BACKGROUND AND AIMS:Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy.APPROACH AND RESULTS:Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways.CONCLUSIONS:Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

ATP6AP2 (also known as the [pro]renin receptor) is a type I transmembrane protein that can be cleaved into two fragments in the Golgi apparatus. While in Drosophila ATP6AP2 functions in the planar cell polarity (PCP) pathway, recent human genetic studies have suggested that ATP6AP2 could participate in the assembly of the V-ATPase in the endoplasmic reticulum (ER). Using a yeast model, we show here that the V-ATPase assembly factor Voa1 can functionally be replaced by Drosophila ATP6AP2. This rescue is even more efficient when coexpressing its binding partner ATP6AP1, indicating that these two proteins together fulfill Voa1 functions in higher organisms. Structure-function analyses in both yeast and Drosophila show that proteolytic cleavage is dispensable, while C-terminus-dependent ER retrieval is required for ATP6AP2 function. Accordingly, we demonstrate that both overexpression and lack of ATP6AP2 causes ER stress in Drosophila wing cells and that the induction of ER stress is sufficient to cause PCP phenotypes. In summary, our results suggest that full-length ATP6AP2 contributes to the assembly of the V-ATPase proton pore and that impairment of this function affects ER homeostasis and PCP signaling.

Silver-enabled fabrics may be transformed during use in ways that may affect their release characteristics and antibacterial efficacy. Here, we assess how chemical transformations of silver in fabrics treated with Ag nanoparticles or AgCl particles, or containing interwoven Ag-0 fibers affect silver leaching and their antibacterial efficacy under different use and end-of-life scenarios. Fabrics were exposed to artificial sweat (use phase) or artificial landfill leachate (sodium chloride, sodium sulfide, or acetic acid; end of life phase). Chemical transformations induced by exposure to sodium chloride, sodium sulfide and acetic acid result in variations in Ag release and corresponding changes in bactericidal properties of the Ag-treated textiles. Exposure to solutions containing chloride ions (sodium chloride and artificial sweat) generally increased leaching compared to deionized water. Conversely, exposure to sodium sulfide and acetic acid solutions decreased Ag release. Exposure to artificial sweat did not affect antibacterial efficacy for fabrics with greater than similar to 10 mu g Ag (g fabric)(-1). Sulfide solution exposure decreased antibacterial performance for all but the 500 mu g Ag (g fabric)(-1). The lower efficacy was consistent with chemical transformation of elemental Ag to AgCl/Ag-0 or Ag2S, respectively for chloride and sulfide exposure. AgCl-coated fabrics were more resilient to chemical attack than Ag-0-enabled fabrics. These results indicate that fabrics with as low as similar to 10 mu g Ag (g fabric)(-1) can maintain high antibacterial efficacy under normal use phase conditions, but below this concentration efficacy significantly decreases. Taken together, the data permit a comparison of the benefits (antimicrobial efficacy) in the context of the impacts (silver release) and inform selection and design of materials and loadings that give the best overall lifecycle benefit.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1 , encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

The function of the vacuolar H+-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V0 domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ) and vma21QQ in combination with voa1Δ, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects.

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V-1 sector catalyzes ATP hydrolysis and the V-0 sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V-0 assembly. We have discovered a fifth V-0 assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V-0 assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V-0 assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V-0-Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V-0 subunits c, c', and c ''. On assembly of the remaining three V-0 subunits (a, d, and e) into the V0 complex, Voa1p dissociates from the now fully assembled V-0-Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V-0 assembly in the ER, but then it dissociates before exit of the V-0-Vma21p complex from the ER for transport to the Golgi compartment.

The animal and human pathogen Listeria monocytogenes secretes several virulence factors, including a phosphatidylinositol-specific phospholipase C (PI-PLC). Sufficient quantities of L. monocytogenes PI-PLC for biophysical studies were obtained by overexpression of the enzyme in Escherichia coli. The purified PI-PLC was examined in enzyme kinetics experiments using a new fluorogenic substrate, methyl-FLIP. Methyl-FLIP is a water-soluble monomeric substrate cleaved in a manner similar to the natural aggregate substrate, phosphatidylinositol (PI). Michaelis–Menten kinetics were observed with KM=61±7 μM and Vmax=120±5 μmol min−1 mg−1, corresponding to kcat=66±3 s−1. The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC6PC). The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC6PC binds to a second site, with an interaction between the two sites. The result is a decrease in KM and an increase in Vmax, producing an overall four to five-fold increase in catalytic efficiency (kcat/KM). The interaction is not a regulatory mechanism, as is the case for multimeric enzymes; rather, it suggests interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site.

