The Vacuolar H+-ATPase subunit C is involved in oligogalacturonide (OG) internalization and OG-triggered immunity.

In plants, the perception of cell wall fragments initiates signal transduction cascades that activate the immune response. Previous research on early protein dynamics induced by oligogalacturonides (OGs), pectin fragments acting as damage-associated molecular patterns (DAMPs), revealed significant phosphorylation changes in several proteins. Among them, the subunit C of the vacuolar H+-ATPase, known as DE-ETIOLATED 3 (DET3), was selected to elucidate its role in the OG-triggered immune response. The Arabidopsis det3 knockdown mutant exhibited defects in H2O2 accumulation, mitogen-activated protein kinases (MAPKs) activation, and induction of defense marker genes in response to OG treatment. Interestingly, the det3 mutant showed a higher basal resistance to the fungal pathogen Botrytis cinerea that, in turn, was completely reversed by the pre-treatment with OGs. Our results suggest a compromised ability of the det3 mutant to maintain a primed state over time, leading to a weaker defense response when the plant is later exposed to the fungal pathogen. Using fluorescently labelled OGs, we demonstrated that endocytosis of OGs was less efficient in the det3 mutant, implicating DET3 in the internalization process of OGs. This impairment aligns with the observed defect in the priming response in the det3 mutant, underscoring that proper internalization and signaling of OGs are crucial for initiating and maintaining a primed state in plant defense responses.

Molecular mechanism of Oxr1p mediated disassembly of yeast V-ATPase.

The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p induces V-ATPase disassembly in vitro. Whether and how Oxr1p is involved in enzyme disassembly in vivo, however, is not known. Here, using yeast genetics and fluorescence microscopy, we show that Oxr1p is essential for efficient V-ATPase disassembly in the cell. Supporting biochemical and biophysical in vitro experiments show that whereas Oxr1p-driven holoenzyme disassembly can occur in the absence of nucleotides, the presence of ATP greatly accelerates the process. ATP hydrolysis is needed, however, for subsequent release of Oxr1p so that the free V1 can adopt the autoinhibited conformation. Overall, our study unravels the molecular mechanism of Oxr1p-induced disassembly that occurs in vivo as part of the canonical V-ATPase regulation by reversible disassembly.

Unveiling Vacuolar H+-ATPase Subunit a as the Primary Target of the Pyridinylmethyl-Benzamide Fungicide, Fluopicolide.

An estimated 240 fungicides are presently in use, but the direct targets for the majority remain elusive, constraining fungicide development and efficient resistance monitoring. In this study, we found that Pcα-actinin knockout did not influence the sensitivity of Phytophthora capsici to fluopicolide, which is a notable oomycete inhibitor. Using a combination of Bulk Segregant Analysis Sequencing and Drug Affinity Responsive Target Stability (DARTS) assays, the vacuolar H+-ATPase subunit a (PcVHA-a) was pinpointed as the target protein of fluopicolide. We also confirmed four distinct point mutations in PcVHA-a responsible for fluopicolide resistance in P. capsici through site-directed mutagenesis. Molecular docking, ATPase activity assays, and a DARTS assay suggested a fluopicolide-PcVHA-a interaction. Sequence analysis and further molecular docking validated the specificity of fluopicolide for oomycetes or fish. These findings support the claim that PcVHA-a is the target of fluopicolide, proposing vacuolar H+-ATPase as a promising target for novel fungicide development.

The study of intercalated cells using ex vivo techniques: primary cell culture, cell lines, kidney slices, and organoids.

