Regulation of the Bcas1 and Baiap3 transcripts in the subthalamic nucleus in mice recovering from MPTP toxicity.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure leads to significant and irreversible damage to dopaminergic neurons in both mice and humans. While MPTP exposure in humans causes permanent symptoms of Parkinson's disease, MPTP treated mice will recover behaviorally over a 3-week period. This mouse specific recovery might be linked to transcriptional changes in the basal ganglia enabling mice to maintain normal motor function in spite of low striatal dopamine levels. Laser microdissection was used to isolate the subthalamic nucleus from mice 7 and 28 days following MPTP exposure. High quality RNA was recovered and expressional analysis was performed on whole mouse genome microarrays. Identified regulated transcripts were validated in a separate batch of animals using quantitative PCR. Two transcripts with a significant regulation from days 7 to 28 in the MPTP treated groups, were identified: the brain specific angiogenesis inhibitor associated protein 3 (Baiap3) and the breast carcinoma amplified sequence 1 (Bcas1). Further studies of the molecular pathways involving these two transcripts may uncover processes in the subthalamic nucleus associated with the behavioral recovery observed after MPTP exposure.

Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor.

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors.

G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.

Pancreatic lipase structure-function relationships by domain exchange.

We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.

Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor.

A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and > 500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in that < or = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.

Construction of a high affinity zinc switch in the kappa-opioid receptor.

Very limited structural information is available concerning the superfamily of G-protein-coupled receptors with their seven-transmembrane segments. Recently a non-peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family.

Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine.

The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.

Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus.

The physiological role of pancreatic lipases has traditionally been assigned solely to triacylglyceride metabolism, while the digestion of phospholipids requires the presence of the pancreatic phospholipase A2, a 14-kDa enzyme unrelated to pancreatic lipases. However, in the guinea pig, it was observed that the pancreatic phospholipase A2 was absent and that a guinea pig pancreatic-lipase-related protein 2 (GPL-RP2) was responsible for phospholipase activity, in contrast to the situation observed in other mammalian species. As the guinea pig is a member of the hystricomorph rodents, it was of interest to investigate if other species within this evolutionary suborder display similar characteristics. The coypu (Myocastor coypus) also a member of the hystricomorph rodents, was chosen for further investigations. The cDNAs encoding two pancreatic lipases and a procolipase from the coypu were cloned, expressed and characterized. One lipase, CoPL-RP2, was identified as belonging to the RP2 subfamily, while the second, CoPL, was found to belong to the classical pancreatic lipase subfamily. Enzymic characterization and sequence data suggest a role for coypu colipase as a specific cofactor for CoPL, while this coypu colipase cannot be an important cofactor for CoPL-RP2 in vivo. Also, the new lipase cDNA sequences were used in a phylogentic analysis to reinvestigate the taxonomical position of the hystricomorph rodents (e.g. coypu and guinea pig) with respect to the myomorph rodents (e.g. rat and mouse).

Structure-function relationships in naturally occurring mutants of pancreatic lipase.

From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.

Evidence for a pancreatic lipase subfamily with new kinetic properties.

Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.

Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2.

Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.

One-step purification and characterization of human pancreatic lipase expressed in insect cells.

A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post-infection using both anti-HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation-exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N-terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re-activation by colipase.