Proton-driven sodium secretion in a saline water animal.

Aquatic animals residing in saline habitats either allow extracellular sodium concentration to conform to environmental values or regulate sodium to lower levels. The latter strategy requires an energy-driven process to move sodium against a large concentration gradient to eliminate excess sodium that diffuses into the animal. Previous studies of invertebrate and vertebrate species indicate a sodium pump, Na+/K+ ATPase, powers sodium secretion. We provide the first functional evidence of a saline-water animal, Aedes taeniorhynchus mosquito larva, utilizing a proton pump to power this process. Vacuolar-type H+ ATPase (VHA) protein is highly expressed on the apical membrane of the posterior rectal cells, and in situ sodium flux across this epithelium increases significantly in larvae held in higher salinity and is sensitive to Bafilomycin A1, an inhibitor of VHA. We also report the first evidence of splice variants of the sodium/proton exchanger, NHE3, with both high and low molecular weight variants highly expressed on the apical membrane of the posterior rectal cells. Evidence of NHE3 function was indicated with in situ sodium transport significantly inhibited by a NHE3 antagonist, S3226. We propose that the outward proton pumping by VHA establishes a favourable electromotive gradient to drive sodium secretion via NHE3 thus producing a hyperosmotic, sodium-rich urine. This H+- driven Na+ secretion process is the primary mechanism of ion regulation in salt-tolerant culicine mosquito species and was first investigated over 80 years ago.

Plasma exosomes impair microglial degradation of α-synuclein through V-ATPase subunit V1G1.

INTRODUCTION: Microglia are the main phagocytes in the brain and can induce neuroinflammation. Moreover, they are critical to alpha-synuclein (α-syn) aggregation and propagation. Plasma exosomes derived from patients diagnosed with Parkinson's disease (PD-exo) reportedly evoked α-syn aggregation and inflammation in microglia. In turn, microglia internalized and released exosomal α-syn, enhancing α-syn propagation. However, the specific mechanism through which PD-exo influences α-syn degradation remains unknown. METHODS: Exosomes were extracted from the plasma of patients with PD by differential ultracentrifugation, analyzed using electron microscopy (EM) and nanoparticle flow cytometry, and stereotaxically injected into the unilateral striatum of the mice. Transmission EM was employed to visualize lysosomes and autophagosomes in BV2 cells, and lysosome pH was measured with LysoSensor Yellow/Blue DND-160. Cathepsin B and D, lysosomal-associated membrane protein 1 (LAMP1), ATP6V1G1, tumor susceptibility gene 101 protein, calnexin, α-syn, ionized calcium binding adaptor molecule 1, and NLR family pyrin domain containing 3 were evaluated using quantitative polymerase chain reaction or western blotting, and α-syn, LAMP1, and ATP6V1G1 were also observed by immunofluorescence. Small interfering ribonucleic acid against V1G1 was transfected into BV2 cells and primary microglia using Lipofectamine® 3000. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. A lentiviral-mediated strategy to overexpress ATP6V1G1 in the brain of MPTP-treated mice was employed. Motor coordination was assessed using rotarod and pole tests, and neurodegeneration in the mouse substantia nigra and striatum tissues was determined using immunofluorescence histochemical and western blotting of tyrosine hydroxylase. RESULTS: PD-exo decreased the expression of V1G1, responsible for the acidification of intra- and extracellular milieu. This impairment of lysosomal acidification resulted in the accumulation of abnormally swollen lysosomes and decreased lysosomal enzyme activities, impairing lysosomal protein degradation and causing α-syn accumulation. Additionally, V1G1 overexpression conferred the mice neuroprotection during MPTP exposure. CONCLUSION: Pathogenic protein accumulation is a key feature of PD, and compromised V-type ATPase dysfunction might participate in PD pathogenesis. Moreover, V1G1 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which may provide opportunities to develop novel therapeutic interventions for PD treatment.

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

ATP6AP2, a regulator of LRP6/ß-catenin protein trafficking, promotes Wnt/ß-catenin signaling and bone formation in a cell type dependent manner.

Wnt/ß-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/ß-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/ß-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired ß-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of ß-catenin phosphorylation, but necessary for LRP6/ß-catenin and N-cadherin/ß-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active ß-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating ß-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/ß-catenin signaling and trabecular bone formation.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

Nanoformulation of the Broad-Spectrum Hydrophobic Antiviral Vacuolar ATPase Inhibitor Diphyllin in Human Recombinant H-ferritin.

