Purification and cryoelectron microscopy structure determination of human V-ATPase.

Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps, which play important roles in many physiological processes by acidifying intracellular vesicles, organelles, and the extracellular milieu. Long-standing challenges in purifying mammalian V-ATPases have limited the biochemical and structural study of mammalian V-ATPase. Here, we provide a protocol for purifying milligrams of human V-ATPase and detail procedures for the reconstruction of its structure by cryo-EM. Our method can be applied to any biochemical and biophysical study of human V-ATPase. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).

ATP6V0d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis.

V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V0d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V0d2 in the biogenesis of pathogen-containing vacuoles using ATP6V0d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V0d2 upregulation. ATP6V0d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V0d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V0d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche.

Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2α-mediated tumor progression.

Macrophages perform key functions in tissue homeostasis that are influenced by the local tissue environment. Within the tumor microenvironment, tumor-associated macrophages can be altered to acquire properties that enhance tumor growth. Here, we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, activated mTORC1 that subsequently suppressed TFEB-mediated expression of the macrophage-specific vacuolar ATPase subunit ATP6V0d2. Atp6v0d2-/- mice were more susceptible to tumor growth, with enhanced HIF-2α-mediated VEGF production in macrophages that display a more protumoral phenotype. We found that ATP6V0d2 targeted HIF-2α but not HIF-1α for lysosome-mediated degradation. Blockade of HIF-2α transcriptional activity reversed the susceptibility of Atp6v0d2-/- mice to tumor development. Furthermore, in a cohort of patients with lung adenocarcinoma, expression of ATP6V0d2 and HIF-2α was positively and negatively correlated with survival, respectively, suggesting a critical role of the macrophage lactate/ATP6V0d2/HIF-2α axis in maintaining tumor growth in human patients. Together, our results highlight the ability of tumor cells to modify the function of tumor-infiltrating macrophages to optimize the microenvironment for tumor growth.

Expression and Transcriptional Regulation of Human ATP6V1A Gene in Gastric Cancers.

Recent studies demonstrate that the invasion and metastasis of gastric cancer (GC) is closely associated with a multi-subunit vacuolar H+-ATPase (V-ATPase). Here we investigated the expression and role of the human ATP6V1A gene that encodes the catalytic subunit A of V-ATPase in GC. We found that ATP6V1A expression level is significantly elevated in GCs compared to normals, but GC patients with higher expression levels of ATP6V1A have a better prognosis. Genomic analysis revealed that APT6V1A copy number is gained in a small fraction of GC patients and lost in a minimum number. Moreover, the ATP6V1A copy number was positively correlated with its mRNA level. To explore additional mechanisms by which ATP6V1A overexpressed in GCs, we investigated the relationship between transcription factor YY1 and ATP6V1A, and found that mRNA expression of YY1 had significant correlation with that of ATP6V1A. To validate that YY1 transcriptionally regulates ATP6V1A, we discovered that the ATP6V1A core promoter region contains three YY1 binding sites. Moreover, RNAi-mediated knockdown of YY1 in GC cells significantly decreased ATP6V1A mRNA and protein expression, while YY1 overexpression increased ATP6V1A expression level. In conclusion, YY1 may play an important regulatory role in ATP6V1A expression with potential mechanistic and clinical implications in GC.

(Pro)renin receptor is crucial for glioma development via the Wnt/ß-catenin signaling pathway.

OBJECTIVE The (pro)renin receptor (PRR) plays an essential role in the early development of the central nervous system by activating the Wnt/ß-catenin signaling pathway. The authors investigated the potential role of the PRR in the pathogenesis of glioma. METHODS The authors performed immunohistochemical analysis to detect both the PRR and isocitrate dehydrogenase 1 with mutations involving arginine 132 ( IDH1R132H) in paraffin sections of 31 gliomas. Expression of the PRR and Wnt pathway components in cultured human glioma cell lines (U251MG, U87MG, and T98G) was measured using Western blotting. The effects of PRR short interfering RNA (siRNA) on glioma cell proliferation (WST-1 assay and direct cell counting) and apoptosis (flow cytometry and the caspase-3 assay) were also examined. RESULTS PRR expression was significantly higher in glioblastoma than in normal tissue or in lower grade glioma, regardless of IDH1R132H mutation. PRR expression was also higher in human glioblastoma cell lines than in human astrocytes. PRR expression showed a significant positive correlation with the Ki-67 labeling index, while it had a significant negative correlation with the survival time of glioma patients. Treatment with PRR siRNA significantly reduced expression of Wnt2, activated ß-catenin, and cyclin D1 by human glioblastoma cell lines, and it reduced the proliferative capacity of these cell lines and induced apoptosis. CONCLUSIONS This is the first evidence that the PRR has an important role in development of glioma by aberrant activation of the Wnt/ß-catenin signaling pathway. This receptor may be both a prognostic marker and a therapeutic target for glioma.

