome fraction and purified yeast V-ATPase, we found that toosendanin directly inhibited V-ATPase activity. By applying cellular thermal shift assay (CETSA), immunoprecipitation-coupled LC-MS/MS analysis, and biotin-toosendanin pull-down assay, we confirmed the direct binding between toosendanin and V-ATPase. Furthermore, toosendanin blocked chemotherapy-induced protective autophagy in cultured cancer cells and xenograft tumor tissues to significantly enhance anti-cancer activity. These results suggest that toosendanin has the potential to be developed into an anti-cancer drug by blocking chemotherapy-induced protective autophagy.

The milk-derived lactoferrin inhibits V-ATPase activity by targeting its V1 domain.

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, herein we aimed to fill this gap by employing a five-stage computational approach. Molecular dynamics simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering, which allowed retrieving representative structures, to then perform protein-protein docking. Subsequently, molecular dynamics simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build a final ranking based on the binding affinities. Detailed atomist analysis of the top ranked complexes clearly indicates that Lf binds to the V1 cytosolic domain of V-ATPase. Particularly, our data suggest that Lf binds to the interfaces between A/B subunits, where the ATP hydrolysis occurs, thus inhibiting this process. The free energy decomposition analysis further identified key binding residues that will certainly aid in the rational design of follow-up experimental studies, hence bridging computational and experimental biochemistry.

Dual Targeting of v-ATPase and mTORC1 Signaling Disarms Multidrug-Resistant Cancers.

Chemotherapeutic treatments are frequently impeded by the development of multidrug resistance (MDR). In this issue of Cell Chemical Biology, Wang et al. (2020) identify the natural product verucopeptin as having therapeutic potential toward MDR cancer cell types by targeting v-ATPase and mTORC1 signaling.

Protective impacts of circular RNA VMA21 on lipopolysaccharide-engendered WI-38 cells injury via mediating microRNA-142-3p.

CircRNA derived from vacuolar ATPase assembly factor (circVMA21) is a newly-researched circRNA, which is reported to adjust the degeneration of intervertebral disc. But, function of circVMA21 in infantile pneumonia is yet to be explored. The research surveyed the role of circVMA21 in lipopolysaccharide (LPS)-caused WI-38 cell inflammatory injury. LPS (10 µg/ml, 12 hr) was exploited to arouse WI-38 cell inflammatory injury. Subsequently, the mediatory impacts of microRNA (miR)-142-3p and circVMA21 in LPS-evoked cell injury were detected after transfection with the inhibited or overexpressed vectors. In above processes, cell behaviors of cell viability, apoptosis, and pro-inflammatory factors were monitored. NF-κB and JNK pathways were elucidated to showcase the feasible molecular mechanisms. Results displayed that LPS engendered WI-38 cell inflammatory injury was alleviated as well as activated NF-κB and JNK pathways was interdicted by miR-142-3p suppression. Importantly, restrained miR-142-3p expression was discovered in WI-38 cells after overexpressing circVMA21. Moreover, overexpressed circVMA21 exerted the similar functions as miR-142-3p suppression in LPS-triggered WI-38 cell injury. But, the influence was clearly reversed by miR-142-3p overexpression. Hindered NF-κB and JNK pathways caused by overexpressed circVMA21 was also crippled by miR-142-3p overexpression. The research discolsed that circVMA21 protected WI-38 cells to resist LPS-triggered inflammatory injury via miR-142-3p-NF-κB/JNK axis.

[Effect of synergistic polarization macrophage modulated by N-terminal domain of a2 vacuolar ATPase and macrophage colony stimulating factor on proliferation of gastric cancer cells].

OBJECTIVE: To investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase (a2NTD) and macrophage colony-stimulating factor (M-CSF) on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells. METHODS: Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups: the control group (RPMI 1640), the experimental group I (M-CSF 100 µg/L), the experimental group II (a2NTD 500 µg/L) and the experimental group III (a2NTD 500 µg/L plus M-CSF 100 µg/L). After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901. RESULTS: The expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups (+). The mean absorbance (A) was 0.092 ± 0.005 in control group, 0.095 ± 0.006 in group I, 0.094 ± 0.005 in group II, 0.094 ± 0.005 in group III, and no significant differences were observed among 4 groups (all P>0.05). Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group (-) with A 0.025 ± 0.004; it was normal in groupI and group II (+) with A 0.191 ± 0.012 in group I and 0.197 ± 0.136 in group II (P=0.212), and it was up-regulated significantly in group III (+++) with A 0.285 ± 0.011. There were significant differences between either two groups except group I and group II (all P<0.01). Secretion of IL-10 in group I and group II [(85.65 ± 13.64) ng/L and (87.77 ± 14.25) ng/L] was significantly higher compared with control group [(71.67 ± 7.56) ng/L, P<0.01]. Secretion of IL-12 in group I and group II [(9.91 ± 1.50) ng/L and (10.15 ± 1.80) ng/L] was significantly lower compared with control group [(16.87 ± 1.10) ng/L, P<0.01]. Secretion of IL-10 in group III [(116.98 ± 14.27) ng/L] was the highest, and secretion of IL-12 [(5.31 ± 0.88) ng/L] was the lowest (all P<0.01). There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group III showed the greatest impact compared with other 3 groups (P<0.01). CONCLUSIONS: a2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.

Inhibition of human endothelial cell proliferation by ShIF, a vacuolar H(+)-ATPase-like protein.

ShIF is a bone marrow stroma cell-derived factor originally identified to support proliferation of bone marrow cells in vitro. This protein shares high sequence homology to the yeast vacuolar H(+)-ATPase subunit, Vph1p, and the 116 kDa proton pump of the rat and bovine synaptic vesicle, Vpp1. We examined the function of ShIF in the proliferation of human umbilical vein endothelial cells (HUVEC). ShIF inhibited HUVEC proliferation in a dose-dependent manner. Recombinant ShIF added at 10 and 20 ng/ml inhibited HUVEC proliferation by 21.6 and 44.3%, respectively and increasing the concentration of ShIF to 100 ng/ml inhibited proliferation by as much as 55.5%. When HUVEC cells were cultured at various concentrations of ShIF in the presence of anti-ShIF antibody, the inhibitory effects of ShIF to HUVEC proliferation were abrogated by 89-91% indicating that the activity of ShIF to HUVEC was specific. HUVEC cultured in the presence of ShIF and bafilomycin, a specific inhibitor of ATPase, resulted to a 90% growth inhibition. Thus, ShIF may act as an antagonist to the ATPase complex by disrupting the production of cellular ATP thereby decreasing the ability of HUVEC to proliferate.