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.

miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway as a critical route affecting the toxic effects of lead on HK-2 cells.

Lead is a widespread environmental pollutant with serious adverse effects on human health, but the mechanism underlying its toxicity remains elusive. This study aimed to investigate the role of miR-584-5p / Ykt6 axis in the toxic effect of lead on HK-2 cells and the related mechanism. Our data suggested that lead exposure caused significant cytotoxicity, DNA and chromosome damage to HK-2 cells. Mechanistically, lead exposure down-regulated miR-584-5p and up-regulated Ykt6 expression, consequently, autophagosomal number and autophagic flux increased, lysosomal number and activity decreased, exosomal secretion increased. Interestingly, when miR-584-5p level was enhanced with mimic, autophagosomal number and autophagic flux decreased, lysosomal number and activity increased, ultimately, exosomal secretion was down-regulated, which resulted in significant aggravated toxic effects of lead. Further, directly blocking exosomal secretion with inhibitor GW4869 also resulted in exacerbated toxic effects of lead. Herein, we conclude that miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway may be a critical route affecting the toxic effects of lead on HK-2 cells. We provide a novel insight into the mechanism underlying the toxicity of lead on human cells.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase.

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex.

Lysosomal dysfunction by inactivation of V-ATPase drives innate immune response in C. elegans.

Pathogens target vacuolar ATPase (V-ATPase) to inhibit lysosomal acidification or lysosomal fusion, causing lysosomal dysfunction. However, it remains unknown whether cells can detect dysfunctional lysosomes and initiate an immune response. In this study, we discover that dysfunction of lysosomes caused by inactivation of V-ATPase enhances innate immunity against bacterial infections. We find that lysosomal V-ATPase interacts with DVE-1, whose nuclear localization serves as a proxy for the induction of mitochondrial unfolded protein response (UPRmt). The inactivation of V-ATPase promotes the nuclear localization of DVE-1, activating UPRmt and inducing downstream immune response genes. Furthermore, pathogen resistance conferred by inactivation of V-ATPase requires dve-1 and its downstream immune effectors. Interestingly, animals grow slower after vha RNAi, suggesting that the vha-RNAi-induced immune response costs the most energy through activation of DVE-1, which trades off with growth. This study reveals how dysfunctional lysosomes can trigger an immune response, emphasizing the importance of conserving energy during immune defense.

The A subunit of vacuolar H+-ATPase gene (CmVHA-A) plays opposite roles in plant growth and drought tolerance of chrysanthemum under different growing conditions.

As the most prominent proton pumps in plants, vacuolar H+-ATPases (VHAs) comprise multiple subunits that are important for physiological processes and stress tolerance in plants. However, few studies on the roles of subunit genes of VHAs in chrysanthemum have been reported to date. In this study, the gene of A subunit of V-ATPase in chrysanthemum (CmVHA-A) was cloned and identified. CmVHA-A was conserved with VHA-A proteins from other plants. Expression analysis showed that CmVHA-A was highly expressed in most tissues of chrysanthemum except for the flower bud, and was readily induced by polyethylene glycol (PEG) treatment. Functional analysis demonstrated that CmVHA-A exerted a negative influence on the growth and development of shoot and root of chrysanthemum under normal conditions. RNA-sequencing (RNA-seq) analysis revealed the possible explanations for phenotypic differences between transgenic and wild-type (WT) plants. Under drought conditions, CmVHA-A positively affected the drought tolerance of chrysanthemum by enhancing antioxidase activity and alleviating photosynthetic disruption. Overall, CmVHA-A plays opposite roles in plant growth and drought tolerance of chrysanthemums under different growing conditions.

Increased V-ATPase activity can lead to chemo-resistance in oral squamous cell carcinoma via autophagy induction: new insights.