Up-regulation of Vps4A promotes neuronal apoptosis after intracerebral hemorrhage in adult rats.

Vps4, vacuolar protein sorting 4, belongs to ATPases Associated with diverse cellular Activities (AAA) protein family which is made up of Vps4A and Vps4B. Previous studies demonstrated that Vps4A plays vital roles in diverse aspects such as virus budding, the efficient transport of H-Ras to the PM (plasma membrane) and the involvement in the MVB (multivesiculate bodies) pathway. Interestingly, Vps4A is also expressed in the brain. However, the distribution and function of Vps4A in ICH diseases remain unclear. In this study, we show that Vps4A may be involved in neuronal apoptosis during pathophysiological processes of intracerebral hemorrhage (ICH). Based on the results of Western blot and immunohistochemistry, we found a remarkable up-regulation of Vps4A expression surrounding the hematoma after ICH. Double labeled immunofluorescence showed that Vps4A was co-expressed with NeuN but rarely with astrocytes and microglia. Morever, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with Vps4A. Additionaly, Vps4A knockdown in vitro specifically leads to decreasing neuronal apoptosis coupled with increased Akt phosphorylation. All datas suggested that Vps4A was involved in promoting neuronal apoptosis via inhibiting Akt phosphorylation after ICH.

Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy.

Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34+ cells to IMO CD34+ patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis.

Bovine parathyroid hormone enhances osteoclast bone resorption by modulating V-ATPase through PTH1R.

The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose­dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling.

Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 µm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

BSND and ATP6V1G3: Novel Immunohistochemical Markers for Chromophobe Renal Cell Carcinoma.

Differentiating between chromophobe renal cell carcinoma (RCC) and other RCC subtypes can be problematic using routine light microscopy. This study aimed to identify novel immunohistochemical markers useful for a differential diagnosis between chromophobe RCC and other RCC subtypes. We selected 3 genes (including BSND and ATP6V1G3) that showed specific transcriptional expression in chromophobe RCC using expression data (n = 783) from The Cancer Genome Atlas (TCGA) database. A subsequent immunohistochemical examination of 186 RCCs obtained in our patient series resulted in a strong diffuse positivity of BSND and ATP6V1G3 proteins (both of which are involved in the regulation of membrane transport) in all the chromophobe RCC specimens (23/23 cases, 100%) but not in the clear cell RCC specimens (0/153 cases, 0%) or the papillary RCC specimens (0/10 cases, 0%). BSND and ATP6V1G3 protein expressions were also detected in renal oncocytoma (13/14 cases, 92.9%) and in the distal nephron, including the collecting duct, in the normal kidney. A computational analysis of TCGA data suggested that DNA methylation was involved in the differential expression pattern of both genes among RCC subtypes. Finally, an immunohistochemical analysis showed lung carcinomas were negative (0/85 cases, 0%) for the expression of both proteins. These results suggest that BSND and ATP6V1G3 are excellent novel immunohistochemical markers for differentiating between chromophobe RCC and other subtypes of RCC, including clear cell and papillary RCCs.

Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells.

The hormonally active form of vitamin D3, 1α,25-(OH)2D3, has an important role in bone metabolism. This study examined the effects of 1α,25-(OH)2D3 on the ability of two cytokines, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1α,25-(OH)2D3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin ß3, V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATc1 also increased 24h after treatment with 1α,25-(OH)2D3. These results suggest that 1α,25-(OH)2D3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts.

Increased expression of T cell immune response cDNA 7 in patients with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.

LASS2/TMSG1 inhibits growth and invasion of breast cancer cell in vitro through regulation of vacuolar ATPase activity.

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2)/tumor metastasis suppressor gene 1 (TMSG1) was a novel tumor metastasis-related gene identified using messenger RNA differential display from non-metastatic human prostate cancer cell variants. The mechanism of LASS2/TMSG1 inhibiting tumor invasion metastasis in breast cancer cells had not been well investigated. In the present study, a full length of 1.2 kb LASS2/TMSG1 complementary DNA (cDNA) coding for a protein of 380 amino acids was cloned. PcDNA3 eukaryotic expression plasmids of LASS2/TMSG1 were constructed and transfected into human breast cancer cell line MCF-7 by lipofectin transfection method. And, the biological effects were observed comparing with control groups. As the result, LASS2/TMSG1 inhibited cell growth in vitro by increasing apoptosis and changing cell cycle distribution. Furthermore, the vacuolar ATPase (V-ATPase) activity and extracellular hydrogen ion concentration were significantly decreased and the activity of secreted matrix metalloproteinase-2 (MMP-2) was downregulated in MCF-7 cells overexpressing LASS2/TMSG1 compared with the controls. Therefore, LASS2/TMSG1 may inhibit growth and invasion of breast cancer cell in vitro through decreasing V-ATPase activity and extracellular hydrogen ion concentration and inactivating secreted MMP-2. The findings provided the evidence that the LASS2/TMSG1 gene had tumor growth and invasion suppressor function in human breast cancer cell and may provide a promising target for cancer metastasis diagnosis and therapy.

Involvement of the receptor-associated prorenin system in the pathogenesis of human conjunctival lymphoma.

PURPOSE: Extranodal marginal zone B-cell lymphoma (EMZL) is the most common subtype of conjunctival lymphoma, though its molecular mechanisms of pathogenesis are largely unknown. We attempted to explore the association of the renin-angiotensin system (RAS) and (pro)renin receptor ([P]RR) in the pathogenesis of conjunctival lymphoma. METHODS: Surgically removed conjunctiva EMZL samples were used for gene expression, and immunohistochemical and immunofluorescence analyses of (P)RR and RAS components. Human B-lymphoblast IM-9 cells were treated with prorenin or angiotensin II (Ang II), and gene expression levels were analyzed using real-time quantitative PCR (qPCR). In addition, immunofluorescence analysis of EMZL samples was used to evaluate the in vivo expression of those components. RESULTS: Gene expression and immunohistochemical analyses revealed the expression of RAS components, including (P)RR and angiotensin II type 1 receptor (AT1R), in EMZL tissues. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for CD20, a marker for B-cells, where they colocalized with prorenin and angiotensinogen, respectively. Prorenin stimulation of human B-lymphoblast IM-9 cells increased mRNA expression levels of fibroblast growth factor 2 (FGF2), while angiotensin II treatment upregulated the expression levels of basigin (BSG), matrix metallopeptidase (MMP)2, 9, and 14, which were abolished by (P)RR and AT1R blockades, respectively. Immunofluorescence analyses of clinical samples showed colocalizations of (P)RR and AT1R with the products of these upregulated genes. CONCLUSIONS: The present study suggests that activation of (P)RR and AT1R is associated with the pathogenesis of conjunctival EMZL by stimulating the production of FGF2 and MMPs.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling. METHODS: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment. RESULTS: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx 2 were up-regulated, while GSK-3ß was down-regulated after HBO treatment. Western blot analysis showed an increased ß-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. CONCLUSIONS: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

The signaling lipid PI(3,5)P2 stabilizes V1-V(o) sector interactions and activates the V-ATPase.

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane V(o) domain. Multiple stresses induce changes in V1-V(o) assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-V(o) assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified V(o) complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of V(o)-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-V(o) interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-V(o) assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1 and vac14 mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.

Silencing of a novel tumor metastasis suppressor gene LASS2/TMSG1 promotes invasion of prostate cancer cell in vitro through increase of vacuolar ATPase activity.

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. Thus, in this study, we explored the effect of small interference RNA (siRNA) targeting LASS2/TMSG1 on the invasion of human prostate carcinoma cell line PC-3M-2B4 and its molecular mechanisms associated with the V-ATPase. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot revealed dramatic reduction of 84.5% and 60% in the levels of LASS2/TMSG1 mRNA and protein after transfection of siRNA in PC-3M-2B4 cells. The V-ATPase activity and extracellular hydrogen ion concentration were significantly increased in 2B4 cells transfected with the LASS2/TMSG1-siRNA compared with the controls. The activity of secreted MMP-2 was up-regulated in LASS2/TMSG1-siRNA treated cells compared with the controls; and the capacity for migration and invasion in LASS2/TMSG1-siRNA treated cells was significantly higher than the controls. Thus, we concluded that silencing of LASS2/TMSG1 may promote invasion of prostate cancer cell in vitro through increase of V-ATPase activity and extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2.

The expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis?

The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin II type 1 and 2 receptors (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-ß1 (TGF-ß1), in early gestation (6-16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-ß1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT(1)R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.

Regulation of TFEB and V-ATPases by mTORC1.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed.