Mg-ATPase Activity Research Articles

Tributyltin-driven enhancement of the DCCD insensitive Mg-ATPase activity in mussel digestive gland mitochondria

The lipophilic pollutant tributyltin (TBT), other than inhibiting the DCCD (N,N′-dicyclohexylcarbodiimide) and oligomycin-sensitive Mg-ATPase activities in digestive gland mitochondria from the Mediterranean mussel Mytilus galloprovincialis, at higher than 1.0 μM concentrations in vitro promotes an increase in the ATPase activity fraction refractory to inhibitors of FO moiety, namely oligomycin and DCCD. By exploring the mechanisms involved in the TBT promoted enzyme desensitization to DCCD, we pointed out intriguing differences in the enzyme desensitization to the two inhibitors. Differently from oligomycin, the TBT promoted enzyme desensitization to DCCD is independent of the redox state of thiol groups of the enzyme complex and strongly temperature dependent, being significantly elicited only at temperatures above the break of the Arrhenius plots (around 18 °C). Such differences may cast light on multiple TBT interaction modes with the enzyme complex. The TBT-driven increase in the activation energy of the Mg-ATPase activities insensitive to inhibitors of FO sector suggests that the temperature-dependent incorporation of the lipophilic toxicant within the lipid bilayer may deeply affect the membrane-bound complex functionality. Accordingly, incorporated TBT may cause structural changes in the intramembrane FO subunits, thus weakening or even preventing DCCD binding to the enzyme complex.

Vascular smooth muscle myosin light chain diphosphorylation: Mechanism, function, and pathological implications

Smooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC(20) ) catalyzed by Ca(2+) /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrin-linked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations ∼3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC(20) homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC(20) diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC(20) diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.

Identification of Calmodulin and MlcC as Light Chains for Dictyostelium Myosin-I Isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.

Drosophila melanogaster Myosin-18 Represents a Highly Divergent Motor with Actin Tethering Properties

The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with Kd values of 2.61 and 1.04 μm, respectively, but only about 50–75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein.

Some Kinetic Singularities of Mg2+-Dependent Mn-ATPase in Rat Brain Synaptic Membranes

Mn(2+) stimulated change of Mg-ATPase activity has been found in the synaptic fraction of rat brain that was named Mn-ATPase. Investigation of the molecular mechanism has shown that Mn-ATPase is a multi-sited enzyme system whose minimum functional unit is a dimer. Its substrate is the MgATP complex. The number of sites for Mn(2+) as for essential activators and that of full-effect inhibitors are equal, n = m = 1. Studying regulation of the Mn-ATPase system by Mg(2+) has shown that Mg(2+) represents a double-sided effect modifier, namely, it activates the enzyme system at low concentration but inhibits at high concentration. Supposedly, binding-release of MgATP and Mg(2+) from the enzyme would be performed by a randomized mechanism. When analyzing experiments by using the kinetic method of complex curves, a "minimal model" for Mn-ATPase has been created.

Interaction Between Troponin and Myosin Enhances Contractile Activity of Myosin in Cardiac Muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.

The Enzymatic Motor Activity of Nonmuscle Myosin II-B is not Critical for Cardiac Myocyte Cytokinesis

