Vacuolar Acidification Research Articles

Meg3 Induces EMT and Invasion of Glioma Cells via Autophagy.

BackgroundGlioma is one of the most common malignant tumors. Glioblastoma (grade IV) is considered the most malignant form of human brain tumors. Maternal expression gene 3 (Meg3) encodes a non-coding RNA (ncRNA) that plays an important role in the development and progression of cancer. However, the role of Meg3 in glioma cells remains largely unclear.MethodsReverse transcription-quantitative (RT-q) PCR was conducted to evaluate the mRNA expression related to cell autophagy and EMT while protein expression was detected by Western blotting. Staining of acidic vacuoles and immunofluorescence staining were used to detect autophagy. The ability of cells to migrate and invade was detected by Transwell migration and invasion assays.ResultsIn the present study, it was found that the overexpression of Meg3 induced EMT, migration and invasion of glioma cells, whereas Meg3 overexpression induced autophagy of glioma cells. More importantly, the inhibition of autophagy impaired the EMT of glioma cells. In addition, Meg3-induced EMT, migration and invasion could be partially reversed by autophagy inhibitors, chloroquine (CQ) and Lys05, in glioma cells.ConclusionAll data suggest that Meg3 induces EMT and invasion of glioma cells via autophagy. Overall, the findings of the present study demonstrate the importance of Meg3 in the molecular etiology of glioma, which also indicate its potential applications in the treatment of glioma.

Identification and impact of stable prognostic biochemical markers for cold-induced sweetening resistance on selection efficiency in potato (Solanum tuberosum L.) breeding programs

Biochemical markers for cold-induced sweetening (CIS) resistance were tested for their stability over years and their use in selection of parents for crossing to achieve high selection efficiency in potato breeding programs.Two regulatory enzymes directly associated with reducing sugar (RS) accumulation during potato tubers cold storage were tested as a predictor for CIS resistance. These enzymes were studied in 33 potato clones from various breeding programs over four years. Clones with the presence of A-II isozymes of UDP-glucose pyrophosphorylase (UGPase) and low activity of vacuolar acid invertase (VAcInv) enzyme had increased resistance to cold-induced sweetening (CIS). Depending on the levels of these enzymes, clones were divided into class A, class B and class C. Clones categorized as class A had average RS of 0.73 mg per g FW after six months at 5.5°C storage. Class B and C had average RS of 1.15 and 3.80 mg per g FW respectively. The enzyme activity was closely associated with RS accumulation over long-term cold storage.The biochemical markers were found to be stable over the years. Repeated-measure analysis showed 75% chance of maintaining class from one year to the next and a 25% chance of switching, No clone switched between class A and class C, even across all four years. Application of these biochemical markers can identify clones with CIS resistance early in the selection process. Biochemical markers were used to select parents for crossing and six families were established. Results showed that with both parents from class A, 95% of their offspring had desirable glucose levels and chip color, which dropped to 52% when one parent was from class A and other from class B. These results suggest that two regulatory enzymes, i.e., UGPase and VAcInv, can be used as stable prognostic biochemical markers for CIS resistance for precise parent selection resulting in progenies with significantly higher percentage of clones with acceptable processing quality.

Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

SummaryThe emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically-related current first-line combination drug piperaquine (PPQ) has now emerged regionally, thwarting its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum chloroquine resistance transporter PfCRT, a 49 kDa member of the drug/metabolite transporter (DMT) superfamily that traverses the membrane of the parasite’s acidic digestive vacuole (DV)3–9. Here we present the 3.2 Å structure of the PfCRT isoform from CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy (cryo-EM) and fragment antigen-binding (Fab) technology. Mutations contributing to CQ and PPQ resistance localize primarily to moderately-conserved sites on distinct helices lining a central negatively-charged cavity, implicating this as the principal site of interaction with positively-charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, with these drugs being mutually competitive. This isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism driving CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest distinct mechanistic features mediating CQ and PPQ resistance in PfCRT variants. These data provide the first atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

Expression of genes involved in phagocytosis in uncultured heterotrophic flagellates

