Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.

Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1

L’ubiquitination est une modification post-traductionnelle qui joue un rôle majeur dans la régulation d’une multitude de processus cellulaires. Dans cette thèse, je discuterai de la caractérisation de deux protéines, BRCA1 et BAP1, soit deux suppresseurs de tumeurs fonctionnellement reliés. BRCA1, une ubiquitine ligase qui catalyse la liaison de l’ubiquitine à une protéine cible, est mutée dans les cancers du sein et de l'ovaire. Il est bien établi que cette protéine aide à maintenir la stabilité génomique suite à un bris double brin de l’ADN (BDB), et ce, à l’aide d’un mécanisme de réparation bien caractérisé appelé recombinaison homologue. Cependant, les mécanismes de régulation de BRCA1 suite à des stresses génotoxiques n’impliquant pas directement un BDB ne sont pas pleinement élucidés. Nous avons démontré que BRCA1 est régulée par dégradation protéasomale suite à une exposition des cellules à deux agents génotoxiques reconnus pour ne pas directement générer des BDBs, soit les rayons UV, qui provoquent la distorsion de l’hélice d’ADN, et le méthyle méthanesulfonate (MMS), qui entraîne l’alkylation de l’ADN. La dégradation de BRCA1 est réversible et indépendante des kinases associées à la voie des PI3 kinase, soit ATM, ATR et DNA-PK, protéines qui sont rapidement activées par les dommages à l’ADN. Nous proposons que la dégradation de BRCA1 prévienne son recrutement intempestif, ainsi que celui des facteurs qui lui sont associés, à des sites de dommages d’ADN qui ne sont pas des BDBs, et que cette régulation coordonne la réparation de l’ADN. L’enzyme de déubiquitination BAP1 a initialement été identifiée comme une protéine capable d’interagir avec BRCA1 et de réguler sa fonction. Elle est également connue pour sa capacité à se lier avec les protéines du groupe Polycomb, ASXL1 et ASXL2. Cependant, l’importance de ces interactions n’a toujours pas été établie. Nous avons démontré que BAP1 forme deux complexes protéiques mutuellement exclusifs avec ASXL1 et ASXL2. Ces interactions sont critiques pour la liaison de BAP1 à l’ubiquitine ainsi que pour la stimulation de son activité enzymatique envers l’histone H2A. Nous avons également identifié des mutations de BAP1 dérivées de cancers qui empêchent à la fois son interaction avec ASXL1 et AXSL2, et son activité de déubiquitinase, ce qui fournit un lien mécanistique direct entre la déubiquitination de H2A et la tumorigenèse. Élucider les mécanismes de régulation de BRCA1 et BAP1 menera à une meilleure compréhension de leurs rôles de suppresseurs de tumeurs, permettant ainsi d’établir de nouvelles stratégies de diagnostic et traitement du cancer.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

The rat Na+-sulfate cotransporter rNaS2: functional characterization, tissue distribution, and gene (slc13a4) structure

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K-M for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0 +/- 0.7, suggesting a Na+:SO42- stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.

Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na+-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate. selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K, for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na+ kinetics determined a Hill coefficient of 1.6 +/- 0.6, suggesting a Na: SO42- stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region. (C) 2004 Elsevier Inc. All rights reserved.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

Functional characterization of GABAergic pallidopallidal and striatopallidal synapses in the rat globus pallidus in vitro

As a central integrator of basal ganglia function, the external segment of the globus pallidus (GP) plays a critical role in the control of voluntary movement. Driven by intrinsic mechanisms and excitatory glutamatergic inputs from the subthalamic nucleus, GP neurons receive GABAergic inhibitory input from the striatum (Str-GP) and from local collaterals of neighbouring pallidal neurons (GP-GP). Here we provide electrophysiological evidence for functional differences between these two inhibitory inputs. The basic synaptic characteristics of GP-GP and Str-GP GABAergic synapses were studied using whole-cell recordings with paired-pulse and train stimulation protocols and variance-mean (VM) analysis. We found (i) IPSC kinetics are consistent with local collaterals innervating the soma and proximal dendrites of GP neurons whereas striatal inputs innervate more distal regions. (ii) Compared to GP-GP synapses Str-GP synapses have a greater paired-pulse ratio, indicative of a lower probability of release. This was confirmed using VM analysis. (iii) In response to 20 and 50 Hz train stimulation, GP-GP synapses are weakly facilitatory in 1 mm external calcium and depressant in 2.4 mm calcium. This is in contrast to Str-GP synapses which display facilitation under both conditions. This is the first quantitative study comparing the properties of GP-GP and Str-GP synapses. The results are consistent with the differential location of these inhibitory synapses and subtle differences in their release probability which underpin stable GP-GP responses and robust short-term facilitation of Str-GP responses. These fundamental differences may provide the physiological basis for functional specialization.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM(2) receptor, whereas when paired with the calcitonin receptor AMY(3) receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT((a))). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM(2) and AMY(3(a)) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Functional characterization of SjB10, an intracellular serpin from Schistosoma japonicum

Serine protease inhibitors (serpin) play essential roles in many organisms. Mammalian serpins regulate the blood coagulation, fibrinolysis, inflammation and complement activation pathways. In parasitic helminths, serpins are less well characterized, but may also be involved in evasion of the host immune response. In this study, a Schistosoma japonicum serpin (SjB10), containing a 1212 bp open reading frame (ORF), was cloned, expressed and functionally characterized. Sequence analysis, comparative modelling and structural-based alignment revealed that SjB10 contains the essential structural motifs and consensus secondary structures of inhibitory serpins. Transcriptional profiling demonstrated that SjB10 is expressed in adult males, schistosomula and eggs but particularly in the cercariae, suggesting a possible role in cercarial penetration of mammalian host skin. Recombinant SjB10 (rSjB10) inhibited pancreatic elastase (PE) in a dose-dependent manner. rSjB10 was recognized strongly by experimentally infected rat sera indicating that native SjB10 is released into host tissue and induces an immune response. By immunochemistry, SjB10 localized in the S. japonicum adult foregut and extra-embryonic layer of the egg. This study provides a comprehensive demonstration of sequence and structural-based analysis of a functional S. japonicum serpin. Furthermore, our findings suggest that SjB10 may be associated with important functional roles in S. japonicum particularly in host-parasite interactions.

Molecular and Functional Characterization of Antimicrobial Peptides in Crustaceans

Most living organisms are constantly exposed to potentially harmful pathogens. It is the immune system of the organism that enables it to survive in an environment loaded with dangerous pathogenic microorganisms. The innate immunity provides organisms with a rapid and non-specific first line of defense against pathogens. It includes physical barriers such as skin and mucous membranes and chemical barriers including the high acidity of gastric juice, and specialized soluble molecules that possess antimicrobial activity. One of the well-known innate immune defense mechanisms is the production of antimicrobial substances by specific cells or tissues of the organisms. Antimicrobial peptides (AMPs) are such natural substances that

Molecular and Functional Characterization of Histone Derived Antimicrobial Peptides from Marine Organisms

Antimicrobial peptides (AMPs) are gene encoded, small sized, generally cationic, amphiphathic peptides characterized by antimicrobial activity against bacteria, fungi, viruses and other pathogens. They are a major component of the innate immune defense system of almost all living organisms, ranging from bacteria to humans and represent the first line of defense against the invading microbial pathogens (Boman, 1995; Zasloff, 2002). Antimicrobial peptides represent a heterogeneous group displaying multiple modes of action that are determined by the sequence and concentration of peptides. Their remarkable specificity for prokaryotes with low toxicity for eukaryotic cells has favored their investigation and exploitation as new antibiotics