Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.     

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO₃ transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO₃ transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO₃⁻ currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO₃-dependent pH recovery from a CO₂-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO₂/HCO₃⁻-free solution that was abolished by Cl⁻ removal from the bath. In the presence of CO₂/HCO₃⁻, however, the outward current produced by D555E decreased only slightly after Cl⁻ removal. Starting from a Cl⁻-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO₂/HCO₃⁻. The substitution of Asp⁵⁵⁵ with an asparagine also produced a Cl⁻ current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO₃⁻. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp⁵⁵⁵ plays a role in HCO₃⁻ selectivity.The electrogenic Na/HCO₃ cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO₃⁻ across the plasma membrane (1–3). NBCe1 plays a role in transepithelial HCO₃⁻ movement and pH_i regulation in many tissues (4–6). NBCe1 is responsible for HCO₃⁻ reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO₃⁻ from the lumen through a series of reactions involving titration of HCO₃⁻ by H⁺ secretion via the apical Na/H exchanger, production of CO₂, and regeneration of HCO₃⁻ and H⁺ in the tubule cells. HCO₃⁻ then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na⁺/HCO₃⁻ exit is the stoichiometry of 1:3 Na⁺:HCO₃⁻, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na⁺:1HCO₃⁻ or 1Na⁺:2HCO₃⁻ causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO₃⁻ reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO₃ transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO₃ transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na⁺-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO₃ transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO₃⁻ permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO₃ transporters but not among electroneutral Na/HCO₃ transporters and examined their role in electrogenic Na/HCO₃ transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp⁵⁵⁵ of NBCe1 plays an important role in HCO₃⁻ selectivity.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO₂⁻) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H₂O and D₂O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H₂O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D₂O than in H₂O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.Denitrification is an anaerobic respiration pathway found in bacteria, archaea, and fungi, in which ATP synthesis is coupled to the sequential reduction of nitrate (NO₃⁻) and nitrite (NO₂⁻) (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (1–3).³ The first committed step in this reaction cascade is the formation of gaseous NO by nitrite reductase (NiR), the key enzyme of this pathway. Two distinct classes of periplasmic NiR are found in denitrifying bacteria, one containing cd₁ hemes as prosthetic groups (4–6) and the other utilizing two copper centers to catalyze the one-electron reduction of nitrite (7). Copper-containing NiRs are divided into two main groups according to the color of their oxidized type 1 copper center (T1Cu), with shades ranging from blue to green (3, 7). NiR from Alcaligenes xylosoxidans subsp. xylosoxidans (NCIMB 11015, AxNiR), which is analyzed in this study, is a member of the blue CuNiR group. The blue and green subclasses show a high degree of sequence similarity (70%) (8) and have similar trimeric structures with each monomer (∼36.5 kDa in AxNiR) consisting of two greek key β-barrel cupredoxin-like motifs as well as one long and two short α-helical regions (7, 9).Each NiR monomer contains two copper-binding sites per catalytic unit. One is a T1Cu center, which receives electrons from a physiological redox partner protein and is buried 7 Å beneath the protein surface (10), and the other copper is a type 2 center (T2Cu), constituting the catalytically active substrate-binding site (11). The physiological electron donor for the blue NiRs are the small copper protein azurin (14 kDa) (7) and cytochrome c₅₅₁ (7, 12, 13). The T1Cu, which is responsible for the color of NiR, serves as the electron delivery center and is coordinated by two histidine residues as well as one cysteine and one methionine residue. The catalytic T2Cu, which like all T2Cu centers has very weak optical bands, is ligated to three His residues and an H₂O/OH⁻ ligand in the resting state. This H₂O/OH⁻ ligand is held in place by hydrogen bonds to the active site residues, Asp-92 (AxNiR numbering) and His-249, and gets displaced by the substrate during catalytic turnover (14). The T2Cu is located at the base of a 13–14-Å substrate access channel at the interface of two monomers with one of the three His residues being part of the adjacent subunit (15, 16). The two copper centers are connected by a 12.6-Å covalent bridge provided by the T1Cu-coordinating Cys and by one of the T2Cu His ligands (17, 18). This linkage has been suggested to constitute the electron transfer (ET) pathway from the T1Cu center to the catalytically active T2Cu center via 11 covalent bonds (19).Intramolecular ET from T1- to T2Cu has been extensively examined using pulse radiolysis studies (7, 19–24). In a variety of NiR species, ET could be measured, both in the presence and absence of substrate, with observed ET rate constants (k_ET(obs)) ranging from ∼150 to ∼2000 s⁻¹. According to the Marcus semi-classical ET theory (25), the redox potentials (E′₀, redox midpoint potential at pH 7.0) of the copper centers affect both the thermodynamic equilibrium and the ET kinetics. In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (E′₀ (T1Cu) ∼240 mV and E′₀ (T2Cu) ∼230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (≥150 s⁻¹) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H₂O/OH⁻ ligand and unable to bind nitrite (26, 27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7, 16, 25, 27–30). However, k_ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitrite-bound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16, 25, 30, 31). These rearrangements include subtle changes in the Cys-His bridge linking T1- and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref. 29). Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19, 27, 29, 32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14, 16, 19, 26, 27, 33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34). This finding, however, contradicts the pulse radiolysis results at room temperature (see above), and the apparent discrepancy between solution studies and x-ray crystallographic data collected at cryogenic temperature remains to be resolved.The one-electron reduction of nitrite to NO involves two protons according to the chemical net equation NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O, if the T2Cu is ligated by an H₂O molecule in the resting state rather than an OH⁻ ion. Although the exact enzymatic mechanism is still somewhat controversial (35, 36), one suggested reaction sequence is given in Scheme 1. The potential participation of active site residues in catalyzing the proton transfer (PT) steps has been investigated by studying the pH dependence of NiR under steady-state conditions as well as by pulse radiolysis. The trends obtained for k_cat and k_ET(obs), are similar with pH optima between 5.2 and 6, indicating the involvement of two amino acid residues (21, 22, 37). Asp-92 and His-249 have been proposed as acid-base catalysts (18, 21, 22, 28, 38), and the abrupt drop in rates at increasing pH may indicate that OH⁻ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35, 40, 41).⁴Open in a separate windowSCHEME 1.A potential reaction mechanism proposed for CuNiRs. Adapted from Ref. 36. Nitrite is shown to bind to the oxidized T2Cu as nitrous acid, thus involving the first protonation step. It coordinates to the oxidized T2Cu center in a bidentate fashion. Following inter-copper ET yielding a reduced T2Cu center, the initially deprotonated Asp-92 accepts a proton, which is subsequently transferred to the substrate. His-249 may be a potential source of this second proton. PT and ET reactions may be reversible and they may be concerted rather than sequential as suggested by the arrows. See text for further information.There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET. An extensive analysis of the solvent kinetic isotope effect (SKIE) has been employed as a means of determining whether solvent-exchangeable protons and/or water molecules play a rate-limiting role in the catalytic turnover and/or in inter-copper ET.     

Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL^−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K_ATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL^−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL^−/− mice. Accordingly, isolated islets from ATGL^−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL^−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.Free fatty acids (FFA)⁵ and other lipid molecules are important for proper glucose-stimulated insulin secretion (GSIS) by β-cells. Thus, deprivation of fatty acids (FA) in vivo (1) diminishes GSIS, whereas a short term exposure to FFA enhances it (1–3). In contrast, a sustained provision of FA, particularly in the presence of high glucose in vitro, is detrimental to β-cells in that it reduces insulin gene expression (4) and secretion (5) and induces β-cell apoptosis (6). The FA supply to the β-cells can be from exogenous sources, such as plasma FFAs and lipoproteins, or endogenous sources, such as intracellular triglyceride (TG) stores. Studies from our laboratory (7–10) and others (11, 12) support the concept that the hydrolysis of endogenous TG plays an important role in fuel-induced insulin secretion because TG depletion with leptin (13) or inhibition of TG lipolysis by lipase inhibitors such as 3,5-dimethylpyrazole (7) or orlistat (11, 12) markedly curtail GSIS in rat islets. Furthermore, mice with β-cell-specific knock-out of hormone-sensitive lipase (HSL), which hydrolyzes both TG and diacylglycerol (DAG), show defective first phase GSIS in vivo and in vitro (14).Lipolysis is an integral part of an essential metabolic pathway, the TG/FFA cycle, in which FFA esterification onto a glycerol backbone leading to the synthesis of TG is followed by its hydrolysis with the release of the FFA that can then be re-esterified. Intracellular TG/FFA cycling is known to occur in adipose tissue of rats and humans (15, 16) and also in liver and skeletal muscle (17). It is generally described as a “futile cycle” as it leads to the net hydrolysis of ATP with the generation of heat (18). However, several studies have shown that this cycle has important functions in the cell. For instance, in brown adipose tissue, it contributes to overall thermogenesis (17, 19). In islets from the normoglycemic, hyperinsulinemic, obese Zucker fatty rat, increased GSIS is associated with increased glucose-stimulated lipolysis and FA esterification, indicating enhanced TG/FFA cycling (10). Stimulation of lipolysis by glucose has also been observed in isolated islets from normal rats (12) and HSL^−/− mice (8) indicating the presence of glucose-responsive TG/FFA cycling in pancreatic β-cells.The identity of the key lipases involved in the TG/FFA cycle in pancreatic islets is uncertain. HSL is expressed in islets (20), is up-regulated by long term treatment with elevated glucose (21), and is associated with insulin secretory granules (22). In addition, our earlier results suggested that elevated HSL expression correlates with augmented TG/FFA cycling in islets of Zucker fatty rats (10). However, it appears that other lipases may contribute to lipolysis and the regulation of GSIS in islet tissue. Thus, results from studies using HSL^−/− mice showed unaltered GSIS (8, 23), except in fasted male mice (8, 9) in which lipolysis was decreased but not abolished. Furthermore, HSL^−/− mice show residual TG lipase activity (8) indicating the presence of other TG lipases.Recently, adipocyte triglyceride lipase (ATGL; also known as Desnutrin, TTS-2, iPLA₂-ζ, and PNPLA2) (24–26) was found to account for most if not all of the residual lipolysis in HSL^−/− mice (26, 27). Two homologues of ATGL, Adiponutrin and GS2, have been described in adipocytes (24). All three enzymes contain a patatin-like domain with broad lipid acyl-hydrolase activity. However, it is not known if adiponutrin and GS2 are actually TG hydrolases. An additional lipase, TG hydrolase or carboxylesterase-3, has been identified in rat adipose tissue (28, 29). Although the hydrolysis of TG is catalyzed by all these lipases, HSL can hydrolyze both TG and DAG, the latter being a better substrate (30).In this study, we observed that besides HSL, ATGL (31), adiponutrin, and GS2 are expressed in rat islets and INS832/13 cells, with ATGL being the most abundant. We then focused on the role of ATGL in fuel-stimulated insulin secretion in two models, INS832/13 β-cells in which ATGL expression was reduced by RNA interference-knockdown (ATGL-KD) and ATGL^−/− mice.     

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca²⁺. The complex formed in the presence of Ca²⁺ was more stable than that without Ca²⁺. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca²⁺ and B. Removal of the Ca²⁺ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca²⁺ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca²⁺ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca²⁺ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca²⁺_, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca²⁺ but also borate and Ca²⁺ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth, although its primary function is not known (Loomis and Durst, 1992). Determining the sites of B in cells is required to identify its function. In cultured tobacco cells more than 80% of cellular B is in the cell wall (Matoh et al., 1993), whereas the membrane fraction (Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not contain a significant amount of B. In radish (Raphanus sativus L. cv Aokubi-daikon) root cell walls, B cross-links two RG-II regions of pectic polysaccharides through a borate-diol ester (Kobayashi et al., 1995, 1996). The association of B with RG-II has been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo (Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and red wine (Pellerin et al., 1996). In cultured tobacco cells the B associated with RG-II accounts for about 80% of the cell wall B (Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in higher plant cell walls (Matoh et al., 1996). Germanic acid, which partly substitutes for B in the growth of the B-deprived plants (Skok, 1957), also cross-links two RG-II chains (Kobayashi et al., 1997). These results suggest that the physiological role of B is to cross-link cell wall pectic polysaccharides in the RG-II region and thereby form a pectic network.It is believed that in the cell wall pectic polysaccharides are cross-linked with Ca²⁺, which binds to carboxyl groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the ability of B and Ca²⁺ to cross-link cell wall pectic polysaccharides needs to be evaluated. In this report we describe the B-RG-II complex of radish root and the role of B-RG-II and Ca²⁺ in the formation of a pectic network.     

Secreted Versus Membrane-anchored Collagenases: RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION*

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13^−/−, Mmp8^−/−, Mmp2^−/−, Mmp9^−/−, Mmp14^−/− and Mmp16^−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14^−/− or Mmp16^−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14^−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1–3). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2, 4). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4–6). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2, 7–13).To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP)² gene families (4, 14–18). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13, 15, 19). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or “melting” of the resulting collagen fragments at physiologic temperatures (4, 15). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of “gelatinases” (4, 15). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20–24).Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20, 25, 26). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13, 27–29). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion.