Christina Karlsson

Human neuropeptide Y signal peptide gain-of-function polymorphism is associated with increased body mass index: Possible mode of function

Neuropeptide Y (NPY) has been implicated in the control of food intake and energy balance based on many observations in animals. We have studied single nucleotide polymorphisms (SNPs) within the regulatory and coding sequences of the human NPY gene. One variant (1128 T>C), which causes an amino acid change from leucine to proline at codon 7 in the signal peptide of NPY, was associated with increased body mass index (BMI) in two separate Swedish populations of normal and overweight individuals. In vitro transcription and translation studies indicated the unlikelihood that this signal peptide variation affects the site of cleavage and targeting or uptake of NPY into the endoplasmic reticulum (ER). However, the mutant, and to a lesser extent the wild-type, signal peptide by themselves markedly potentiated NPY-induced food intake, as well as hypothalamic NPY receptor signaling. Our findings in humans strongly indicate that the NPY signaling system is implicated in body weight regulation and suggest a new and unexpected functional role of a signal peptide.

Avian alcohol dehydrogenase: the chicken liver enzyme. Primary structure, cDNA-cloning, and relationships to other alcohol dehydrogenases.

The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to gamma chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 microM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I less than III less than II, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme-binding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.

Zinc binding of alcohol and sorbitol dehydrogenases.

Zinc is an essential component of many enzymes, serving a role for catalytic activity or structural stability. The removal of catalytic zinc results in an inactive apoenzyme which, however, often retains the native tertiary structure. Structural zinc frequently contributes to the maintenance of the structure of oligomeric enzymes. The removal of zinc from such proteins therefore prevents subunit association. As discerned from zinc analysis of structurally investigated zinc metalloenzymes, the characteristics of a catalytic zinc-binding motif, in many cases, is a combination of three His/Glu/Asp/Cys residues and an activated H2O-molecule (Vallee & Auld, 1990). The spacers between the first and second ligands are short, typically 1–3 amino acids long (alcohol dehydrogenase and sorbitol dehydrogenase are exceptions with 21–25 residues). The second spacer, longer in nature, separates the second and third ligands by about 20–120 amino acid residues (Vallee & Auld, 1989). The structural zinc is necessary for activity only to the extent that the overall conformation of the enzyme affects its action. It serves as a cross-linking agent to stabilize structures (Berg, 1987). The observed pattern of ligands frequently encompasses four cysteine residues, closely spaced in the linear amino acid sequence (Vallee & Auld, 1990). Sorbitol dehydrogenase (SDH) harbours one catalytic zinc atom per subunit (Jeffery et al., 1984a). Because of the structural relationship between SDH and alcohol dehydrogenase (ADH), two of the three ligands to the zinc could be established early in SDH (Cys44 and His69, Jeffery et al., 1984b). The homology in the area around the third zinc ligand in SDH, though, was low toward ADH. The modelled structure of sheep SDH using the crystal structure of horse ADH as reference, gave the best fit with a glutamic acid residue (Glu 155) as the third zinc ligand (Eklund et al., 1985). In order to establish the exact nature of the third zinc ligand, a set of five different potential ligands were mutated to Ala or Gln, residues not able to ligand zinc, and the proteins were expressed in E. coli with subsequent purification and determination of enzyme activities and zinc contents (Karlsson, 1994). ADH contains two zinc atoms per subunit, one catalytic and one structural (Akeson, 1964; Drum et al., 1969; Eklund et al., 1976). The binding site of the structural zinc atom in ADH involves four cysteine residues at positions 97, 100, 103, and 111. Though it has been designated as structural since long, the exact functions, as well as the question which structural property it maintains, are unclear. To investigate the contribution of each of the second zinc ligands to the overall conformation, we have performed in vitro mutagenesis of class I and III ADH (Jelokova et al., 1994). The ligands were mutated, in separate constructs, to non-zinc liganding counterparts, Ala or Ser. Proteins expressed were found to be labile and therefore, were detectable only from crude extracts upon Western blot analysis. Confirmation of correctly working transcription processes was ascertained by positive Northern blot analyses. The recently published crystal structure of glucose dehydrogenase from the archaeon Thermoplasma acidophilum, reveals structural homology (in spite of low sequence identity) to ADH from horse liver and SDH from sheep liver (John et al., 1994). Glucose dehydrogenase is a tetramer and posesses a structural zinc atom, contained within a loop similar to that of ADH. However, the orientation of this structural loop with respect to the subunit is markedly different from that of ADH. This further illustrates the role of the zinc loop in the quaternary structures of several of the enzymes within the medium- chain dehydrogenase/reductase super-family, MDR (Persson et al. 1994)

Site-Directed Mutagenesis of Mammalian Alcohol and Sorbitol Dehydrogenases Map Functional Differences within the Enzyme Family

Mammalian alcohol dehydrogenases (ADH) and sorbitol dehydrogenase (SDH) belong to the same protein super-family of zinc-containing ADHs (Jornvall et al., 1989). ADHs have two zinc atoms per subunit, one catalytic and one structural, while SDH only harbors the catalytic zinc atom (Jeffery et al., 1983). The human class I ADH consists of several isozymes that are formed from three different types of subunit, α, β, and γ (Smith et al., 1971; cf. Jornvall et al., 1989). They combine to form active homo- and heterodimers, which constitute the enzymes responsible for the main ethanol metabolism in liver but exhibit differences in substrate specificity and catalytic efficiency. Steroid dehydrogenase activity has only been shown for isozymes containing the γ subunit (McEvily et al., 1988), and these isozymes are the only ones that can be inhibited by testosterone (Mardh et al., 1986). Moreover, ββ but not γγ ADH has a large pH dependence when ethanol is the substrate (Bosron et al., 1983). In addition to these class I ADHs, at least four more classes of mammalian ADH exist, differing enzymatically, structurally and in organ distribution (Jornvall et al., 1989; Pares et al., 1992).

Crystallizations of Novel Forms of Alcohol Dehydrogenase

Alcohol dehydrogenases in nature are derived from different protein families (cf Jornvall et al., 1993). The mammalian alcohol dehydrogenases constitute what presently appears to be six classes within a large family of medium-chain dehydrogenases and reductases, MDR, with at least seven characterized activity types (Persson et al., 1994). The classes of the mammalian alcohol dehydrogenases differ in structure (55–68% sequence identity), substrate pockets, subunit interactions, and other properties. Much of these aspects has been interpreted by comparisons and modelling studies based on crystallographic analyses of just two of the enzymes of one class, the class I horse (Eklund et al., 1976) and human (Hurley et al., 1991) enzymes, constituting the classical liver enzyme with ethanol activity. It is desirable to get direct crystallographic data on the conformations of more of the enzymes, especially since differences exist between the classes, class-hybrid properties have been found in early enzyme forms (of fish), and the two well-characterized classes, I and III, differ in internal variability patterns (Danielsson et al., 1994a). We have therefore crystallized five novel forms of these dehydrogenases, which should enable further structural characterizations and hence evaluation of the conclusions from modelling and from natural variants.