Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.     

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.     

Cytotoxicity of Anthrax Lethal Toxin to Human Acute Myeloid Leukemia Cells Is Nonapoptotic and Dependent on Extracellular Signal-Regulated Kinase 1/2 Activity

In this study, we attempt to target the mitogen-activated protein kinase (MAPK) pathway in acute myeloid leukemia (AML) cells using a recombinant anthrax lethal toxin (LeTx). LeTx consists of protective antigen (PrAg) and lethal factor (LF). PrAg binds cells, is cleaved by furin, oligomerizes, binds three to four molecules of LF, and undergoes endocytosis, releasing LF into the cytosol. LF cleaves MAPK kinases, inhibiting the MAPK pathway. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic responses to LeTx. Cytotoxicity of LeTx was mimicked by the specific mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the MEK1/2-extracellular signal-regulated kinase (ERK1/2) branch of the MAPK pathway. The four LeTx-resistant cell lines were sensitive to the phosphatidylinositol 3-kinase inhibitor LY294002. Co-treatment of AML cells with both LeTx and LY294002 did not lead to increased sensitivity, showing a lack of additive/synergistic effects when both pathways are inhibited. Flow cytometry analysis of MAPK pathway activation revealed the presence of phospho-ERK1/2 only in LeTx-sensitive cells. Staining for Annexin V/propidium iodide and active caspases showed an increase in double-positive cells and the absence of caspase activation following treatment, indicating that LeTx-induced cell death is caspase-independent and nonapoptotic. We have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the MAPK pathway. Furthermore, we have demonstrated that LeTx-induced cytotoxicity in AML cells is nonapoptotic and dependent on phospho-ERK1/2 levels.     

Direct Proteolytic Cleavage of NLRP1B Is Necessary and Sufficient for Inflammasome Activation by Anthrax Lethal Factor

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.     

Cytosolic Sulfotransferase 1A3 Is Induced by Dopamine and Protects Neuronal Cells from Dopamine Toxicity: ROLE OF D1 RECEPTOR-N-METHYL-d-ASPARTATE RECEPTOR COUPLING*

Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.     

Low Shear Stress Induced HMGB1 Translocation and Release via PECAM-1/PARP-1 Pathway to Induce Inflammation Response

