Yeqi Yao

Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction.

Vacuolar H+-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC50 of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage‐dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell‐cell interaction, and cell spreading. Treatment with EGF increased cell adhesion‐regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion‐regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle‐associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF‐induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion. J. Cell. Biochem. 77:569–583, 2000.

Luteolin inhibition of V‐ATPase a3–d2 interaction decreases osteoclast resorptive activity

V‐ATPase‐mediated acid secretion is required for osteoclast bone resorption. Osteoclasts are enriched in V‐ATPase a3 and d2 subunit isoforms, and disruption of either of their genes impairs bone resorption. Using purified fusion proteins of a3 N‐terminal domain (NTa3) and full‐length d subunits we determined in a solid‐phase binding assay that half‐maximal binding of d1 or d2 to immobilized NTa3 occurs at 3.1 ± 0.4 or 3.6 ± 0.6 nM, respectively, suggesting equally high‐affinity interactions. A high‐throughput modification of this assay was then used to screen chemical libraries for a3–d2 interaction inhibitors, and luteolin, a naturally occurring flavonoid, was identified, with half‐maximal inhibition at 2.4 ± 0.9 µM. Luteolin did not significantly affect NIH/3T3 or RAW 264.7 cell viability, nor did it affect cytokine‐induced osteoclastogenesis of RAW 264.7 cells or bone marrow mononuclear cells at concentrations ≤40 µM. Luteolin inhibited osteoclast bone resorption with an EC50 of approximately 2.5 µM, without affecting osteoclast actin ring formation. Luteolin‐treated osteoclasts produced deeper resorption pits, but with decreased surface area, resulting in overall decreased pit volume. Luteolin did not affect transcription, or protein levels, of V‐ATPase subunits a3, d2, and E, or V1V0 assembly. Previous work has shown that luteolin can be effective in reducing bone resorption, and our studies suggest that this effect of luteolin may be through disruption of osteoclast V‐ATPase a3–d2 interaction. We conclude that the V‐ATPase a3–d2 interaction is a viable target for novel anti‐resorptive therapeutics that potentially preserve osteoclast–osteoblast signaling important for bone remodeling. J. Cell. Biochem. 114: 929–941, 2013.

Topology, glycosylation and conformational changes in the membrane domain of the vacuolar H+‐ATPase a subunit

Published topological models of the integral membrane a subunit of the vacuolar proton‐translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino‐terminus and carboxyl‐terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V‐ATPase substrate, MgATP, and the V‐ATPase‐specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V‐ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle. J. Cell. Biochem. 114: 1474–1487, 2013.

N-Linked Glycosylation Is Required for Vacuolar H(+) -ATPase (V-ATPase) a4 Subunit Stability, Assembly, and Cell Surface Expression.

The a subunit is the largest of 14 different subunits that make up the V‐ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1–a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N‐glycosylation sites, and the a3 isoform has been directly shown to be N‐glycosylated. Here we ask if human a4 (ATP6V0A4) is N‐glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression‐vector system, to express cDNA constructs of epitope‐tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase‐treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4N489D, demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse‐chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N‐glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co‐immunoprecipitation studies suggested that a4N489D does not assemble with the V‐ATPase V1 domain. Taken together, these data show that N‐glycosylation plays a crucial role in a4 stability, and in V‐ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757–2768, 2016.

N-linked Glycosylation of a Subunit Isoforms is Critical for Vertebrate Vacuolar H+-ATPase (V-ATPase) Biosynthesis†

The a subunit of the V0 membrane‐integrated sector of human V‐ATPase has four isoforms, a1‐a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N‐glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N‐glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope‐tagged human a1‐a3 subunits, with or without mutations that convert Asn to Gln at putative N‐glycosylation sites. Expression and N‐glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N‐glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild‐type a subunits after enzymatic deglycosylation. Cycloheximide‐chase experiments showed that unglycosylated subunits were turned over at a higher rate than N‐glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co‐immunoprecipitation studies showed that they were unable to associate with the V‐ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N‐glycosylation is crucial in all four human V‐ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V‐ATPase complexes.

Molecular mechanisms of cutis laxa and distal renal tubular acidosis-causing mutations in V-ATPaseasubunits, ATP6V0A2 and ATP6V0A4

The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1–a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease–causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L. Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.