The respiratory proteins hemoglobin and hemocyanin share the function of oxygen transport, but the proteins belong to separate gene families, and their active sites and the metal ions that bind the oxygen differ. Either hemoglobin or hemocyanin, but not both, is expressed in the hemolymph of many arthropod crustaceans. Hemoglobin is present in Branchiopoda, Ostracoda, Copepoda, rhizocephalan Cirripedia and one suborder of amphipodan Malacostraca, while hemocyanin has been described in Malacostraca. Recent work by several laboratories have provided new information on the gene structure, exon-intron patterns, site of synthesis and expression of hemoglobins in the branchiopods Artemia and Daphnia. These studies suggest the branchiopods are excellent model organisms for studies of oxygen sensors and hypoxia inducible transcription factors during developmental and adult stages. The focus in our laboratory on the ontogeny of hemocyanin in the Dungeness crab, Cancer magister, has demonstrated that both structure and function of hemocyanin change from megalopa to adult crab. The hemocyanin of an oceanic megalopa contains four subunits. Another subunit appears about the time of metamorphosis to first juvenile instar, and expression of a sixth subunit begins four or five molts later. The timing of onset of adult hemocyanin can be altered experimentally by food levels and temperature. Gene expression and functional properties of both red and blue oxygen transport proteins of crustaceans change during ontogeny to insure oxygen delivery appropriate for each developmental stage.

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4. 10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the H-1 NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine H-delta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pK(a) of 8.0 at 6 degreesC, which is identified with the pK(a) of His32. The H-delta1 signal is modulated by rapid exchange of the H-is an element of2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 Angstrom at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor greater than or equal to 100.

Cryptocyanin, a copper-free hexameric protein in crab ( Cancer magister ) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family—hemocyanin, cryptocyanin, phenoloxidase, and hexamerins—may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.

The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol‐specific phospholipase C (PI‐PLC). In this report, we present the first study of the p K a values of histidines of a PI‐PLC. All six histidines of Bacillus cereus PI‐PLC were studied by 2D NMR spectroscopy and site‐directed mutagenesis. The protein was selectively labeled with 13 C ϵ1 ‐histidine. A series of 1 H‐ 13 C HSQC NMR spectra were acquired over a pH range of 4.0‐9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1 H‐ 13 C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild‐type activity. The active site histidines, His32 and His82, display p K a values of 7.6 and 6.9, respectively. His92 and His227 exhibit p K a values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The p K a value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high p K a .

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.

The synthesis of a chromogenic substrate for mammalian PI-PLC, d,l-myo-inositol 4,5-bis(dihydrogen phosphate) 1-(4-nitrophenyl hydrogen phosphate) tetrasodium salt (13), is described. Phosphoinositol bisphosphate 13 (NPIP-P2) is shown to be a substrate for human PI-PLCδ1 in a non-radioactive, continuous assay. © 1997 Elsevier Science Ltd.

Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography. Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme. The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position. The most hydrophobic compound, dioctyl methyl phosphate 14, was also the best inhibitor with an IC50 of 12 microM. In a series of dihexyl lipids, compounds with phosphonoinositol head groups inhibited more strongly than those that do not contain inositol but are otherwise identical. Compound 29, a short-chain lipid with a phosphonoinositol head group, was found to be a competitive inhibitor and the most potent in this series with an IC50 of 18 microM (Ki = 14 microM). Analogs with dihexyl chains were better inhibitors than those with dihexanoyl chains, presumably because the ether-linked lipids are more hydrophobic than the ester-linked lipids. No appreciable difference in inhibition was found between a phosphonoinositol lipid and the corresponding difluorophosphonoinositol lipid. Inositols and inositol derivatives that do not contain lipid moieties show IC50s about 3 orders of magnitude above those of the short-chain lipids. In this group, glucosaminyl(alpha 1-->6)-D-myo-inositol inhibited more strongly than myo-inositol, which in turn is a better inhibitor than inositol phosphate. The addition of polyethylene glycol (PEG-600) resulted in a marked decrease in inhibition by the short-chain lipids, but had little effect on the water-soluble head group analogs. This is accounted for in terms of solubilization of the amphipathic inhibitors by PEG. Since PEG is required in the crystallization, these data indicate that the best strategy for obtaining enzyme inhibitor complexes is to start by cocrystallizing PI-PLC with the head group analogs. The next step is to synthetically add the shortest possible hydrophobic moieties to the analogs and cocrystallize these with the enzyme. This strategy may be applicable to other lipolytic enzymes.