AbstractEnvironmental molecular sequencing has revealed an abundance of microorganisms that were previously unknown, mainly because most had not been cultured in the laboratory. Within this novel diversity, there are the uncultured MAST clades (MArine STramenopiles), which are major components of marine heterotrophic flagellates (HFs) thought to be active bacterial grazers. In this study, we investigated the gene expression of natural HFs in a mixed community where bacterivory was promoted. Using fluorescence in situ hybridization and 18S rDNA derived from metatranscriptomics, we followed the taxonomic dynamics during the incubation, and confirmed the increase in relative abundance of different MAST lineages. We then used single cell genomes of several MAST species to gain an insight into their most expressed genes, with a particular focus on genes related to phagocytosis. The genomes of MAST‐4A and MAST‐4B were the most represented in the metatranscriptomes, and we identified highly expressed genes of these two species involved in motility and cytoskeleton remodeling, as well as many lysosomal enzymes. Particularly relevant were the cathepsins, which are characteristic digestive enzymes of the phagolysosome and the rhodopsins, perhaps used for vacuole acidification. The combination of single cell genomics and metatranscriptomics gives insights on the phagocytic capacity of uncultured and ecologically relevant HF species.

UNDERSTANDING AGING IN TERMS OF PHYSIOLOGICAL STATES

The “network” of homeostatic systems fails in distinct ways in individual isogenic animals during the aging process. We believe that understanding these distinct physiological states, the transitions between them, and how they relate to homeostatic system functions will allow us to better affect change in the aging process. Work in yeast showed that fixing an initial system failure, loss of vacuole acidification capacity, could increase cellular lifespan. Here we showed how the long-lived physiological state conferred by high expression of the hsp-16.2 promoter based lifespan/penetrance biomarker correlates with differences in the expression of other genes, and the structure and function of lysosomes. We found that vacuole acidification failure is not a major initial proximal cause of aging in C. elegans – at least not in their intestine cells.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

CsPH8, a P-type proton pump gene, plays a key role in the diversity of citric acid accumulation in citrus fruits

Citric acid homeostasis patterns and its content are diversified among the fruits of citrus cultivars, but the cause remained unclear. In this study we showed that changes of citric acid content were highly associated with the expression profiles of a P-type proton pump gene (CsPH8) in the fruits of six citrus cultivars; moreover, analysis of 21 different fruit samples indicated that the correlation coefficient between titratable acid content and CsPH8 transcript level was 0.5837 with a significant level (P < 0.05). Overexpression of CsPH8 in acidless pumelo juice sacs, strawberry fruit, and tomato fruit significantly increased the titratable acid or citric acid content besides the gene transcript level. On another hand, RNA interference of CsPH8 in acidic pumelo juice sacs significantly decreased the CsPH8 transcript level and the titratable acid or citric acid content as well. In addition, severe drought significantly increased the CsPH8 transcript level besides the titratable acid content. Taken together, these findings address the function of CsPH8 in citrus vacuolar acidification, confirm that CsPH8 plays a key role in the variation of citric acid content, and supported that the acid fluctuation influenced by drought, is at least partly due to the change of CsPH8 transcript level.

The new psychoactive substance 3-methylmethcathinone (3-MMC or metaphedrone) induces oxidative stress, apoptosis, and autophagy in primary rat hepatocytes at human-relevant concentrations.

3-Methylmethcathinone (3-MMC or metaphedrone) has become one of the most popular recreational drugs worldwide after the ban of mephedrone, and was recently deemed responsible for several intoxications and deaths. This study aimed at assessing the hepatotoxicity of 3-MMC. For this purpose, Wistar rat hepatocytes were isolated by collagenase perfusion, cultured and exposed for 24h at a concentration range varying from 31nM to 10mM 3-MMC. The modulatory effects of cytochrome P450 (CYP) inhibitors on 3-MMC hepatotoxicity were evaluated. 3-MMC-induced toxicity was perceived at the lysosome at lower concentrations (NOEC 312.5µM), compared to mitochondria (NOEC 379.5µM) and cytoplasmic membrane (NOEC 1.04mM). Inhibition of CYP2D6 and CYP2E1 diminished 3-MMC cytotoxicity, yet for CYP2E1 inhibition this effect was only observed for concentrations up to 1.3mM. A significant concentration-dependent increase of intracellular reactive species was observed from 10μM 3-MMC on; a concentration-dependent decrease in antioxidant glutathione defences was also observed. At 10μM, caspase-3, caspase-8, and caspase-9 activities were significantly elevated, corroboratingthe activation of both intrinsic and extrinsic apoptosis pathways. Nuclear morphology and formation of cytoplasmic acidic vacuoles suggest prevalence of necrosis and autophagy at concentrations higher than 10μM. No significant alterations were observed in the mitochondrial membrane potential, but intracellular ATP significantly decreased at 100μM. Our data point to a role of metabolism in the hepatotoxicity of 3-MMC, which seems to be triggered both by autophagic and apoptotic/necrotic mechanisms. This work is the first approach to better understand 3-MMC toxicology.

