Rescue of the temperature-sensitive, autosomal-recessive mutation R298S in the sodium-bicarbonate cotransporter NBCe1-A characterized by a weakened dimer and abnormal aggregation.

BACKGROUND: Band keratopathy, an ocular disease that is characterized by hypercalcemia and opaque bands across the cornea, has been associated with kidney disease. Type-II renal tubular acidosis (RTA), a condition in which the kidneys fail to recover bicarbonate (HCO3-) in the proximal tubule of the nephron, results in HCO3- wastage in the urine and low blood pH. The development of these diseases is associated with autosomal-recessive mutations in the Na+-coupled HCO3- cotransporter NBCe1-A located at the basolateral membranes of either cell type. METHODS: We provide insight into the devastating R298S mutation found in type-II RTA-afflicted individuals using confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in human corneal endothelial and proximal tubule cells and from in-depth biophysical studies of their cytoplasmic N-terminal domains (Nt and Nt-R298S), including Nt crystal structure, melting-temperature, and homodimer dissociation constant (KD) analyses. RESULTS: We illuminate and rescue trafficking defects of the R298S mutation of NBCe1-A. The KD for Nt monomer-dimer equilibrium is established. The KD for Nt-R298S is significantly higher, but immeasurable due to environmental factors (pH, temperature, concentration) that result in dimer instability leading to precipitation. The crystal structure of Nt-dimer shows that R298 is part of a putative substrate conduit and resides near the dimer interface held together by hydrogen-bond networks. CONCLUSIONS: The R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation. GENERAL SIGNIFICANCE: Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation.

Clinical significance of a point mutation in DNA polymerase beta (POLB) gene in gastric cancer.

Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC.

Direct evidence for calcineurin binding to the exon-7 loop of the sodium-bicarbonate cotransporter NBCn1.

The NaHCO3 cotransporter NBCn1 plays a role in neutralizing intracellular acid loads at the basolateral membrane in cells of the medullary thick ascending limb (mTAL). Calcineurin inhibitors (Cn-Is) are known to both downregulate NBCn1 expression in the distal nephron and cause renal tubular acidosis (RTA), a risk factor for nephrocalcinosis and nephrolithiasis. In this report, we provide a new perspective on concurrent studies of NBCn1 in various tissues by using cell-free binding assays to investigate the interaction of NBCn1 with the calcineurin (Cn) isoform PPP3CA. Surface plasmon resonance (SPR) analyses show that the protein domain Exon 7 (translated from cassette II of NBCn1) binds Cn with an equilibrium dissociation constant (KD) of 30 +/- 15 nm. Linked-reaction tests suggest that the binding involves a conformational change. Nested PCR reactions were used to show that NBCn1-Exon 7 splice variants with alternative N-termini regions are expressed in the kidney, as well as other tissues. Additionally, we discuss NBCn1-Exon 7 implication in acid-base balance and calcium crystallization in the kidney.

Epigenetics of progression of chronic kidney disease: fact or fantasy?

Epigenetic modifications are important in the normal functioning of the cell, from regulating dynamic expression of essential genes and associated proteins to repressing those that are unneeded. Epigenetic changes are essential for development and functioning of the kidney, and aberrant methylation, histone modifications, and expression of microRNA could lead to chronic kidney disease (CKD). Here, epigenetic modifications modulate transforming growth factor ß signaling, inflammation, profibrotic genes, and the epithelial-to-mesenchymal transition, promoting renal fibrosis and progression of CKD. Identification of these epigenetic changes is important because they are potentially reversible and may serve as therapeutic targets in the future to prevent subsequent renal fibrosis and CKD. In this review we discuss the different types of epigenetic control, methods to study epigenetic modifications, and how epigenetics promotes progression of CKD.

Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium-bicarbonate cotransporter NBCn1 (SLC4A7).

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus--MEAD in rat only, MERF in human only--as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3(-) transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.

X-ray diffraction studies on merohedrally twinned Δ1-62NtNBCe1-A crystals of the sodium/bicarbonate cotransporter.

NBCe1-A membrane-embedded macromolecules that cotransport sodium and bicarbonate ions across the bilayer serve to maintain acid-base homeostasis throughout the body. Defects result in a number of renal and eye disorders, including type-II renal tubular acidosis and cataracts. Here, crystals of a human truncated mutant of the cytoplasmic N-terminal domain of NBCe1 (Δ1-62NtNBCe1-A) are reported that diffract X-rays to 2.4 Å resolution. The crystal symmetry of Δ1-62NtNBCe1-A is of space group P31 with pseudo-P3121 symmetry and it has a hemihedral twin fraction of 33.0%. The crystals may provide insight into the pathogenic processes observed in a subset of patients with truncating and point mutations in the gene encoding NBCe1.

pH-sensitive self-associations of the N-terminal domain of NBCe1-A suggest a compact conformation under acidic intracellular conditions.

NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3 - ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane.

Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals.

The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with approximately 84% of their surface area buried) and to be surrounded by partially polar microenvironments (with approximately 43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (approximately 18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed.

Structural insights into the exchange domain of sec2p: expression, purification, crystallization, and preliminary x-ray diffraction data analysis.

Sec2p is an essential yeast gene and is part of the cell polarization process that leads to budding. The N-terminal domain of sec2p (Sec2pN)--the guanine-nucleotide exchange factor for sec4p--has been expressed in Escherichia coli, purified, and crystallized. Crystals belong to the space group P2(1) with unit cell dimensions 178.1 x 98.4 x 180.0 A, beta = 91.7( degrees ), and diffract synchrotron-generated X-rays to better than 3.6 A resolution. Pseudo-precession plots reveal a Laue symmetry of 2/m, corresponding to the aforementioned space group, and unusual weak diffraction in the approximately 5-7 A resolution range. The Matthews number calculations for a typical crystal density suggest a range of 28 to 64 molecules per asymmetric unit. Self-rotation and native Patterson calculations demonstrate a pure helical array of protein subunits. Based on the X-ray diffraction data analysis and amino-acid sequence alignments, the paper presents a hypothetical model of the exchange domain of sec2p as a pair of coiled-coil helices that binds to sec4p and facilitates nucleotide disassociation.

Preliminary X-ray diffraction analysis of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-A.

The N-terminal cytoplasmic domain of the Na+-coupled HCO_3- cotransporter NBCe1-A (NtNBCe1) has been linked with proximal renal tubular acidosis. In a previous purification study of recombinant NtNBCe1, crystal growth at a suboptimal protein concentration (<1 mg ml(-1)) yielded small single diamond-shaped crystals that diffracted poorly. In the present study, by increasing the protein concentration 50-fold, the crystal size was doubled and robustness was also improved. Crystal annealing made the crystals suitable for X-ray diffraction. The crystals either belong to space group P3(1)21 or P3(1) with pseudo P3(1)21 symmetry, with unit-cell parameters a = 51.7, b = 51.7, c = 200.6 A, alpha = beta = 90, gamma = 120 degrees , and diffract X-rays to 3.0 A resolution. The calculated Matthews number is 1.9 A3 Da(-1), with two monomers of molecular weight approximately 83 kDa in the asymmetric unit. The molecular- replacement packing solution shows that the molecules form dimers by a domain-swapping mechanism.

Expression and purification of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-A: structural insights from a generalized approach.

The cytoplasmic, N-terminal domain (Nt) of the electrogenic sodium/bicarbonate cotransporter--NBCe1--over-expresses in Escherichia coli and yields a large amount of soluble protein. A novel purification strategy, which involves a streptomycin precipitation, overcomes obstacles of instability and copurifying proteins, and leads to the first seen Nt-NBCe1 crystals. The purification procedure generally lends itself to the purification of Nts from other classes of the SLC4 family. Size-exclusion chromatography suggests that the Nt of NBCe1 as well as the Nt of other SLC4 members form dimers. A comparison of Nt-NBCe1 to SLC4 member Nt-AE1, based on purification properties and predicted secondary-structure sequence alignments, suggests a similar mechanism for dimer stabilization.

Effect of human carbonic anhydrase II on the activity of the human electrogenic Na/HCO3 cotransporter NBCe1-A in Xenopus oocytes.

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA.

The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.

The TB structural genomics consortium: providing a structural foundation for drug discovery.

Structural genomics, the large-scale determination of protein structures, promises to provide a broad structural foundation for drug discovery. The tuberculosis (TB) Structural Genomics Consortium is devoted to encouraging, coordinating, and facilitating the determination of structures of proteins from Mycobacterium tuberculosis and hopes to determine 400 TB protein structures over 5 years. The Consortium has determined structures of 28 proteins from TB to date. These protein structures are already providing a basis for drug discovery efforts.

Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation.

The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.