Low shear stress (LSS) plays a critical role in the site predilection of atherosclerosis through activation of cellular mechanosensors, such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that regulates the expression of various inflammatory cytokines. The nuclear enzyme high mobility group box 1 (HMGB1) can induce inflammation response by binding to toll-like receptor 4 (TLR4). In the present study, we aimed to investigate the role and mechanism of HMGB1 in LSS induced inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated by undisturbed shear stress (USS, 1 Pa) and LSS (0.4 Pa) in our experiments. Gene expression was inhibited by small interfering RNA (siRNA). ICAM-1 expression was regulated by LSS in a time dependent manner. LSS can induce HMGB1 translocation from nucleus to cytoplasm and release. Compared with the USS, LSS could increase the protein expression of PECAM-1 and PARP-1 as well as the secretion of TNF-α and IL-1β. LSS induced the translocation of HMGB1 from nucleus to cytoplasm. Inhibition of HGMB1 reduced LSS-induced inflammatory response. Inhibition of PARP-1 suppressed inflammatory response through inhibiting TLR4 expression and HMGB1 translocation. PECAM-1 inhibition reduced LSS-induced ICAM-1 expression, TNF-α and IL-1β secretion, and monocytes adhesion. LSS can induce inflammatory response via PECAM-1/PARP-1/HMGB1 pathway. PARP-1 plays a fundamental role in HMGB1 translocation and TLR4 expression. Inhibition of PARP-1 may shed light on the treatment of HMGB1 involved inflammation during atherosclerosis.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus highly dependent upon a myriad of cellular mechanisms for successful virus replication. Cholesterol plays a pivotal role throughout the HIV-1 life cycle (23, 40, 41, 64). HIV-1 entry, assembly, and budding processes occur at cholesterol-enriched membrane microdomains known as lipid rafts, and depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Virion-associated cholesterol is required for fusion and subsequent infection of susceptible cells (41), and cholesterol-sequestering drugs, such as β-cyclodextrin, render the virus incompetent for cell entry (4, 25, 57). Therefore, intracellular cholesterol trafficking pathways that allow nascent HIV-1 particles to acquire lipids appear critical for virus replication.Recent evidence supports a critical role for cholesterol trafficking and homeostasis in viral replication, showing that the HIV-1 accessory protein Nef increases synthesis and transport of cholesterol to both lipid rafts and progeny virions and induces multiple genes involved in cholesterol synthesis (80, 88). More recent studies have revealed that binding of Nef to the ATP-binding cassette transporter A1 (ABCA1) leads to impairment of ABCA1-dependent cholesterol efflux and an accumulation of lipids within the cell (51).Mammalian cells acquire cholesterol primarily from endocytosed low-density lipoproteins (LDL). The Niemann-Pick type C-1 (NPC1) protein is well known for its role in intracellular trafficking of LDL-derived free unesterified cholesterol. Dysfunctional NPC1 activity leads to development of NPC disease, a rare, autosomal recessive, neurodegenerative disorder characterized by the massive accumulation of cholesterol and glycosphingolipids in late endosomal/lysosomal (LE/L) compartments (61). In normal cells, endocytosed LDLs are delivered to the LE/Ls, where they are hydrolyzed and free cholesterol is released. Homeostasis is achieved when cholesterol is then rapidly transported out of the LE/Ls to the plasma membrane and endoplasmic reticulum (ER) (17, 19, 42, 73, 85), or first to the trans-Golgi (TG) network (TGN) and then to the ER (76). In NPC1-deficient (NPCD) cells, the cholesterol does not exit the endocytic pathway, resulting in its accumulation within LE/L structures.In 95% of NPC patients, the disease is caused by mutations in the NPC1 gene, while the remaining 5% harbor mutations in the NPC2 gene (50, 72, 79). One of the most frequently found and extensively characterized NPC1 mutations is the I1061T mutation (37, 38, 86). This mutation results in