NMR conformational studies of micelle-bound orexin-B: a neuropeptide involved in the sleep/awake cycle and feeding regulation

The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH(2)) has been determined by (1)H and (13)C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d(25) (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu(7) to Ser(18) and helix II spans Ala(22) to Leu(26). Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831-839 (1999)) determined the [Phe(1)]-orexin-B conformation in water solution by NMR and showed that helix II extends from Ala(23) to Met(28). The C-terminal dipeptide, Thr(27)-Met(28), is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr(27)-Met(28) in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al., Bioorg. Med. Chem. Lett. 13, 111-113, 2003). Asahi et al. have shown that Ala substitution from Gly(24) to Met(28) or D-amino acid substitution from Ala(23) to Met(28) causes a significant reduction in the potency of orexin-B for both OX(1)R and OX(2)R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr(27)-Met(28) to adopt the correct receptor-binding conformation.     

Role of proline residues in the structure and function of a membrane transport protein

By use of site-directed mutagenesis, each prolyl residue in the lac permease of Escherichia coli at positions 28 (putative helix I), 31 (helix I), 61 (helix II), 89 (helix III), 97 (helix III), 123 (helix IV), 192 (putative hydrophilic region 7), 220 (helix VII), 280 (helix VIII), and 327 [helix X; Lolkema, J. S., et al. (1988) Biochemistry 27, 8307] was systematically replaced with Gly, Ala, or Leu or deleted by truncation of the C-terminus [i.e., Pro403 and Pro405; Roepe, P.D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992]. Replacements were chosen on the basis of side-chain helical propensity: Gly, like Pro, is thought to be a "helix breaker", while Ala and Leu are "helix makers". With the exception of Pro28, each prolyl residue can be replaced with Gly or Ala, and Pro403 and -405 can be deleted with the C-terminal tail, and significant lac permease activity is retained. In contrast, when Pro28 is replaced with Gly, Ala, or Ser, lactose transport is abolished, but permease with Ser28 binds p-nitrophenyl alpha-D-galactopyranoside and catalyzes active transport of beta-galactopyranosyl-1-thio-beta-D- galactopyranoside. Replacement of Pro28, -31, -123, -280, or -327 with Leu abolishes lactose transport, while replacement of Pro61, -89, -97, or -220 with Leu has relatively minor effects. None of the alterations in permease activity is due to inability of the mutant proteins to insert into the membrane or to diminished lifetimes after insertion, since the concentration of each mutant permease in the membrane is comparable to that of wild-type permease as judged by immunological analyses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement of intact disulfide bonds in orexin-A-induced stimulation of gastric acid secretion that is mediated by OX1 receptor activation

Orexin-A is a neuropeptide consisting of 33 amino acids with two intrachain disulfide bonds, namely Cys6-Cys12 and Cys7-Cys14, and is a potent stimulator of food consumption and gastric acid secretion. In contrast, orexin-B, a peptide containing 28 amino acids without disulfide bond, which has no stimulatory action of gastric acid. The objective of the present study was to characterize the receptor-mediated mechanism of orexin-A-induced stimulation of gastric acid secretion using orexin-A-related peptides with modification of disulfide bonds. Intracisternal injection of orexin-A, but not orexin-B or orexin-A (15-33), that does not contain both disulfide bonds stimulated gastric acid secretion in pylorus-ligated conscious rats. The ability of the stimulation of gastric acid output was less in three alanine-substituted orexin-A, [Ala(6,12)]orexin-A, [Ala(7,14)]orexin-A, and [Ala(6,7,12,14)]orexin-A, than orexin-A. Orexins-induced calcium increase was measured in CHO-K1 cells expressing OX1R or OX2R. Orexin-A induced a transient increase in [Ca(2+)]i in CHO-K1/OX1R cells in a dose-dependent manner. EC50 values for OX1R of orexin-A, orexin-B, or orexin-A (15-33) was 0.068, 0.69 or 4.1 nM, respectively, suggesting that peptides containing no disulfide bonds have lower potency for the receptor. Agonistic activity for OX1R of the three orexin-A analogues with modification of one or both disulfide bonds was significantly reduced as compared with that of orexin-A. EC50 values for OX2R of orexin-A and orexin-B was almost equal but potency for the receptor of orexin-A (15-33) and three alanine substituted orexin-A was less than that of orexin-A. A significant inverse relationship between gastric acid output and EC50 values for OX1R, but not OX2R, was observed. These results suggested that the orexin-A-induced acid stimulation requires OX1R activation and that disulfide bonds in orexin-A may have a key role in the receptor activation.     

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.     