This review summarizes methods to study kidney intercalated cell (IC) function ex vivo. While important for acid-base homeostasis, IC dysfunction is often not recognized clinically until it becomes severe. The advantage of using ex vivo techniques is that they allow for the differential evaluation of IC function in controlled environments. Although in vitro kidney tubular perfusion is a classical ex vivo technique to study IC, here we concentrate on primary cell cultures, immortalized cell lines, and ex vivo kidney slices. Ex vivo techniques are useful in evaluating IC signaling pathways that allow rapid responses to extracellular changes in pH, CO2, and bicarbonate (HCO3-). However, these methods for IC work can also be challenging, as cell lines that recapitulate IC do not proliferate easily in culture. Moreover, a "pure" IC population in culture does not necessarily replicate its collecting duct (CD) environment, where ICs are surrounded by the more abundant principal cells (PCs). It is reassuring that many findings obtained in ex vivo IC systems signaling have been largely confirmed in vivo. Some of these newly identified signaling pathways reveal that ICs are important for regulating NaCl reabsorption, thus suggesting new frontiers to target antihypertensive treatments. Moreover, recent single-cell characterization studies of kidney epithelial cells revealed a dual developmental origin of IC, as well as the presence of novel CD cell types with certain IC characteristics. These exciting findings present new opportunities for the study of IC ex vivo and will likely rediscover the importance of available tools in this field.NEW & NOTEWORTHY The study of kidney intercalated cells has been limited by current cell culture and kidney tissue isolation techniques. This review is to be used as a reference to select ex vivo techniques to study intercalated cells. We focused on the use of cell lines and kidney slices as potential useful models to study membrane transport proteins. We also review how novel collecting duct organoids may help better elucidate the role of these intriguing cells.

Significance of the plasma membrane H+-ATPase and V-ATPase for growth and pathogenicity in pathogenic fungi.

Pathogenic fungi are widespread and cause a variety of diseases in human beings and other organisms. At present, limited classes of antifungal agents are available to treat invasive fungal diseases. With the wide use of the commercial antifungal agents, drug resistance of pathogenic fungi are continuously increasing. Therefore, exploring effective antifungal agents with novel drug targets is urgently needed to cope with the challenges that the antifungal area faces. pH homeostasis is vital for multiple cellular processes, revealing the potential for defining novel drug targets. Fungi have evolved a number of strategies to maintain a stable pH internal environment in response to rapid metabolism and a dramatically changing extracellular environment. Among them, plasma membrane H+-ATPase (PMA) and vacuolar H+-ATPase (V-ATPase) play a central role in the regulation of pH homeostasis system. In this chapter, we will summarize the current knowledge about pH homeostasis and its regulation mechanisms in pathogenic fungi, especially for the recent advances in PMA and V-ATPase, which would help in revealing the regulating mechanism of pH on cell growth and pathogenicity, and further designing effective drugs and identify new targets for combating fungal diseases.

Tender love and disassembly: How a TLDc domain protein breaks the V-ATPase.

Vacuolar ATPases (V-ATPases, V1 Vo -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V1 -ATPase disengages from Vo proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V1 and Vo subcomplexes.

Structural and Functional Diversity of Two ATP-Driven Plant Proton Pumps.

Two ATP-dependent proton pumps function in plant cells. Plasma membrane H+-ATPase (PM H+-ATPase) transfers protons from the cytoplasm to the apoplast, while vacuolar H+-ATPase (V-ATPase), located in tonoplasts and other endomembranes, is responsible for proton pumping into the organelle lumen. Both enzymes belong to two different families of proteins and, therefore, differ significantly in their structure and mechanism of action. The plasma membrane H+-ATPase is a member of the P-ATPases that undergo conformational changes, associated with two distinct E1 and E2 states, and autophosphorylation during the catalytic cycle. The vacuolar H+-ATPase represents rotary enzymes functioning as a molecular motor. The plant V-ATPase consists of thirteen different subunits organized into two subcomplexes, the peripheral V1 and the membrane-embedded V0, in which the stator and rotor parts have been distinguished. In contrast, the plant plasma membrane proton pump is a functional single polypeptide chain. However, when the enzyme is active, it transforms into a large twelve-protein complex of six H+-ATPase molecules and six 14-3-3 proteins. Despite these differences, both proton pumps can be regulated by the same mechanisms (such as reversible phosphorylation) and, in some processes, such as cytosolic pH regulation, may act in a coordinated way.

Clathrin-dependent endocytosis plays a critical role in larval and pupal development, and female oocyte production in the red flour beetle (Tribolium castaneum).