Nonmuscle myosin (NM) II is believed to drive contractile ring constriction during cytokinesis and NM II-B is the major nonmuscle myosin II isoform expressed in cardiac myocytes. Ablation of NM II-B in mice resulted in premature binucleation in cardiac myocytes consistent with a role for NM II-B in cytokinesis. Surprisingly binucleation can be rescued by expressing a motor-impaired mutant form of NM II-B (R709C) in the mouse heart, suggesting that NM II-B enzymatic motor activity is not essential for cytokinesis. We tested this hypothesis directly by using a COS-7 cell line which resembles cardiac myocytes in containing only NM II-B and small quantities of NM II-C (<7%) but no NM II-A. Previous work had shown that using siRNA to deplete NMHC II-B in this cell line resulted in multinucleation due to a failure in cytokinesis. We now show that COS-7 cell multinucleation can be rescued by transfecting with mutant forms of NM II-B (R709C) or NM II-A (N93K). These two mutant NM IIs have previously been shown to have marked reductions in their actin-activated MgATPase activity (70% in the case of NM II-B and 96% in the case of NM II-A) and neither could propel actin filaments in an in vitro motility assay. Importantly, cytokinesis is inhibited by the myosin II inhibitor blebbistatin in COS-7 cells expressing either wild-type NM II-B or the mutant form of NM II-A (or II-B). Whereas blebbistatin blocks myosin in an actin-detached state, the mutant NM IIs (R709C II-B and N93K II-A) have no defect in their actin-binding activity. Our results therefore argue that the role of NM II in cytokinesis depends more on its actin binding (cross-linking) property than its enzymatic Mg2+-ATPase activity.

Cardiac Actin Capping Protein Reduction and Protein Kinase C Inhibition Maintain Myofilament Function During Cardioplegic Arrest

Background: Heart transplantation is associated with cold, cardioplegic arrest that impairs myocardial function. Protein Kinase C (PKC) suppression of myofilaments may contribute to this dysfunction. CapZ-deficient cardiac myofilaments are unresponsive to PKC. We hypothesized that myofilaments from CapZ-deficient transgenic hearts are resistant to cardioplegic dysfunction and that PKC inhibition improves function. Methods: Heart function was assessed using a Langendorff apparatus. Myofilaments isolated from murine hearts were assessed with an actomyosin MgATPase assay and protein phosphorylation gels. PKC activation was examined by immunoblotting. Results: Wildtype hearts showed impaired function after cardioplegic arrest. CapZ-deficient transgenic mouse hearts performed significantly better after 1 h cardioplegia than wildtype hearts, but not after 4 h cardioplegic arrest. Wildtype myofilaments had depressed activation at 1 and 4 h cardioplegic arrest, as demonstrated by reduced actomyosin MgATPase activity. CapZ-deficient myofilaments showed no reduced actomyosin MgATPase activity at either time. Troponin I (TnI) phosphorylation increased by approximately 20% at 1 and 4 h in wildtype mice. Myosin binding protein C (MyBP-C),and troponin T (TnT) phosphorylation increased by less than 10% at 1 h, and tended to rise at 4 h. Myofilament protein phosphorylation was largely unchanged in CapZ-deficient hearts at 1 h, but MyBP-C tended to be dephosphorylated at 4 h cardioplegic arrest. Myofilament-associated PKC-α, -βII, -δ, and -ε increased at 1 and 4 h cardioplegia in wildtype hearts, whereas only PKC-α increased in transgenic myofilaments at 1 h. PKC inhibition abolished the cardioplegic-dependent changes in actomyosin MgATPase activity and TnI phosphorylation of wildtype myofilaments. Conclusions: We demonstrate a direct link between PKC activation and myofilament dysfunction associated with cold, cardioplegic arrest. Moreover, we show for the first time a cardioprotective benefit of decreased cardiac CapZ.

Dual Regulatory Functions of the Thin Filament Revealed by Replacement of the Troponin I Inhibitory Peptide with a Linker

Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.

C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration

Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca2+-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland

Tri- n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di- n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC 50 = 0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F 0, either in the form of free enzyme or of enzyme–substrate complex (K i = K′ i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg 2+ cofactor, may bind strongly to F 1 subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation.

Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay

Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for ‘ pCa–velocity’ relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin.

Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function

The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.

Tributyltin inhibits the oligomycin-sensitive Mg-ATPase activity in Mytilus galloprovincialis digestive gland mitochondria

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filter-feeding molluscs. TBT is known to act as a membrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM (203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μM TBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.

Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally

Nonmuscle myosin II plays a crucial role in a variety of cellular processes (e.g., polarity formation, cell motility, and cytokinesis). It is composed of two heavy chains, two regulatory light chains and two essential light chains. The ATPase activity of the myosin II motor domain is regulated through phosphorylation of the regulatory light chain (RLC) by myosin light chain kinase. To study myosin function and localization in cellular processes, GFP-fused RLCs are widely used; however, the exact kinetic properties of myosins with bound GFP-RLC are poorly described. More importantly, it has not been shown that a regulatory light chain fused at its N-terminus with GFP can maintain the normal phosphorylation-dependent regulation of nonmuscle myosin or serve as a substrate for myosin light chain kinase. We coexpressed N-terminal GFP-RLC with a heavy meromyosin (HMM)-like fragment of nonmuscle myosin IIA and essential light chain to characterize the phosphorylation dynamics and in vitro kinetic properties of the resulting HMM. Myosin light chain kinase phosphorylates the GFP-RLC bound to HMM IIA with the same V(max) as it does the wild type RLC bound to HMM IIA, but the K(m) is about two fold higher for the GFP fusion protein, meaning that it is a somewhat poorer substrate. The steady-state actin-activated MgATPase activity of the GFP-RLC HMM is very low in the absence of phosphorylation demonstrating that the GFP moiety does not prevent formation of the off state. The actin-activated MgATPase activity of phosphorylated GFP-RLC-HMM and is about half that of wild type phosphorylated HMM. The ability of phosphorylated GFP-RLC-HMM to move actin filaments in the actin gliding assay is also slightly compromised. These data indicate that despite some kinetic differences the N-terminal GFP fusion to the regulatory light chain is a reasonable model system for studying myosin function in vivo.

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

3P135 ホッキガイ牽引筋たんぱく質成分およびMg-ATPase活性のCa依存性の季節変化(筋肉(筋蛋白質・収縮),第48回日本生物物理学会年会)

3P135 ホッキガイ牽引筋たんぱく質成分およびMg-ATPase活性のCa依存性の季節変化(筋肉(筋蛋白質・収縮),第48回日本生物物理学会年会)

HGAL, a Lymphoma Prognostic Biomarker, Regulates Lymphocyte and Lymphoma Cell Motility by Modulation of RhoA Signaling Pathway.

Abstract 316Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein. Disclosures:No relevant conflicts of interest to declare.

Conserved Polar Residues Stabilize Transmembrane Domains and Promote Oligomerization in Human Nucleoside Triphosphate Diphosphohydrolase 3

Polar residues play essential roles in the functions of transmembrane helices by mediating and stabilizing their helical interactions. To investigate the structural and functional roles of the conserved polar residues in the N- and C-terminal transmembrane helices of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) (N-terminus, S33, S39, T41, and Q44; C-terminus, T490, T495, and C501), each was singly mutated to alanine. The mutant proteins were analyzed for enzymatic activities, glycosylation status, expression level, and Triton X-100 detergent sensitivity. The Q44A mutation decreased Mg-ATPase activity by approximately 70% and abolished Triton X-100 detergent inhibition of Ca-dependent nucleotidase activities while greatly attenuating Triton X-100 inhibition of Mg-dependent nucleotidase activities. The polar residues were also mutated to cysteine, singly and in pairs, to allow a disulfide cross-linking strategy to map potential inter- and intramolecular hydrogen bond interactions. The results support the centrality of Q44 for the strong intermolecular interactions driving the association of the N-terminal helices of two NTPDase3 monomers in a dimer, and the possibility that T41 may play a role in the specificity of this interaction. In addition, S33 and C501 form an intramolecular association, while S39 and T495 may contribute to helical interactions involved in forming higher-order oligomers. Lastly, Tween 20 substantially and selectively increases NTPDase3 activity, mediated by the transmembrane helices containing the conserved polar residues. Taken together, the data suggest a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, oligomeric NTPDase3. These interactions are important for proper protein expression, full enzymatic activity, and susceptibility to membrane perturbations.

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.