misfolding of the NPC1 protein, leading to its degradation and causing an 85% decrease in cellular NPC1 expression (20). Cells with such low levels of functional NPC1 maintain only 38% of normal sphingomyelinase activity and have impaired cholesterol esterification and trafficking.NPC1 is a large, multispanning protein that resides in the limiting membrane of the LE and binds cholesterol via its N-terminal domain (31). While the complete physiological function of NPC1 is still unclear, NPC1 does share homology with the resistance-nodulation-division family of prokaryotic permeases and may function as a transmembrane efflux pump to transport cargos in LEs (9, 75). Other studies suggest that NPC1 might also function in vesicle-mediated pathways for cargo transportation from LEs to other intracellular sites (21, 33). Recent studies by Infante et al. have propelled forward our understanding of how NPC1 works together with NPC2, also known to bind cholesterol, to support cholesterol efflux from the LE (32). Their findings provide a basis for either of two possible models, with respect to cholesterol trafficking: (i) NPC1 binds cholesterol found within the LE and mediates either direct export or transfer to NPC2 for delivery to a cholesterol efflux transporter, such as ABCA1; or (ii) NPC2 is the first to bind cholesterol and then mediate its delivery to NPC1 for direct export or transfer to ABCA1. These recent findings underscore the highly critical role of these proteins in maintaining intracellular cholesterol homeostasis.In addition to its role in sterol trafficking, some studies suggest that the NPC pathway may be directly involved in trafficking multiple proteins from LE/L compartments. LEs act as sorting stations to deliver endocytosed molecules to L''s for degradation, while at the same time retrieving other classes of proteins and lipids for transport back to nondegradative compartments (3, 14, 15, 28, 63, 69, 78). LE compartments also serve as sorting stations for HIV-1 viral proteins and represent a major site for HIV-1 assembly and budding (7, 12, 16, 22, 24, 57, 59).The endosomal trafficking defects observed in NPCD cells extend to proteins such as IGF2/MPR, NPC1, and annexin II, all of which utilize the endosomal recycling pathway (42, 74). Electron microscopy studies have shown that within the LEs of NPCD cells these proteins are trapped in the cholesterol-enriched membrane-bound vesicular structures (47). Cholesterol and glycosphingolipid accumulation within NPCD cells appears to disrupt Rab9 GTPase function in LE-to-TGN transport, trapping Rab9-associated proteins, such as vimentin, Tip47, and the mannose-6-phosphate receptor in LEs (18, 83). Overexpression of Rab7 and Rab9 GTPases can reverse the cholesterol accumulation phenotype caused by NPCD (8, 84). These observations suggest that NPC1, directly or indirectly, plays a role in protein export from LEs. It is unknown whether NPC1 is involved in the export of HIV-1 proteins from LEs; however, the Rab9 GTPase-mediated pathway is known to be required for HIV-1 replication (53). This strongly suggests that HIV assembly will be hindered when the NPC pathway is disrupted.Given the function of NPC1 in mediating intracellular cholesterol trafficking within the LE and given the need of HIV-1 for cholesterol, NPC1 involvement in HIV-1 biogenesis is highly likely. In the present study, using cells treated with U18666A or NPCD cells, we show that impaired NPC1 function results in profound suppression of HIV-1 replication. Further, our findings demonstrate that the NPC1 protein is essential for proper trafficking of the HIV-1 Gag protein during the late stages of assembly and budding. It appears that in NPCD cells, in which cholesterol and cellular proteins accumulate in LE/L compartments, the viral Gag protein fails to traffic properly and accumulates within these compartments, resulting in decreased particle production. Our findings not only reinforce the dependence of HIV-1 on cholesterol homeostasis but also support a role for NPC1 in HIV-1 viral protein trafficking and particle release from infected cells.     

ZmGT1基因转运谷胱甘肽轭合物功能研究及除草剂阿特拉津诱导表达分析

还原型谷胱甘肽在植物抵御生物压和非生物压过程中扮演着重要的角色．利用RACE-PCR技术从玉米体内克隆得到一个编码谷胱甘肽转运蛋白的基因ZmGT1．利用缺失谷胱甘肽转运子基因的突变型酵母菌(hgt1Δ)研究ZmGT1基因的生理功能发现,ZmGT1基因能够修复hgt1Δ突变型酵母菌在谷胱甘肽(GSH)作为唯一硫源的培养基中的生长,并且具有调控谷胱甘肽轭合物GS-N-ethylmaleimide (GS-NEM)吸收的功能．ZmGT1基因在玉米幼苗的各个不同器官均有表达,其中在叶片中的表达量更高．玉米ZmGT1基因能够被除草剂阿特拉津强烈地诱导,经过阿特拉津处理96 h后,玉米叶片中ZmGT1基因的表达量提高约4～5倍,该结果表明,谷胱甘肽转运蛋白在玉米解毒外源有害物质的过程中可能发挥着作用．     

Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

Growth plate abnormalities, associated with impaired hypertrophic chondrocyte apoptosis, are observed in humans and animals with abnormalities of vitamin D action and renal phosphate reabsorption. Low circulating phosphate levels impair hypertrophic chondrocyte apoptosis, whereas treatment of these cells with phosphate activates the mitochondrial apoptotic pathway. Because phosphate-mediated apoptosis of chondrocytes is differentiation-dependent, studies were performed to identify factors that contribute to hypertrophic chondrocyte apoptosis. An increase in the percentage of cells with low mitochondrial membrane potential, evaluated by JC-1 fluorescence, was observed during hypertrophic differentiation of primary murine chondrocytes in culture. This percentage was further increased by treatment of hypertrophic, but not proliferative, chondrocytes with phosphate. Phosphate-mediated apoptosis was observed as early as 30 min post-treatment and was dependent upon Erk1/2 phosphorylation. Inhibition of Erk1/2 phosphorylation in vivo confirmed an important role for this signaling pathway in regulating hypertrophic chondrocyte apoptosis in growing mice. Murine embryonic metatarsals cultured under phosphate-restricted conditions demonstrated a 2.5-fold increase in parathyroid hormone-related protein mRNA expression accompanied by a marked attenuation in phospho-Erk immunoreactivity in hypertrophic chondrocytes. Thus, these investigations point to an important role for phosphate in regulating mitochondrial membrane potential in hypertrophic chondrocytes and growth plate maturation by the parathyroid hormone-related protein signaling pathway.     

sodC Is an α-COP-Related Gene Which Is Essential for Establishing and Maintaining Polarized Growth in Aspergillus nidulans

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.     

Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

Subcellular Distribution of Homer 1b/c in Relation to Endoplasmic Reticulum and Plasma Membrane Proteins in Purkinje Neurons

The subcellular distribution of endoplasmic reticulum proteins (IP₃R1 and RYR), plasma membrane(PM) proteins (mGluR1 and PMCA Ca²⁺-pump), and scaffolding proteins, such as Homer 1b/c, was assessed by laser scanning confocal microscopy of rat cerebellum parasagittal sections. There appeared to be two classes of Ca²⁺ stores, nonjunctional Ca²⁺ stores and junctional Ca²⁺ stores, possibly referable to central cisternae/tubules and sub-PM cisternae, respectively, in soma, dendrites, and dendritic spines. Only some IP₃R1s appeared to be part of multimeric, junctional Ca²⁺ signaling networks, whose composition is shown to include PMCA, mGluR1, Homer 1b/c and, not always, RYR1.     

Release of prostaglandin F1α and F2α from superfused platelets: Quantitative evaluation of the inhibitory effects of some aspirin-like drugs

The release of PGF_1α and PGF_2α from superfused blood platelets was studied by the combined use of two radioimmunoassay systems with different specificities. PGF_1α only accounted for approximately 30% of the total immunoreactivity. A substantially similar pattern of release was obtained with platelets of rat and human origin, although the latter released considerably lower amounts of both compounds. Indomethacin, Fenoprofen, Ditazole and Aspirin all inhibited PGF_2α release from rat platelets in descending order of potency. Hydrocortisone was practically inactive. The release of PGF_1α and PGF_2α was inhibited to the same extent by both Indomethacin and Fenoprofen. Moreover, a quite similar inhibitory effect by the same drug on rat and human platelets was found in preliminary experiments. In agreement with a previous similar finding, Aspirin displayed a higher inhibitory activity than that reported in other tissues. The use of superfused platelets seems to provide a simple and reproducible model for studying pharmacologic influences upon PG formation.     