BACKGROUND: Clathrin-dependent endocytosis is a vesicular transport process by which cells take macromolecules from the extracellular space to the intracellular space. It plays important roles in various cellular functions, but its biological significance in insect development and reproduction has not been well studied. RESULTS: We characterized and functionally analyzed four major clathrin-dependent endocytic pathway genes (TcChc, TcAP50, TcVhaSFD, TcRab7) in Tribolium castaneum. RNA interference (RNAi) by injecting double-stranded RNA (dsRNA) targeting each gene at three doses (50, 100, or 200 ng per insect) in 20-day-old larvae led to 100% larval mortality. When the expressions of TcChc, TcVhaSFD, and TcRab7 were suppressed by injecting their respective dsRNAs at each dose in 1-day-old pupae, the adults that emerged from the dsRNA-injected pupae were deformed, with the absence of wing development. The deformed adults died within 2 days after eclosion. When the expression of TcAP50 was suppressed by injecting its dsRNA into 1-day-old pupae, although no apparent deformed adults were observed, all the adults died within 35 days after eclosion. In addition, when the expressions of TcChc and TcVhaSFD were suppressed by injecting their respective dsRNAs at a reduced dose (10 ng per insect) in 5-day-old pupae, the ovarian development and oocyte production in the resultant females were completely inhibited. CONCLUSION: Our results indicate that clathrin-dependent endocytosis is essential for insect development and reproduction. The results from this study can help researchers identify potential molecular targets for developing novel strategies for insect pest management. © 2023 Society of Chemical Industry.

Subunit C of V-ATPase-VmaC Is Required for Hyphal Growth and Conidiation in A. fumigatus by Affecting Vacuolar Calcium Homeostasis and Cell Wall Integration.

Aspergillus fumigatus is a widespread airborne fungal pathogen in humans. However, the functional genes in A. fumigatus that may contribute to its pathogenesis have not yet been fully identified. Vacuolar H+-ATPase is universal in eukaryotic organisms but exhibits specific roles in various species. Here, we identified VmaC as a putative subunit of vacuolar H+-ATPase in A. fumigatus that is widely conserved through evolution. The C-terminal hydrophobic domain of VmaC plays a critical role in its vacuolar localization and growth and conidiation. Deletion or turn-off of VmaC encoding gene-AfvmaC expression is not lethal but leads to a very sick and tiny colony phenotype, which is different from that of yeast with conditional ScvmaC defects. Furthermore, we found that AfvmaC not only participates in maintaining calcium homeostasis and vacuolar acidity but is also involved in cell wall integration pathway regulation, highlighting the importance of the vacuole as a storage organelle associated with many aspects of cellular homeostasis. This study indicates that fungal VmaC is relatively conserved. When compared to that in model yeasts, VmaC in A. fumigatus is required for hyphal growth and conidiation, suggesting that specific motifs in VmaC might be functioned in Aspergilli.

Reduced gene dosage is a common mechanism of neuropathologies caused by ATP6AP2 splicing mutations.

BACKGROUND: Mutations that alter splicing of X-linked ATP6AP2 cause a spectrum of neurodevelopmental and neurodegenerative pathologies including parkinsonism in affected males. All previously reported splicing mutations increase the level of a minor isoform with skipped exon 4 (Δe4) that encodes a functionally deficient protein. OBJECTIVES: We investigated the pathogenic mechanism of a novel c.168+6T>A variant reported in a family with X-linked intellectual disability, epilepsy, and parkinsonism. We also analyzed ATP6AP2 splicing defects in brains of carriers of a c.345C>T variant associated with X-linked spasticity and parkinsonism. METHODS: We generated induced pluripotent stem cells from patients with c.168+6T>A, reprogrammed them to neural progenitor cells and analyzed them by RNA-Seq and qRT-PCR. We also quantified ATP6AP2 isoforms in the brains of c.345C>T carriers by Nanostring nCounter. RESULTS: The c.168+6T>A increased skipping of ATP6AP2 exon 2 and usage of cryptic intronic donor splice sites. This results in out-of-frame splicing products and a reciprocal 50% reduction in functional full-length ATP6AP2 transcripts. Neural progenitors of patients with c.168+6T>A exhibited downregulated neural development gene networks. Analysis of blood transcriptomes of c.168+6T>A carriers identified potential biomarkers of ATP6AP2 deficiency in non-neural tissues. The c.345C>T variant increased exon 4 skipping with concomitant decrease of full length ATP6AP2 in brains of carriers. CONCLUSION: A common pathogenic consequence of splicing mutations affecting inclusion of different ATP6AP2 exons is reduction of the functional full-length transcript. The exacerbated ATP6AP2 splicing defect in brains of c.345C>T carriers is consistent with their CNS-restricted clinical presentations.

