Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae

The uncoupling protein (UCP) from mammalian brown adipose tissue is an integral component of the mitochondrial inner membrane where it dissipates the proton electrochemical gradient. UCP is transported into mitochondria from the cytosol but lacks a cleavable targeting peptide. We have expressed the rat UCP in Saccharomyces cerevisiae and shown that this protein, which is not normally found in yeast, is targeted to the mitochondria where it disrupts mitochondrial function, probably by uncoupling oxidative phosphorylation. The observed growth defect is dependent upon the level of expression of UCP. When the unmodified UCP cDNA is expressed in yeast under the control of the GAL10 promoter no defect in growth is observed. We have inserted the UCP coding sequence behind the strong phosphoglycerate kinase promoter under the control of the GAL1-10 upstream activation site and introduced a yeast consensus sequence (ATAATG) at the translation start site. We have found that UCP expressed in S. cerevisiae is targeted to mitochondria and that its expression induces a marked growth defect on non-fermentable carbon sources in a manner dependent on induction with galactose.     

Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes

Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [³⁵S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS citrate synthase - EDTA ethylenediaminetetraacetic acid,-acetate - GAPDH NADP⁺-glyceraldehyde-3-phosphate dehydrogenase - rRNA ribosomal RNA - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - Tris 2-amino-2(hydroxymethyl)-1,3-propanediol     

The Importance of Tryptophan B28 in H2 Relaxin for RXFP2 Binding and Activation

H2 relaxin (relaxin) is a member of the insulin–relaxin superfamily and exhibits several non-reproductive functions in addition to its well-known properties as a pregnancy hormone. Over the years, the therapeutic potential of relaxin has been examined for a number of conditions. It is currently in phase III clinical trials for the treatment of acute heart failure. The 53 amino acid peptide hormone consists of two polypeptide chains (A and B) which are cross-linked by two inter-chains and one intra-A chain disulfide bridge. Although its cognate receptor is relaxin family peptide receptor (RXFP) 1, relaxin is also able to cross-react with RXFP2, for which the native ligand is INSL3. The “RXXXRXXI” motif in the B-chain of H2 relaxin is responsible for primary binding to LRR of the RXFP1 receptor (Büllesbach and Schwabe, J Biol Chem 280:14051–14056, 2005). Previous RXFP2 receptor mutation and molecular modelling studies strongly suggest that, in addition to this motif, the Trp-B28 residue in the B-chain is responsible for H2–RXFP2 interaction. To confirm this finding, here we have mutated H2 relaxin in which Trp-B28 was replaced with alanine. The synthetic relaxin analogue was then tested on cells expressing either RXFP1 or 2 to determine the affinity and potency for the respective receptors. Our results confirm that Trp-B28 in the B-chain is crucial for binding and activating RXFP2, but not for RXFP1.     

Preliminary Structure–Function Relationship Studies on Insulin-Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide bonded member of insulin/relaxin superfamily of peptides that includes insulin, insulin-like growth factor I and II (IGFI and IGFII), insulin-like peptide 3, 4, 5 and 6 (INSL3, 4, 5 and 6), relaxin-1 (H1 relaxin), -2 (H2 relaxin) and -3 (H3 relaxin). Although it is expressed in relatively high levels in the gut, its biological function remains unclear. However, recent reports suggest a significant orexigenic action and a role in the regulation of insulin secretion and β-cell homeostasis, which implies that both agonists and antagonists of the peptide may have significant therapeutic applications. Modern solid phase synthesis techniques together with regioselective disulfide bond formation were employed for a preliminary structure–function relationship study of mouse INSL5. Two point mutated analogues, mouse INSL5 A-B(R24A, W25A) and mouse INSL5 A-B(K6A, R14A, Y18A) were chemically prepared, where the residues in the B-chain that may be involved in receptor activation and affinity binding, were respectively mutated. Synthetic mouse INSL5 A-B(R24A, W25A) analogue was inactive on RXFP4, the native receptor for INSL5, suggesting Arg^B24 and Trp^B25 are probably directly involved in INSL5 receptor activation. Mouse INSL5 A-B(K6A, R14A, Y18A) analogue had both decreased affinity and potency on RXFP4 (pIC₅₀ 7.7 ± 0.2, pEC₅₀ 7.87 ± 0.18) which indicated that one or more of these residues are critical for the binding to the receptor.     

The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.     

Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.     

INSL5 is a high affinity specific agonist for GPCR142 (GPR100)

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.     

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Relaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.     

Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.     

Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents: A Viral Vector-based Approach to Investigate Neuropeptide Function

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.