The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membrane-bound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V₀ subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V₁ and V₀(−d) that failed to form V₁V₀, and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V₁V₀ assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V₁V₀ was delivered to the vacuole and active. It retained 63–71% of the wild-type activity and was responsive to glucose. Tether-less V₁V₀ disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V₁ subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V₀ assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.V-ATPase⁴ proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1–6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1–3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V₁ (peripheral) and V₀ (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V₁V₀ assembly (8). Deletion of a peripheral V₁ subunit leads to disruption of the entire V₁ domain in yeast. Loss of a V₀ subunit does not affect V₁ assembly but disrupts the entire V₀ domain, which also prevents V₁ from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requires disruption of both genes (9).Eight different subunits (A-H) compose the V₁ domain where ATP hydrolysis takes place at a catalytic hexamer A₃B₃ (1). Six subunits (a, c, c′, c′′, d, e) form V₀, the membrane intrinsic domain that holds V₁ and forms the path for proton transport via a hydrophobic ring structure (c-ring). V₁ and V₀ subunits contribute to the formation of one central and three peripheral stalks that connect the c-ring and the catalytic hexamer A₃B₃ (1). ATP hydrolysis drives rotation of the central stalk (connected to the c-ring) (10). Protons are transferred from the cytosol to each subunit of the c-ring and from the c-ring to the other side of the membrane passing through subunit a (11). As many protons, as subunits forming the c-ring, get transferred against a concentration gradient when hydrolysis of three ATP molecules powers 360° rotation.V-ATPases are related to F-ATP (F₁F₀ ATP) synthases. Both proteins work as molecular motors (10, 12–14). It is postulated that the asymmetry imposed by having a 3-fold symmetry in F₁ (and V₁) and an apparent 10-fold symmetry in the c-ring of F₀ (and V₀) requires energy to be transiently stored. The energy of coupling is thought to be stored in the peripheral (stator) and central (rotor) stalk structures of F₁F₀ (15–17). Subunit a is the only peripheral stalk component of the V-ATPase complex that is secured in the membrane (18). It is key for maintaining structural stability when relative rotation of subunits occurs during catalysis. Thus, the tether of subunit a in V₀ could be functionally comparable with the tether of subunit b in F₀ (Escherichia coli), although subunits a (V₀) and b (F₀) do not share sequence homology.Subunit a is a 95-kDa protein that consists of two domains that are structurally and functionally distinguishable. The hydrophilic N-terminal domain (∼45 kDa) is oriented toward the cytosolic side of the membrane and contains the information necessary to deliver V-ATPases to different compartments (19). The N terminus interacts with multiple V₁ subunits, including the catalytic subunit A (20) and peripheral stalk-forming subunits H, C, E, and G of V₁ (18, 21). It is through these interactions that the N-terminal domain serves as a stator, which prevents rotation of the A₃B₃ hexamer during catalysis. The other half of subunit a, the C-terminal domain (∼50 kDa), is buried in the membrane by multiple transmembrane-spanning regions (9). The C-terminal domain interacts with the periphery of the c-ring (22) and contributes to the path for proton transport (11, 19) by providing access to cytosolic protons and directing their exit to the luminal side of the membrane.In contrast to its role as stator during catalysis, the N-terminal domain of subunit a is a movable element that switches positions when V₁V₀ is regulated by disassembly and reassembly in vivo (1, 2, 23, 24). Inactivation of V-ATPases by disassembly is a rapid response to glucose starvation in yeast (23, 25). In the absence of glucose the V-ATPase complex dissociates into three parts: V₁ subunit C, V₁ (without subunit C), and V₀ (23). Disassembly is reversible, and the three components reassociate immediately after glucose addition, restoring ATP hydrolysis and proton transport. As V₁V₀ disassembles and reassembles, the N-terminal domain of subunit a alternates between V₁V₀ and V₀ (26, 27). In V₁V₀ it contributes to stabilizing the stator-forming V₁ subunits (1, 18). In V₀, its role has yet to be determined.As its functional and regulatory roles emerge, it becomes clear that the cytosolic N terminus of V₀ subunit a is key for V₁V₀ activity, assembly, and regulation. In this study deletions were made at amino acids that connect the N-terminal and C-terminal domains of subunit a Vph1. Shrinking of the tether that anchors subunit a to the membrane harmed assembly of subunit d into V₀, making yeast cells sensitive to pH (vma growth phenotype). Growth defects were rescued by exogenous VMA6, the gene encoding subunit d. Remarkably, subunit d restored assembly and significant function of V-ATPase proton pumps that had up to 46 residues of the tether removed. Because V₁V₀ containing tether-less vph1 assembled with peripheral V₁ subunits and with the glycolytic enzyme phosphofructokinase, we concluded that no major structural changes were generated at the N- and C-terminal domains. Early steps of V₀ assembly, and trafficking were likely impaired by shorter tethers and rescued by VMA6. The potential mechanisms by which overexpression of subunit d rescued subunit a deletions are discussed.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.