Vacuolar (H+ )-ATPase subunit c is essential for the survival and systemic RNA interference response in Locusta migratoria.

BACKGROUND: Vacuolar (H+ )-ATPase (V-ATPase) is a multi-subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V-ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V-ATPase-c functions in insects. RESULTS: We identified and characterized the V-ATPase-c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV-ATPase-c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV-ATPase-c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV-ATPase-c. In addition, silencing of LmV-ATPase-c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double-stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV-ATPase-c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria. CONCLUSION: Our results indicate that LmV-ATPase-c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV-ATPase-c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.

Ameliorating cancer cachexia by inhibiting cancer cell release of Hsp70 and Hsp90 with omeprazole.

BACKGROUND: Cancer cachexia, characterized by muscle and fat tissue wasting, is a major determinant of cancer-related mortality without established treatment. Recent animal data revealed that cancer cells induce muscle wasting by releasing Hsp70 and Hsp90 as surface proteins on extracellular vesicles (EVs). Here, we test a therapeutic strategy for ameliorating cancer cachexia by inhibiting the release of Hsp70 and Hsp90 using proton pump inhibitor omeprazole. METHODS: Omeprazole effect on Hsp70/90 release through EVs by Lewis lung carcinoma (LLC) cells in vitro, serum levels of Hsp70/90 and Hsp70/90-carrying EVs in LLC tumour-bearing mice, and LLC-induced muscle protein degradation pathways in C2C12 myotubes and mice were determined. Omeprazole effect on endolysosomal pH and Rab27b expression in LLC cells were analysed. RESULTS: Omeprazole treatment of LLC cells inhibited Hsp70/90 and Hsp70/90-carrying EV release in a dose-dependent manner (1 to 10 µM) and attenuated the catabolic activity of LLC cell-conditioned medium on C2C12 myotubes. Systemic omeprazole administration to LLC tumour-bearing mice (5 mg/kg/day subcutaneously) for 2 weeks blocked elevation of serum Hsp70, Hsp90, and Hsp70/90-carrying EVs, abrogated skeletal muscle catabolism, and prevented loss of muscle function as well as muscle and epididymal fat mass without altering tumour growth. Consequently, median survival increased by 23.3%. Mechanistically, omeprazole increased cancer cell endolysosomal pH level dose-dependently (0.1 to 1 µM) by inhibiting vacuolar H+ -ATPase. Further, omeprazole suppressed the highly elevated expression of Rab27b, a key regulator of EV release, in LLC cells. CONCLUSIONS: Omeprazole ameliorates cancer cachexia by inhibiting cancer cell release of Hsp70 and Hsp90.

Proton pump inhibitors interfere with the anti-tumor potency of RC48ADC.

Antibody-drug conjugates (ADCs) are a promising modality for cancers, but the interaction between them and proton pump inhibitors (PPIs), the common adjuvant drugs for cancer treatment, has not been understood. Here, the interactions between PPIs and RC48ADC, a novel HER2-targeting ADC, were quantified in vitro. CCK-8 assay showed that RC48ADC displayed a significant inhibitory effect on the proliferation of SK-BR-3, NCI-N87 and SK-OV-3 cells with the IC50 values of 4.91 ± 1.15 ng/mL, 14.54 ± 0.85 ng/mL and 11.28 ± 0.68 ng/mL respectively. PPIs alone had no significant anti-tumor effect in the dose range of 1.37-1000 ng/mL. When used together, PPIs inhibited the anti-tumor activity of RC48ADC in a dose-dependent manner. And 1000 ng/mL (~Cmax) PPIs significantly recovered RC48ADC-inhibited cell proliferation by (32.85 ± 2.81) % (p < 0.05). However, cimetidine, a non-PPIs gastric acid secretion inhibitor, had no significant inhibitory effect on RC48ADC. Furthermore, omeprazole, rather than cimetidine, significantly reduced the activity of vacuolar H+-ATPase and Cathepsin B compared with the control cells. These results, if confirmed in vivo, indicate that PPIs are antagonists of RC48ADC, even all ADCs, appearing to be due to inhibition of vacuolar H+-ATPase activity. Moreover, cimetidine combined with ADCs instead of PPIs can prevent an adverse drug interaction.

V-ATPase subunit a is required for survival and midgut development of Locusta migratoria.

The vacuolar-type H+ -ATPase (V-ATPase) is an ATP-dependent proton pump, which regulates various cellular processes. To date, most functional studies on V-ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV-ATPase a were identified in Locusta migratoria. RT-qPCR analysis revealed that LmV-ATPase a1 and LmV-ATPase a2 are differentially expressed in various tissues and developmental stages. Injection of dsRNA for the common region of LmV-ATPase a1 and LmV-ATPase a2 into third-instar nymphs resulted in a significant suppression of LmV-ATPase a. The injected nymphs ceased feeding, lost body weight and finally died at a mortality of 98.6%. Furthermore, aberrations of midgut epithelial cells, the accumulation of electron-lucent vesicles in the cytoplasm, and a partially damaged brush border were observed in dsLmV-ATPase a-injected nymphs using transmission electron microscopy. Especially, the mRNA level of wingles, and notch genes were dramatically down-regulated in the dsLmV-ATPase a-injected nymphs. Taken together, our results suggest that LmV-ATPase a is required for survival and midgut development of L. migratoria. Hence, this gene could be a good target for RNAi-based control against locusts.

Elevated (Pro)renin Receptor Expression by Anti-Cancer Drugs, Carboplatin and Paclitaxel, in Cultured Cancer Cells: Possible Involvement of Apoptosis and Autophagy.

(Pro)renin receptor [(P)RR] is a component of the renin-angiotensin system and plays an essential role in the activity of vacuolar H+-ATPase and autophagy. (P)RR is expressed in cancer cells. However, the relationship among (P)RR, apoptosis and autophagy in the treatment of anti-cancer drugs has not been clarified. The aim of this study was to clarify the effects of anti-cancer drugs with autophagy-promoting activity on (P)RR expression in cancer cells. MCF-7 breast cancer cells and A549 lung cancer cells were treated with carboplatin or paclitaxel, and the expression of (P)RR, apoptosis markers and autophagy markers were assessed by RT-qPCR, western blot analysis and immunocytochemistry. Expression levels of (P)RR mRNA and soluble (P)RR protein were increased by carboplatin or paclitaxel in a dose-dependent manner. Immunofluorescence staining of (P)RR was increased in both MCF-7 and A549 cells treated by carboplatin or paclitaxel. Apoptosis induction was shown by elevated BAX/BCL2 mRNA levels and increased active caspase3-positive cells. Moreover, autophagy induction was confirmed by increased levels of autophagy-associated mRNAs and LC3B-II proteins. (P)RR knockdown by (P)RR-specific siRNA suppressed the cell viability in MCF-7 cells and A549 cells under the treatment of carboplatin or paclitaxel, suggesting that (P)RR deficiency inhibits the proliferation of cancer cells in a pathway different from carboplatin or paclitaxel. The present study showed that the expression of (P)RR mRNA and soluble (P)RR was increased by anti-cancer drugs with autophagy-promoting activity. Upregulated (P)RR and autophagy may constitute a stress adaptation that protects cancer cells from apoptosis.

Plasma Membrane Receptors Involved in the Binding and Response of Osteoclasts to Noncellular Components of the Bone.

Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell-cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and other bone cells interact with the bone has emerged, increasingly sophisticated efforts have been made to create bone biomimetics that reproduce both the structural properties of the bone and the bone's ability to regulate osteoclasts and other bone cells. A more complete understanding of the interactions between osteoclasts and the bone may lead to new strategies for the treatment of bone diseases and the production of bone biomimetics to repair defects.

Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.

OBJECTIVE: The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H+-ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal deacidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal deacidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance, and contractile dysfunction. Hence, the v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves the binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for the activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally; specifically, whether AA induces v-ATPase reassembly in a mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart. METHODS: Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity, and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation, and coimmunoprecipitation. RESULTS: In lipid-overexposed cardiomyocytes, lysine/leucine/arginine reinternalized CD36 to the endosomes, prevented/reversed lipid accumulation, preserved/restored insulin sensitivity, and contractile function. These beneficial AA actions required the mTORC1-v-ATPase axis, adaptor protein Ragulator, and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes and partially reversed cardiac hypertrophy. CONCLUSION: Specific AAs acting through v-ATPase reassembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.

Purification of active human vacuolar H+-ATPase in native lipid-containing nanodiscs.

Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme's isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain-containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.

Novel vertebrate- and brain-specific driver of neuronal outgrowth.

During the process of neuronal outgrowth, developing neurons produce new projections, neurites, that are essential for brain wiring. Here, we discover a relatively late-evolved protein that we denote Ac45-related protein (Ac45RP) and that, surprisingly, drives neuronal outgrowth. Ac45RP is a paralog of the Ac45 protein that is a component of the vacuolar proton ATPase (V-ATPase), the main pH regulator in eukaryotic cells. Ac45RP mRNA expression is brain specific and coincides with the peak of neurogenesis and the onset of synaptogenesis. Furthermore, Ac45RP physically interacts with the V-ATPase V0-sector and colocalizes with V0 in unconventional, but not synaptic, secretory vesicles of extending neurites. Excess Ac45RP enhances the expression of V0-subunits, causes a more elaborate Golgi, and increases the number of cytoplasmic vesicular structures, plasma membrane formation and outgrowth of actin-containing neurites devoid of synaptic markers. CRISPR-cas9n-mediated Ac45RP knockdown reduces neurite outgrowth. We conclude that the novel vertebrate- and brain-specific Ac45RP is a V0-interacting constituent of unconventional vesicular structures that drives membrane expansion during neurite outgrowth and as such may furnish a tool for future neuroregenerative treatment strategies.

Vacuolar H(+)-ATPase is not restricted to clear cells of the epididymal epithelium in cattle.

Communication among epididymal epithelial cells creates the best luminal condition where spermatozoa mature, transport and are stored. Vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) have been used as signal indicators for clear and basal cells of the epididymal epithelium, respectively, in mice, rats, bats, and pigs; however, these two markers have not yet been described in the epididymis of bulls. Here, we examined the presence and distribution of the B1 subunit of V-ATPase (B1-VATPase) and KRT5 in the distinct regions of adult bovine epididymides, specifically, the caput, corpus, and cauda. Immunofluorescence staining and confocal microscopy showed that narrow shaped-clear cells were placed in the caput and corpus regions of the bovine epididymis; however, they were absent in the cauda epididymis. In addition, B1-VATPase was highly expressed in the cauda spermatozoa; however, it was rarely detected in the caput spermatozoa. On the other hand, KRT5-positive cells, basal cells, were maintained beneath the basal lamina and they had the traditional form with a dome-shaped morphology from the caput to cauda region of the bovine epididymis. The co-expression of B1-VATPase and KRT5 was confined to basal cells placed in the basal region of the epithelium. In summary, 1) clear cells were present with region-specific localization, 2) B1-VATPase was present in the corpus and cauda spermatozoa but absent in the caput, 3) co-expressed cells with B1-VATPase and KRT5 were present in the adult bovine epididymis, and 4) B1-VATPase was not a specific marker for clear cells in the bovine epididymis. Therefore, the perfect epididymal luminal condition created by the specific expression and localization patterns of B1-VATPase might be necessary to obtain fertilizing capacity of spermatozoa in the bovine epididymis.