Drug Sensing Protein Crystals Doped with Luminescent Lanthanide Complexes

Hen egg white lysozyme (HEWL) crystals are a model system for protein crystallization due to economical production and facile crystal growth. Here we grew small, rod-like HEWL crystals and proceeded with stepwise synthesis to dope the protein crystals with luminescent lanthanide complexes [(Eu(TTA)3phen] (TTA = 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedionato, phen = 1,10-phenanthroline). Several compounds (coumarin, tinidazole, and acridine orange) were observed to quench the lanthanide complexes in solution or embedded within crystals. Coumarin is a precursor for anticoagulant drugs and tinidazole is used to combat bacterial or protozoan infections. In contrast, acridine orange is a widely used, versatile fluorescent dye used to stain acidic vacuoles, RNA, and DNA in living cells. These results suggest that protein crystals may provide a feasible matrix for entrapping luminescent species with practical biosensing applications.

Expression patterns of genes involved in sugar metabolism and accumulation during peach fruit development and ripening

In fleshy fruits, sugars (glucose, fructose, sucrose and sorbitol) are most important for fruit growth and development but also play a central role in fruit quality. In Peach (Prunus persica), we observed the sugars content at six developmental stages through high pressure liquid chromatography (HPLC). There was variation during fruit development and ripening, sucrose first increased then decreased; glucose and fructose first decreased, and then increased while sorbitol always decreased. In past, understanding about the mechanisms which control the sugar metabolism and accumulation in peach remains quite limited. We studied the transcript profiles of total 30 genes encoding key enzymes and transporters, which were involved in sucrose metabolism, resynthesis and transport. 13 putative genes including sucrose phosphatase synthase (PpSPS4 and PpSPS2), Hexokinases (PpHK1 and PpHK3), neutral/natural invertase (PpNINV1and PpNINV2), vacuolar acid invertase (PpVAINV2), sucrose transporter (PpSUT2), vacuolar glucose transporters (PpVGT1, PpVGT2 and PpVGT3) and tonoplast monosaccharides transporters (PpTMT1 and PpTMT2) highly expressed during peach fruit ripening. PpSPS4 had significantly positive correlation with glucose and fructose. Our results suggested that more sucrose accumulated in cytosol because of upregulation of (PpSPS4 and PpSPS2), that sucrose imported into vacuole through upregulation of PpSUT2, PpVGT1, PpVGT2, PpVGT3, PpTMT1 and PpTMT2 transporters, where cleaving of sucrose is done by upregulation of PpVAINV2. Hence, level of sucrose decreased as fruit ripe. As a result, the concentration of glucose and fructose increased at ripening stage (S4 II to S4 III). So, these marker genes can be involved in peach fruit ripening. This study will improve our understanding and lay foundation to explore the function of these genes and improvement of peach fruit quality.

Genome-wide screening of Saccharomyces cerevisiae deletion mutants reveals cellular processes required for tolerance to the cell wall antagonist calcofluor white

We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify nonessential genes associated with increased sensitivity to or resistance against the cell wall antagonist calcofluor white. Through a genome-wide screen, we isolated 537 strains that had an altered growth rate relative to wild type, of which 485 showed increased sensitivity and 52 showed increased resistance to calcofluor white. The MAPK signaling pathway, N-glycan biosynthesis, endocytosis, vacuole acidification, autophagy, and the sulfur relay system were identified as being associated with calcofluor white sensitivity. Resistance genes were mainly involved in chitin metabolism and the RIM101 pathway or encoded several components of the ESCRT complexes or related to cysteine and methionine metabolism and RNA degradation. Further investigation indicated a clear global response network that S. cerevisiae relies on in the presence of the cell wall antagonist calcofluor white, which may help us to understand fungal cell wall remodeling and the mechanisms of toxicity of calcofluor white with respect to eukaryotic cells.

OsVIN2 encodes a vacuolar acid invertase that affects grain size by altering sugar metabolism in rice.

