Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

α4β2 nicotinic acetylcholine receptors in the early postnatal mouse superior cervical ganglion

Heteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4-containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4-containing receptors constitute about 20% of hetero-oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4-containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2-double knockout (KO) mice removes all α4-containing receptors, suggesting that in the postnatal mouse SCG, α4 co-assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up-regulated in the SCG of P3 α5β4-double KO mice, where they make up about 50% of receptors that bind [(3) H]-epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4-double KO mice maintained for one to two days in culture comprise <10% of α4β2 and >90% of α3β2, as determined by patch clamp recordings with α4β2- and α3β2-specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (±α5) represent about 62% of receptors, whereas 17% are α3β2β4, and 21% are α4β2 (±α5) receptors.     

The differential amino acid requirement within osteopontin in α4 and α9 integrin-mediated cell binding and migration

Osteopontin (OPN) contains at least two major integrin recognition domains, Arg159-Gly-Asp161 (RGD) and Ser162-Val-Val-Tyr-Gly-Leu-Arg168 (SVVYGLR), recognized by αvβ3 and α5β1 and α4 and α9 integrins, respectively. OPN is specifically cleaved by thrombin and matrix metalloproteinase (MMP)-3 or MMP-7 at a position of Arg168/Ser169 (R/S) and Gly166/Leu167 (G/L), respectively. We in this study examined the requirement of residues within SVVYGLR for the α4 and α9 integrin recognition and how MMP-cleavage influences the integrin recognition. The residues, Val164, Tyr165, and Leu167 are critical for α4 and α9 integrin recognition in both cell adhesion and cell migration. The residue Arg168 is additionally required for α9 integrin recognition in cell adhesion and this explains why α9 integrin binds to only thrombin cleaved form of OPN. α4 integrin is able to bind to SVVYG (MMP-cleaved form of RAA OPN-N half), while α9 integrin is not, supporting the above notion that Arg168 is additionally required for α9 integrin-mediated cell adhesion. The residue Val163 is important for α4, but not for α9 integrin recognition in cell migration. Importantly, we found that the replacement of Arg168 by Ala (R168A mutant) induces the augmentation of cell migration via α4 and α9 integrins.     

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation

BackgroundGamma-aminobutyric acid A (GABA_A) receptors have been implicated in anxiety and epileptic disorders.Hypothesis/PurposeThis study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders.MethodsStigmasterol was evaluated on various recombinant GABA_A receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2β2γ2L, α4β3δ, and α4β3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABA_A α2β2γ2L, α4β3, and α4β3δ complexes by in silico docking.ResultsStigmasterol enhanced GABA-induced currents at ternary α2β2γ2L, α4β3δ, and binary α4β3 GABA_AR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4β3δ was significantly higher compared to the binary α4β3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4β3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of β3 at α4β3δ complex. In in vivo studies, Stigmasterol (0.5–3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism.ConclusionTo our knowledge, this is the first study reporting the positive modulation of GABA_A receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.     

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.     

α-cyano-4-hydroxycinnamate impairs pancreatic cancer cells by stimulating the p38 signaling pathway

Multiple studies are currently targeting dysregulated cancer cell metabolism with distinct combinations of inhibitors. In this study, we evaluated in pancreatic cancer cells metformin, which blocks oxidative phosphorylation, in combination with α-cyano-4-hydroxycinnamate, which has been reported to inhibit the export of lactate from the cytosol. The combination of metformin with α-cyano-4-hydroxycinnamate had a major inhibitory effect on the migration of 6606PDA cells. Monotherapy with α-cyano-4-hydroxycinnamate and especially the combination with metformin also caused significant inhibition of cell proliferation and induced cell death. α-cyano-4-hydroxycinnamate in combination with metformin reduced the export of lactate significantly, whereas α-cyano-4-hydroxycinnamate monotherapy only modestly influenced lactate export. None of these two drugs inhibited the expression of distinct glycolytic enzymes. Interestingly, α-cyano-4-hydroxycinnamate rather inhibited the ERK and very strongly stimulated the p38 signaling pathway in 6606PDA as well as in 7265PDA cells. In addition, the inhibition of the p38 signaling pathway by PH-797804 partially reversed the effect of α-cyano-4-hydroxycinnamate on cell apoptosis in both cell lines. We conclude that α-cyano-4-hydroxycinnamate monotherapy and especially the combinatorial therapy with metformin has strong anti-cancerous effects. α-cyano-4-hydroxycinnamate causes cancer cell apoptosis by a novel mechanism for this drug, namely the stimulation of the p38 signaling pathway.     

The CC' and DE loops in Ig domains 1 and 2 of MAdCAM-1 play different roles in MAdCAM-1 binding to low- and high-affinity integrin alpha4beta7

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4β7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4β7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4β7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4β7. The Asp-42 in the CC' loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4β7. The other CC' loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4β7 and α4β7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4β7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4β7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4β7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4β7. Moreover, DE loop residues were important for stabilizing the low-affinity α4β7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC' and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4β7 and suggest that the inactive α4β7 and α4β7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.     

Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high-pressure Fourier transform infrared spectroscopy

It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.     

NFAT2 regulates COX-2 expression and modulates the integrin repertoire in endothelial cells at the crossroads of angiogenesis and inflammation

The mechanisms controlling the switch between the pro-angiogenic and pro-inflammatory states of endothelial cells are still poorly understood. In this paper, we show that: (a) COX-2 expression induced by VEGF-A is NFAT2-dependent; and (b) the integrin profile in endothelial cells induced by the pro-angiogenic VEGF-A is distinct from that brought on by the inflammatory cytokine TNF-α. Two groups of integrin subunits specifically upregulated over time by both cytokines were identified using RT-PCR and Western Immunoblotting. The first group included α4, α5, α6, and β5 subunits that were upregulated by VEGF-A; the second group consisted of αV and β3 induced by TNF-α. Both cytokines significantly enhanced the expression of β1 and modulated α2 mRNA. In contrast to TNF-α, VEGF-A induction of integrin subunits depended on the activation of the calcineurin/NFAT pathway. Both calcineurin inhibitors (cyclosporineA and 11R-VIVIT) and downregulation of NFAT2 with specific siRNA decreased induction of integrin subunits. This process of induction could be increased by upregulation of NFAT2 by pBJ5-NFAT2 transfection. This suggests that NFAT2 mediates VEGF-induced upregulation of integrin subunit synthesis by providing a constant supply of newly synthesized “refreshed” mature integrin receptors, particularly α2β1, α5β1, α4β1, α6β1 and αVβ5, which are involved at different stages of angiogenesis.     

The α-glucosidases of Dictyostelium discoideum: I. Identification and characterization

We have identified four isozymes of α-glucosidase in the cellular slime mold, Dictyostelium discoideum. The isozymes can be distinguished by their physical and enzymatic properties. α-Glucosidase-1, α-glucosidase-2, and α-gluocosidase-3 are all present in vegetative cells, while α-glucosidase-4 is present only after the cells have proceeded through aggregation. Three of the four enzymes, α-glucosidase-1, α-glucosidase-3, and α-glucosidase-4, have acidic pH optima of 3.5, 2.2, and 4.0, respectively. In contrast, α-glucosidase-2 has a neutral pH optimum, 7.25. α-Glucosidase-1, α-glucosidase-2, and α-glucosidase-3 are distinguishable by electrophoresis in native polyacrylamide gels. α-Glucosidase-4 comigrates with α-glucosidase-2 on native gels but they can be resolved by isoelectric focusing. The isozymes also differ with respect to affinity for the substrates p-nitrophenyl-α-d-glucoside and 4-methyl-umbelliferyl-α-d-glucopyranoside and the relative maximal rates of hydrolysis of these substrates. α-Glucosidases-1, -2, and -4 have apparent K_m's in the millimolar range while the apparent K_m of α-glucosidase-3 for p-nitrophenyl-α-d-glucoside is much higher. This may suggest that isozyme 3 is an endoglycosidase or may have greater affinity for other sugar substrates. α-Glucosidase-1 is the major isozyme in vegetative cells.     

Altered chaperone-like activity of alpha-crystallins promotes cataractogenesis

Despite the enormous number of studies demonstrating changes in the chaperone-like activity of α-crystallins in vitro, little is known about how these changes influence life-long lens transparency in vivo. Using the γB-crystallin I4F mutant protein as a target for αA-crystallins, we examined how cataract phenotypes are modulated by interactions between α-crystallins with altered chaperone-like activities and γB-I4F proteins in vivo. Double heterozygous α-crystallin knock-out αA(+/-) αB(+/-) mice with a decreased amount of α-crystallins were used to simulate reduced total α-crystallin chaperone-like activity in vivo. We found that triple heterozygous αA(+/-) αB(+/-) γB(I4F/+) mice developed more severe whole cataracts than heterozygous γB(I4F/+) mice. Thus, total chaperone-like activity of α-crystallins is important for maintaining lens transparency. We further tested whether mutant αA-crystallin Y118D proteins with increased chaperone-like activity influenced the whole cataract caused by the γB-I4F mutation. Unexpectedly, compound αA(Y118D/+) γB(I4F/+) mutant lenses displayed severe nuclear cataracts, whereas the lens cortex remained unaffected. Thus, the synergistic effect of αA-Y118D and γB-I4F mutant proteins is detrimental to the transparency only in the lens core. α-Crystallins with different chaperone-like activities are likely required in the lens cortex and nucleus for maintaining transparency.     

Mnk mediates integrin α6β4-dependent eIF4E phosphorylation and translation of VEGF mRNA

It was previously shown that integrin α6β4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6β4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6β4 and mediates the α6β4 signaling, important for translational control. Integrin α6β4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6β4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6β4 level is high.     

An extracellular RRR motif flanking the M1 transmembrane domain governs the biogenesis of homomeric neuronal nicotinic acetylcholine receptors

We have previously demonstrated that the highly conserved R209, that flanks the M1 transmembrane segment of nicotinic acetylcholine (ACh) receptors, is required for the transport of assembled homomeric neuronal α7 nicotinic ACh receptors to the cell surface. In the present paper we show that basic residues at positions 208 and 210 are necessary for the assembly of α7 receptors. On the contrary, a basic residue at position 210 of α3 subunit decreases the assembly of heteromeric neuronal α3β4 nicotinic ACh receptors. A basic residue at position 210 of the β4 subunit slightly decreases α3β4 receptor expression. We conclude that a pre-M1 RRR motif is necessary for the biogenesis of homomeric α-bungarotoxin-sensitive neuronal α7 nicotinic ACh receptors.     

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

Background

AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).

Methods

Recombinant AHSP, WT α-Hb:AHSP and α-Hb^HE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.

Results

AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.

Conclusion

α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.

General significance

The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.     

Methionine sulfoxide reductase A (MsrA) restores α-crystallin chaperone activity lost upon methionine oxidation

Background

Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and α-crystallin which is a two-subunit (αA and αB) chaperone. Here, we tested whether PMSO formation damages α-crystallin chaperone function and whether MsrA could repair PMSO-α-crystallin.

Methods

Total α-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-α-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of α-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined.

Results

PMSO oxidation of total α-crystallin (met 138 of αA and met 68 of αB) resulted in loss of α-crystallin chaperone activity. MsrA treatment of PMSO-α-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-α-crystallin.

Conclusions

Methionine oxidation damages α-crystallin chaperone function and MsrA can repair PMSO-α-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of α-crystallin methionines in the lens.

Significance

Methionine oxidation of α-crystallin in combination with loss of MsrA repair causes loss of α-crystallin chaperone function. Since increased PMSO levels and loss of α-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.     

Structural specializations of α(4)β(7), an integrin that mediates rolling adhesion

The lymphocyte homing receptor integrin α(4)β(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)β(1) and α(4)β(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)β(7). A long binding groove at the α(4)-β(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)β(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)β(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.