Role of amino acid residues in left-handed helical conformation for the conformational stability of a protein

Our previous study of six non-Gly to Gly/Ala mutant human lysozymes in a left-handed helical region showed that only one non-Gly residue at a rigid site had unfavorable strain energy as compared with Gly at the same position (Takano et al., Proteins 2001; 44:233-243). To further examine the role of left-handed residues in the conformational stability of a protein, we constructed ten Gly to Ala mutant human lysozymes. Most Gly residues in human lysozyme are located in the left-handed helix region. The thermodynamic parameters for denaturation and crystal structures were determined by differential scanning calorimetry and X-ray analysis, respectively. The difference in denaturation Gibbs energy (DeltaDeltaG) for the ten Gly to Ala mutants ranged from + 1.9 to -7.5 kJ/mol, indicating that the effect of the mutation depends on the environment of the residue. We confirm that Gly in a left-handed region is more favorable at rigid sites than non-Gly, but there is little difference in energetic cost between Gly and non-Gly at flexible sites. The present results indicate that dihedral angles in the backbone conformation and also the flexibility at the position should be considered for analyses of protein stability, and protein structural determination, prediction, and design.     

Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues. Conformation determined by 1H NMR and CD spectroscopies and distance geometry in 25% methanol solution.

The conformation of pituitary adenylate cyclase activating polypeptide with 27 residues (PACAP27) has been determined by two-dimensional NMR and CD spectroscopies and distance geometry in 25% methanol. Residues 9-20 and 22-25 have well-defined conformations but other residues do not show ordered conformations. The conformation of residues 9-20 is composed of three distinct regions of beta turn-like conformation (residues 9-12), alpha helix (residues 12-14) and the looser helical conformation (residues 15-20), while residues 22-24 form alpha helix. PACAP27 has a 2 helices separated by a disordered region similar to a VIP analog reported by Fry et al. but is distinct from the VIP analog in the position of the first helix, which is shifted by 2 residues toward the C-terminus, and in the form of the second helix [Fry, D.C., Madison, V.S., Bolin, D.R., Greeley, D.N., Toome, V. and Wegrzynski, B.B. (1989) Biochemistry 28, 2399-2409].     

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Design of ET(B) receptor agonists: NMR spectroscopic and conformational studies of ET7-21[Leu7, Aib11, Cys(Acm)15

In a previous report we have shown that the endothelin-B receptor-selective linear endothelin peptide, ET-1[Cys (Acm)1,15, Ala3, Leu7, Aib11], folds into an alpha-helical conformation in a methanol-d3/water co-solvent [Hewage et al. (1998) FEBS Lett., 425, 234-238]. To study the requirements for the structure-activity relationships, truncated analogues of this peptide were subjected to further studies. Here we report the solution conformation of ET7-21[Leu7, Aib11, Cys(Acm)15], in a methanol-d3/water co-solvent at pH 3.6, by NMR spectroscopic and molecular modelling studies. Further truncation of this short peptide results in it displaying poor agonist activity. The modelled structure shows that the peptide folds into an alpha-helical conformation between residues Lys9-His16, whereas the C-terminus prefers no fixed conformation. This truncated linear endothelin analogue is pivotal for designing endothelin-B receptor agonists.     

Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY

The structure of [Ala(31), Pro(32)]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y(5)-receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y(5)-receptor-selective agonist [Ala(31), Aib(32)]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala(31), Aib(32)]-NPY, the N-terminal residues Tyr(1)-Asp(16) are disordered in solution. Starting from residue Leu(17), an alpha helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala(31)-Tyr(36) indicates that the alpha helix has become interrupted through the [Ala(31), Pro(32)] mutation. This finding is further supported by (15)N-relaxation data through which we can demonstrate that the well-defined alpha helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased (3)J(HN)(alpha) scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y(5)-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala(31), Pro(32)]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn(29) and Leu(30) were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala(31), Pro(32)]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala(31), Pro(32)]-NPY.     

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.     

Helix-forming tendencies of nonpolar amino acids predicted by Monte Carlo simulated annealing

Monte Carlo simulated annealing is applied to the study of the α-helix-forming tendencies of seven nonpolar amino acids, Ala, Leu, Met, Phe, Ile, Val, and Gly. Homooligomers of 10 amino acids are used and the helix tendency is calculated by folding α-helicies from completely random initial conformations. The results of the simulation imply that Met, Ala, and Leu are helix formers and that Val, Ile, and Gly are helix breakers, while Phe comes in between the two groups. The differences between helix formers and breakers turned out to be large in agreement with the recent experiments with short peptides. It is argued from the energy distributions of the obtained conformations that the helix tendency is small for the helix breakers because of steric hindrance of side chains. Homoglycine is shown to favor a random coil conformation. The β-strand tendencies of the same homooligomers are also considered, and they are shown to agree with the frequencies of amino acids in β-sheet from the protein data base. © 1994 Wiley-Liss, Inc.     

Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3

A glucose dehydrogenase gene was isolated from Bacillus megaterium IWG3, and its nucleotide sequence was identified. The amino acid sequence of the enzyme deduced from the nucleotide sequence is very similar to the protein sequence of the enzyme from B. megaterium M1286 reported by Jany et al. (Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6-10). The isolated gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones (H35, H39, F18, F20, F191, F192, N1, N13, N14, N28, N71, and N72) containing mutant genes for thermostable glucose dehydrogenase were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions: H35 and H39, Glu-96 to Gly; F18 and F191, Glu-96 to Ala; F20, Gln-252 to Leu; F192, Gln-252 to Leu and Ala-258 to Gly; N1, Glu-96 to Lys and Val-183 to Ile; N13 and N14, Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His; N28, Glu-96 to Lys, Asp-108 to Asn, Pro-194 to Gln, and Glu-210 to Lys; and N71 and N72, Tyr-253 to Cys. These mutant enzymes have higher stability at 60 degrees C than the wild-type enzyme. The results of this study indicate that the tetrameric structure of glucose dehydrogenase is stabilized by several kinds of mutation, and at least one of the following amino acid substitutions stabilizes the enzyme: Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.     

氮氧自由基自旋探针R1在特定空间位点上对bacteriorhodopsin产生颜色调谐效应的机理分析

蛋白质结构研究中的氮氧自由基自旋探针R1,在特定的空间位置上会对bacteriorhodopsin的结构与功能产生影响。该文作者发现:将bacteriorhodopsin中远离视黄醛的第192位Gly替换为自旋探针R1后,产生了显著的颜色调谐,最大吸收峰有28nm的蓝移。但用R1替换Gly192邻近的Gly195和Ala196,未产生颜色调谐效应;替换螺旋E末端的Val130和Tyr131,以及螺旋B末端的Leu62,均无颜色调谐。进一步表征bacteriorhodopsin光循环中间体O和M的衰减过程发现:R1替换前,Gly192Cys(+)的O态衰减寿命约为847.7ms;但替换后,其衰减显著加快至308.2ms。M态的衰减寿命在R1替换后反而显著延长,尤其是慢组分Ms从24.8ms延长至83.6ms。Gly192R1的顺磁共振信号揭示:该位置上的R1处于低运动性状态,表明R1可能与局部环境的结构组分发生了直接作用。晶体结构分析发现:R1可能直接与质子传递通道中的三体水分子作用。由于三体水分子通过长程的氢键网络与视黄醛活性中心相连,并且参与质子在bacteriorhodopsin中的传递,因此自旋探针R1...     

Orexin-A/OX1R is involved in regulation of autophagy to promote cortisol secretion in adrenocortical cell

BackgroundHypercortisolism has emerged as a prominent clinical condition worldwide caused by biochemical cortisol excess in patients, and optimization treatment is needed urgently in the clinic. Previously, we observed that orexin-A/orexin type 1 receptor (OX1R) promoted cell proliferation, inhibited apoptosis, and increased cortisol release in adrenocortical cells. However, the functions of orexin-A/OX1R on autophagy and its molecular mechanism are not known.MethodsTransmission electron microscopy and confocal microscope were performed to detect autophagosomes. Western blot were performed to detect autophagy proteins. The cortisol concentration was assessed with an ELISA.FindingsOur data demonstrated that orexin-A/OX1R activated the mammalian target of rapamycin/p70 ribosomal protein S6 kinase-1 pathway, thereby inhibiting autophagy in H295R cells and Y-1 cells. Furthermore, the orexin-A/OX1R-mediated suppression of autophagy played a crucial role in cortisol secretion. Mechanistically, the expression of 3β-hydroxysteroid dehydrogenase/isomerase, the rate-limiting enzyme in cortisol synthesis, was increased with autophagy inhibition mediated by orexin-A/OX1R.InterpretationThis study provided the evidence that orexin-A/OX1R participated in modulating mTOR/p70S6K1/autophagy signaling pathway to promote cortisol secretion in adrenocortical cell. The findings suggest the mechanistic basis for disorders of cortisol secretion, providing the potential therapeutic targets for hypercortisolism treatment.FundThis work was supported by National Natural Science Foundation of China (32170603, 31871286), the Doctoral Start-up Foundation of Liaoning Province (20180540008, 2019-BS-298), the Natural Science Foundation of Liaoning Province (2019-ZD-0779), and Shenyang Science and Technology Plan Fund Projects (21-173-9-28).     

An unusual C-H...O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H. O hydrogen bond between Ala(4) C(alpha)H and (D)Leu(9) CO. The parameters for C-H. O interaction are Ala(4) C(alpha)H. O=C (D)Leu(9) distance 3.27 A, C(alpha)-H. O angle 176 degrees, and O. H(alpha) distance 2.29 A. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.