OsVIN2, a vacuolar invertase, affects grain size and yield by altering sugar composition, transport, and starch accumulation in rice. Grain size, a major determinant of rice yield, is influenced by many developmental and environmental factors. Sugar metabolism plays vital roles in plant development. However, the way in which sugar metabolism affects rice grain size remains largely elusive. In this study, we characterized the small grain-size rice mutant sgs1. Histological analyses showed that reduced spikelet hull and endosperm size results from decreased cell size rather than cell number. Map-based cloning and complementation tests revealed that a DaiZ7 transposon insertion in a vacuolar invertase gene OsVIN2 is responsible for the mutant phenotype. Subcellular distribution and biochemical analysis indicated that OsVIN2 is located in the vacuolar lumen, and that its sucrose hydrolysis activity is maintained under acidic conditions. Furthermore, an altered sugar content with increased sucrose and decreased hexose levels, as well as changes in invertase and sucrose synthase activities, sugar transport gene expression, and starch constitution in sgs1 implies that OsVIN2 affects sucrose metabolism, including sugar composition, transport, and conversion from the source to the sink organs. Collectively, OsVIN2 is involved in sugar metabolism, and thus regulates grain size; our findings provide insights into grain development and also suggest a potential strategy to improve grain quality and yield in rice.

Abstract 279: c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer

Abstract Breast cancer is the most prevalent type of cancer among women. Triple negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer. It is the most challenging subtype to treat mainly due its aggressive nature and lack of targets such as HER2 (human epidermal growth factor 2), PR (progesterone receptor), and ER (estrogen receptor) found in other subtypes. We have shown that inhibiting both EGFR (epidermal growth factor receptor)/HER2 with lapatinib and JNK with JNK-IN-8 causes synergistic cell death in 11 out of 12 TNBC cell lines. EGFR is expressed in up to 90% tumors and JNK activation is linked to TNBC phenotype, metastasis, and stem cell character, among others. Synergism was achieved in the resistant, low-EGFR expressing cell line when a FGFR inhibitor (BGJ398) was used instead of lapatinib in combination with JNK-IN-8. Combination drug effects were accompanied by elevated oxidative stress. Suppression of oxidative stress restored cell viability in cells treated with the drug combination. JNK-IN-8 treatment alone or in combination with lapatinib induced massive cytoplasmic vacuolization with little apoptotic blebbing. The vacuoles were shown to be membrane-derived and acidic using Lucifer Yellow and Acridine Orange, respectively. Pre-treatment of cells with bafilomycin A1 (lysosome inhibitor), 5-(N-Ethyl-N-isopropyl) amiloride (macropinocytosis inhibitor), or N-acetylcysteine (antioxidant) reduced vacuolization. A similar effect was observed when cells were serum-starved. JNK1 knockout mouse mammary tumor cells, in comparison with JNK2 knockout, were resistant to drug synergism and displayed significantly less oxidative stress and vacuolization. Our studies demonstrated that concomitant blockade of JNK and individual tumor-appropriate targets induce synergistic cell death in TNBC cells in response to oxidative stress. Moreover, JNK blockade resulted in the formation of serum-dependent, acidic vacuoles that were reversible by lysosome or macropinocytosis inhibition. Loss-of-function studies indicated that JNK1 was the key isoform responsible for the drug response in TNBC cells. These findings suggest that JNK is involved in a novel cell death pathway and is a robust target in combination therapies against TNBC. Citation Format: Milad Soleimani, Ritvik Bhattacharjee, Uma Giri, Tamer S. Kaoud, Kevin N. Dalby, S. Gail Eckhardt, Carla L. Van Den Berg. c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 279.

The MYB5-driven MBW complex recruits a WRKY factor to enhance the expression of targets involved in vacuolar hyper-acidification and trafficking in grapevine.

The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB-bHLH-WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY-type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper-acidification, such as the P-type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper-acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated.

Sinensetin induces apoptosis and autophagy in the treatment of human T-cell lymphoma.

The present study was carried out to explore the effect of sinensetin in human T-cell lymphoma Jurkat cells and to reveal the underlying molecular mechanisms. We found that sinensetin significantly impeded Jurkat cell proliferation in a dose-dependent and time-dependent manner. Additionally, sinensetin treatment triggered apoptosis and autophagy in Jurkat cells. The apoptosis induction was related to a loss of mitochondrial membrane potential and to increased caspase-3/-8/-9 and poly(ADP-ribose) polymerase (PARP) cleavage. Sinensetin also induced autophagy, as evidenced by the formation of acidic vacuoles, the upregulation of LC3-II and beclin-1, and the downregulation of p62. In addition, the inhibition of autophagy by 3-methyladenine significantly enhanced the apoptosis rate and improved the sensitivity of the Jurkat cells to sinensetin. Moreover, sinensetin induced cell death, apoptosis, and autophagy through the activation of the reactive oxygen species/ c-Jun N-terminal kinase signaling pathway and the inhibition of the Akt/mTOR signaling pathways. In summary, our results revealed that sinensetin induced apoptosis and autophagy in human T-cell lymphoma Jurkat cells by activating reactive oxygen species/ c-Jun N-terminal kinase and blocking the Akt/mTOR signaling pathways. Thus, sinensetin might be a potential candidate in the development of antitumor drugs targeting T-cell leukemia.