Oral squamous cell carcinoma (OSCC) is a cancer type with a high rate of recurrence and a poor prognosis. Tumor chemo-resistance remains an issue for OSCC patients despite the availability of multimodal therapy options, which causes an increase in tumor invasiveness. Vacuolar ATPase (V-ATPase), appears to be one of the most significant molecules implicated in MDR in tumors like OSCC. It is primarily responsible for controlling the acidity in the solid tumors' microenvironment, which interferes with the absorption of chemotherapeutic medications. However, the exact cellular and molecular mechanisms V-ATPase plays in OSCC chemo-resistance have not been understood. Uncovering these mechanisms can contribute to combating OSCC chemo-resistance and poor prognosis. Hence, in this review, we suggest that one of these underlying mechanisms is autophagy induced by V-ATPase which can potentially contribute to OSCC chemo-resistance. Finally, specialized autophagy and V-ATPase inhibitors may be beneficial as an approach to reduce drug resistance to anticancer therapies in addition to serving as coadjuvants in antitumor treatments. Also, V-ATPase could be a prognostic factor for OSCC patients. However, in the future, more investigations are required to demonstrate these suggestions and hypotheses.

The soluble (pro)renin receptor promotes a preeclampsia-like phenotype both in vitro and in vivo.

Preeclampsia is classified as new-onset hypertension coupled with gross endothelial dysfunction. Placental (pro)renin receptor ((P)RR) and plasma soluble (P)RR (s(P)RR) are elevated in patients with preeclampsia. Thus, we aimed to interrogate the role (P)RR may play in the pathogenesis of preeclampsia. Human uterine microvascular endothelial cells (HUtMECs, n = 4) were cultured with either; vehicle (PBS), 25-100 nM recombinant s(P)RR, or 10 ng/ml TNF-a (positive control) for 24 h. Conditioned media and cells were assessed for endothelial dysfunction markers via qPCR, ELISA, and immunoblot. Angiogenic capacity was assessed through tube formation and adhesion assays. Additionally, pregnant rats were injected with an adenovirus overexpressing s(P)RR from mid-pregnancy (day 8.5), until term (n = 6-7 dams/treatment). Maternal and fetal tissues were assessed. HUtMECs treated with recombinant s(P)RR displayed increased expression of endothelial dysfunction makers including vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and endothelin-1 mRNA expression (P = 0.003, P = 0.001, P = 0.009, respectively), along with elevated endothelin-1 protein secretion (P < 0.001) compared with controls. Recombinant s(P)RR impaired angiogenic capacity decreasing the number of branches, total branch length, and mesh area (P < 0.001, P = 0.004, and P = 0.009, respectively), while also increasing vascular adhesion (P = 0.032). +ADV rats exhibited increased systolic (P = 0.001), diastolic (P = 0.010), and mean arterial pressures (P = 0.012), compared with -ADV pregnancies. Renal arteries from +ADV-treated rats had decreased sensitivity to acetylcholine-induced relaxation (P = 0.030), compared with -ADV pregnancies. Our data show that treatment with s(P)RR caused hypertension and growth restriction in vivo and caused marked endothelial dysfunction in vitro. These findings demonstrate the significant adverse actions of s(P)RR on vascular dysfunction that is characteristic of the preeclamptic phenotype.

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.

Ammonia transport in the excretory system of mosquito larvae (Aedes aegypti): Rh protein expression and the transcriptome of the rectum.

The role of the mosquito excretory organs (Malpighian tubules, MT and hindgut, HG) in ammonia transport as well as expression and function of the Rhesus (Rh protein) ammonia transporters within these organs was examined in Aedes aegypti larvae and adult females. Immunohistological examination revealed that the Rh proteins are co-localized with V-type H+-ATPase (VA) to the apical membranes of MT and HG epithelia of both larvae and adult females. Of the two Rh transporter genes present in A. aegypti, AeRh50-1 and AeRh50-2, we show using quantitative real-time PCR (qPCR) and an RNA in-situ hybridization (ISH) assay that AeRh50-1 is the predominant Rh protein expressed in the excretory organs of larvae and adult females. Further assessment of AeRh50-1 function in larvae and adults using RNAi (i.e. dsRNA-mediated knockdown) revealed significantly decreased [NH4+] (mmol l-1) levels in the secreted fluid of larval MT which does not affect overall NH4+ transport rates, as well as significantly decreased NH4+ flux rates across the HG (haemolymph to lumen) of adult females. We also used RNA sequencing to identify the expression of ion transporters and enzymes within the rectum of larvae, of which limited information currently exists for this important osmoregulatory organ. Of the ammonia transporters in A. aegypti, AeRh50-1 transcript is most abundant in the rectum thus validating our immunohistochemical and RNA ISH findings. In addition to enriched VA transcript (subunits A and d1) in the rectum, we also identified high Na+-K+-ATPase transcript (α subunit) expression which becomes significantly elevated in response to HEA, and we also found enriched carbonic anhydrase 9, inwardly rectifying K+ channel Kir2a, and Na+-coupled cation-chloride (Cl-) co-transporter CCC2 transcripts. Finally, the modulation in excretory organ function and/or Rh protein expression was examined in relation to high ammonia challenge, specifically high environmental ammonia (HEA) rearing of larvae. NH4+ flux measurements using the scanning-ion selective electrode (SIET) technique revealed no significant differences in NH4+ transport across organs comprising the alimentary canal of larvae reared in HEA vs freshwater. Further, significantly increased VA activity, but not NKA, was observed in the MT of HEA-reared larvae. Relatively high Rh protein immunostaining persists within the hindgut epithelium, as well as the ovary, of females at 24-48 h post blood meal corresponding with previously demonstrated peak levels of ammonia formation. These data provide new insight into the role of the excretory organs in ammonia transport physiology and the contribution of Rh proteins in mediating ammonia movement across the epithelia of the MT and HG, and the first comprehensive examination of ion transporter and channel expression in the mosquito rectum.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Myocardin reverses insulin resistance and ameliorates cardiomyopathy by increasing IRS-1 expression in a murine model of lipodystrophy caused by adipose deficiency of vacuolar H+-ATPase V0d1 subunit.

Aim: Adipose tissue (AT) dysfunction that occurs in both obesity and lipodystrophy is associated with the development of cardiomyopathy. However, it is unclear how dysfunctional AT induces cardiomyopathy due to limited animal models available. We have identified vacuolar H+-ATPase subunit Vod1, encoded by Atp6v0d1, as a master regulator of adipogenesis, and adipose-specific deletion of Atp6v0d1 (Atp6v0d1AKO) in mice caused generalized lipodystrophy and spontaneous cardiomyopathy. Using this unique animal model, we explore the mechanism(s) underlying lipodystrophy-related cardiomyopathy. Methods and Results: Atp6v0d1AKO mice developed cardiac hypertrophy at 12 weeks, and progressed to heart failure at 28 weeks. The Atp6v0d1AKO mouse hearts exhibited excessive lipid accumulation and altered lipid and glucose metabolism, which are typical for obesity- and diabetes-related cardiomyopathy. The Atp6v0d1AKO mice developed cardiac insulin resistance evidenced by decreased IRS-1/2 expression in hearts. Meanwhile, the expression of forkhead box O1 (FoxO1), a transcription factor which plays critical roles in regulating cardiac lipid and glucose metabolism, was increased. RNA-seq data and molecular biological assays demonstrated reduced expression of myocardin, a transcription coactivator, in Atp6v0d1AKO mouse hearts. RNA interference (RNAi), luciferase reporter and ChIP-qPCR assays revealed the critical role of myocardin in regulating IRS-1 transcription through the CArG-like element in IRS-1 promoter. Reducing IRS-1 expression with RNAi increased FoxO1 expression, while increasing IRS-1 expression reversed myocardin downregulation-induced FoxO1 upregulation in cardiomyocytes. In vivo, restoring myocardin expression specifically in Atp6v0d1AKO cardiomyocytes increased IRS-1, but decreased FoxO1 expression. As a result, the abnormal expressions of metabolic genes in Atp6v0d1AKO hearts were reversed, and cardiac dysfunctions were ameliorated. Myocardin expression was also reduced in high fat diet-induced diabetic cardiomyopathy and palmitic acid-treated cardiomyocytes. Moreover, increasing systemic insulin resistance with rosiglitazone restored cardiac myocardin expression and improved cardiac functions in Atp6v0d1AKO mice. Conclusion: Atp6v0d1AKO mice are a novel animal model for studying lipodystrophy- or metabolic dysfunction-related cardiomyopathy. Moreover, myocardin serves as a key regulator of cardiac insulin sensitivity and metabolic homeostasis, highlighting myocardin as a potential therapeutic target for treating lipodystrophy- and diabetes-related cardiomyopathy.