Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H-ATPase.

The vacuolar ATPase (V-ATPase; VV) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase V proton channel subcomplex.

A nanobody against the V-ATPase c subunit inhibits metastasis of 4T1-12B breast tumor cells to lung in mice.

The vacuolar H-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells . We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit.

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses.

Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement.

Tender love and disassembly: How a TLDc domain protein breaks the V-ATPase.

Vacuolar ATPases (V-ATPases, V V -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V -ATPase disengages from V proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex.

Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.

The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V -ATPase and V proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted V and mutant V . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V to V provides sufficient free energy to overcome V autoinhibition.

Purification of active human vacuolar H-ATPase in native lipid-containing nanodiscs.

Vacuolar H-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes.

A "Sugar-Coated" Proton Pump Comes into Focus: High-Resolution Structure of a Human V-ATPase.

In this issue of Molecular Cell, Wang et al. (2020a) present near-atomic resolution cryoEM structures of a proton-pumping vacuolar ATPase from human cells, illuminating the glycosylation of integral subunits and identifying a novel and conserved glycolipid ligand.

Functional reconstitution of vacuolar H-ATPase from V proton channel and mutant V-ATPase provides insight into the mechanism of reversible disassembly.

The vacuolar H-ATPase (V-ATPase; VV-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V and V, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase Here, using the model organism , we show that a key impediment for binding of V to V is the conformation of the inhibitory C-terminal domain of subunit H (H).

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

Vacuolar H-ATPases (V-ATPases; VV-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V and V H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V However, how H fulfills these two functions is poorly understood.

Breaking up and making up: The secret life of the vacuolar H -ATPase.

The vacuolar ATPase (V-ATPase; V V -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation.

Crystal structure of yeast V1-ATPase in the autoinhibited state.

Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site.

Identification of a novel variant of LMP-1 of EBV in patients with endemic Burkitt lymphoma in western Kenya.

Background: Epstein Barr virus (EBV) is a gammaherpesvirus that is associated with nasopharyngeal carcinoma (NPC) and endemic Burkitt lymphoma (eBL). EBV carries several latent genes that contribute to oncogenesis including the latent membrane protein 1 (LMP-1), a known oncogene and constitutively active CD40 homolog. Variation in the C terminal region of LMP-1 has been linked to NPC pathogenesis, but little is known regarding LMP-1 variation and eBL.

Crystal structure of the yeast vacuolar ATPase heterotrimeric EGC(head) peripheral stalk complex.

Vacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V₁-ATPase and V₀ proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit "a" and three "peripheral stalks," each made of a heterodimer of E and G subunits. Here, we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (C(head)).

Subunit interactions at the V1-Vo interface in yeast vacuolar ATPase.

Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V(1)-ATPase and V(o)-proton channel domains. We found previously that the head domain of the single copy C subunit (C(head)) binds one subunit EG heterodimer with high affinity (Oot, R.A.

Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts.

In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome∼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5.

Domain characterization and interaction of the yeast vacuolar ATPase subunit C with the peripheral stator stalk subunits E and G.

The proton pumping activity of the eukaryotic vacuolar ATPase (V-ATPase) is regulated by a unique mechanism that involves reversible enzyme dissociation. In yeast, under conditions of nutrient depletion, the soluble catalytic V(1) sector disengages from the membrane integral V(o), and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V(1) subunit, C, is released into the cytosol.

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces.

Aim: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot.

Methods And Results: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample.

Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep.

Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically.

Cholesterol granuloma of the petrous apex: MR and CT evaluation.

We reviewed the clinical, CT, and MR findings in seven consecutive patients who had a total of nine cholesterol granulomas. Preoperative MR scans were available for five of the seven patients; two patients were studied with MR after treatment only (one had a recurrent lesion and the other was asymptomatic at the time of study). Preoperative CT scans were available for all patients, except one patient who was examined after developing a symptomatic recurrence.

The role of MR and CT in evaluating clival chordomas and chondrosarcomas.

Sixteen chordomas and nine chondrosarcomas of the clivus were evaluated with CT and MR either before (22 cases) or after (three cases) treatment with proton beam irradiation. The ability of these imaging techniques to provide information necessary to direct patient treatment was studied. The tumor was detected and its gross margins were identified by both techniques in all instances.

Regrowth patterns of glioblastoma multiforme related to planning of interstitial brachytherapy radiation fields.

Recognition of the local nature of glioblastoma has generated an increasing interest in treatment using radioactive implants (interstitial brachytherapy). A key issue in such implantation is the configuration of the radiation field in relation to the resected tumor. In particular, should radiation be provided to the area from which the tumor has been resected? To clarify this issue, we evaluated patterns of tumor regrowth into this resected area in 62 patients.

Susceptibility induced MR line broadening: applications to brain iron mapping.

Magnetic susceptibility variations due to the presence of iron in neural tissue can result in a shift of local resonance frequency, decreased T2 resulting from water diffusion through local field gradients, and line broadening due to field inhomogeneity within a voxel. In this study, modified spin echo phase contrast pulse sequences were used to map proton resonance line widths in phantoms and in vivo. In agar gels containing varying concentrations of Fe3O4 (magnetite), line broadening mechanisms permitted accurate spatial localization of iron deposits from measurement of local resonance line widths.

The evolution of neonatal herpes encephalitis as demonstrated by cranial ultrasound with CT correlation.

Three neonates with herpes encephalitis had serial cranial ultrasound and CT studies performed during their hospitalization. Initially, subtle changes of diffuse brain edema were present but with no ventricular compression. A second phase revealed progressive brain edema with ventricular compression.