Mechanism underlying N-(3-oxo-dodecanoyl)-L-homoserine lactone mediated intracellular calcium mobilization in human platelets

Acyl-homoserine lactones (AHLs), are the key autoinducer molecules that mediate Pseudomonas aeruginosa associated quorum sensing. P. aeruginosa produces two types of AHLs; N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 HSL) and N-butyryl-L-homoserine lactone (C4 HSL). AHLs are not only regulating the virulence gene of bacteria but also influence the host cell functions by interkingdom signaling. In this study, we explored the mechanism of AHLs induced calcium mobilization in human platelets. We found that 3-oxo-C12 HSL but not C4 HSL induces intracellular calcium release. 3-oxo-C12 HSL induced calcium mobilization was majorly contributed from the dense tubular system (DTS). Furthermore, 3-oxo-C12 HSL also stimulates the store-operated Ca2+ entry (SOCE) in platelet. Intracellular calcium rise was significantly lowered in rotenone, and bafilomycin pre-treated platelets suggesting partial involvement of mitochondria and acidic vacuoles. The significant effect of 3-oxo-C12 HSL on calcium mobilization can alter the platelet functions that might results in thrombotic disorders in individuals infected with P. aeruginosa.

Novel c.G630A TCIRG1 mutation causes aberrant splicing resulting in an unusually mild form of autosomal recessive osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a severe genetic bone disease characterized by high bone density due to mutations that affect formation or function of osteoclasts. Mutations in the a3 subunit of the vacuolar-type H+ -ATPase (encoded by T-cell immune regulator 1 [TCIRG1]) are responsible for ~50% of all ARO cases. We identified a novel TCIRG1 (c.G630A) mutation responsible for an unusually mild form of the disease. To characterize this mutation, osteoclasts were differentiated using peripheral blood monocytes from the patient (c.G630A/c.G630A), male sibling (+/+), unaffected female sibling (+/c.G630A), and unaffected parent (+/c.G630A). Osteoclast formation, bone-resorbing function, TCIRG1 protein, and mRNA expression levels were assessed. The c.G630A mutation did not affect osteoclast differentiation; however, bone-resorbing function was decreased. Both TCIRG1 protein and full-length TCIRG1 mRNA expression levels were also diminished in the affected patient's sample. The c.G630A mutation replaces the last nucleotide of exon 6 and may cause splicing defects. We analyzed the TCIRG1 splicing pattern between exons 4 to 8 and detected deletions of exons 5, 6, 7, and 5-6 (ΔE56). These deletions were only observed in c.G630A/c.G630A and +/c.G630A samples, but not in +/+ controls. Among these deletions, only ΔE56 maintained the reading frame and was predicted to generate an 85 kDa protein. Exons 5-6 encode an uncharacterized portion of the cytoplasmic N-terminal domain of a3, a domain not involved in proton translocation. To investigate the effect of ΔE56 on V-ATPase function, we transformed yeast with plasmids carrying full-length or truncated Vph1p, the yeast ortholog of a3. Both proteins were expressed; however, ΔE56-Vph1p transformed yeast failed to grow on Zn2+ -containing plates, a growth assay dependent on V-ATPase-mediated vacuolar acidification. In conclusion, our results show that the ΔE56 truncated protein is not functional, suggesting that the mild ARO phenotype observed in the patient is likely due to the residual full-length protein expression.

Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs

Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.

Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2.

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

Induction of a non-apoptotic mode of cell death associated with autophagic characteristics with steroidal maleic anhydrides and 7β-hydroxycholesterol on glioma cells

Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic, deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell death was studied on C6 rat glioma cells, and 7β-hydroxycholesterol was used as positive cytotoxic control. The highest cytotoxicity was observed with lithocholic and chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5β-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death associated with mitochondrial membrane potential loss and reactive oxygen species overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found with 7β-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7β-hydroxycholesterol. Compound 1b and 7β-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7β-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1, Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7β-hydroxycholesterol-induced cell death, respectively, supporting that 7β-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7β-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a non apoptotic mode of cell death, involving the mitochondria and associated with several characteristics of autophagy.