Solution structure of Ser14Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease

The NMR solution study of Ser14Gly-humanin (S14G-HN), a 1000-fold more potent derivative of humanin (HN), is reported. HN is 24-residue peptide that selectively suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults and offers hope for the development of a cure against AD. In aqueous solution the NMR data show that S14G-HN is a flexible peptide with turn-like structures in its conformational ensemble distributed over an extensive part of its sequence from Pro3 to Glu15. In the more lipophilic environment of 30% TFE, an alpha-helical structure spanning residues Phe6 to Thr13 is identified. Comparison of these findings to the NMR structure of the parent HN and to existing structure-function relationship literature data outlines the important for activity structural features for this class of neuroprotective peptides, and brings forth flexibility as an important characteristic that may facilitate interactions with functional counterparts of the neuroprotection pathway.     

Identification of essential amino acids in Humanin,a neuroprotective factor against Alzheimer's disease-relevant insults

Humanin (HN) is a secretory peptide that inhibits neurotoxicity by various Alzheimer's disease-relevant insults. We have so far identified that the substitution of Leu9 for Arg nullifies the extracellular secretion of HN. Here we comprehensively investigate the amino acid requirement of HN essential for its secretion and for its neuroprotective function. Intracellulary expressed HN-EGFP (EGFP N-terminally fused with HN) was extracellularly secreted, whereas neither EGFP nor (L9R)HN-EGFP was secreted at all. While Ala substitution of neither residue affected HN secretion, Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN. In the Leu9-Leu11 domain, the Leu10 residue turned out to play a central role in this function, because the Asp substitution of Leu10, but not Leu9 or Leu11, nullified the secretion of HN. Utilizing Ala-scanned HN constructs, we also investigated a comprehensive structure-function relationship for the neuroprotective function of full-length HN, which revealed (i) that Pro3, Ser7, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19 were essential for this function and (ii) that Ser7 and Leu9 were essential for self-dimerization of HN. These findings indicate that HN has activity similar to a signal peptide, for which the Leu9-Leu11 region, particularly Leu10, functions as a core domain, and suggest that self-dimerization of HN is a process essential for its neuroprotective function.     

A humanin derivative reduces amyloid beta accumulation and ameliorates memory deficit in triple transgenic mice

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APP(swe), tau(P310L), and PS-1(M146V) that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.     

The biological activity of Humanin analogs correlates with structure stabilities in solution

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10 °C in 10 mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10 °C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37 °C. While S14G-HN showed small changes in both solutions at 37 °C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37 °C was faster for S7A-HN than HN. These results show that the structure stability at 37 °C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities.     

Humanin derivative,HNG, enhances neurotransmitter release

BackgroundHumanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated.MethodsThe extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells.ResultsThe hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells.ConclusionsThese findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions.General significanceThe regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.     

Synthesis, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b-f) group. The analogs 1b-f were evaluated for their inhibition of [(125)I]iodo-MLA binding at rat brain alpha7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b-f were evaluated for inhibition of binding to rat brain alpha,beta nAChR using [(3)H]epibatidine. At the alpha7 nAChR, MLA showed a K(i) value of 0.87 nM, analogs 1b-e possessed K(i) values of 1.67-2.16 nM, and 1f showed a K(i) value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K(i)=1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for alpha,beta nAChRs. MLA antagonized nicotine-induced seizures with an AD(50)=2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine's antinociceptive effects in the tail-flick assay. Compound 1c (K(i)=1.78 nM at alpha7 nAChR) with an AD(50) value of 1.8 mg/kg was 6.7 times more potent than MLA (AD(50)=12 mg/kg) in antagonizing nicotine's antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against beta-amyloid(1-42), these new analogs which have high alpha7 nAChR affinity and good selectivity relative to alpha,beta nAChRs will be useful biological tools for studying the effects of alpha7 nAChR antagonist and neuroprotection.     

Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes

Humanin (HN) has been reported to be an endogenous peptide that exerts highly selective neuroprotection against cell death induced by various types of Alzheimer's disease-related insults. We previously proposed the much broader cytoprotective potential of HN from the result that HN suppressed serum-deprivation-induced death of rat pheochromocytoma cells. In this study, we showed that HN also suppressed death of human lymphocytes cultured under serum-deprived condition. Further, we revealed, by assaying metabolic activity and survival rate, that HN was a potent factor capable of increasing the metabolic activity of individual serum-deprived lymphocytes. To our knowledge, there is no report described about a rescue factor that increases the metabolic activity of individual serum-deprived cells and prolongs their survival. This novel feature of HN may enable us to apply this peptide for the management of diseases involving poor metabolic activity, such as mitochondria-related disorders and brain ischemia.     

d-Ser-containing humanin shows promotion of fibril formation

Humanin (HN), a peptide of 24 amino acid residues, suppresses the neuronal cell death that is induced by the gene products of Alzheimer’s disease. HN contains two Ser residues at positions 7 and 14. Because the proportion of d-Ser isomerized from l-Ser in proteins appears to increase as cellular organs age, we explored the structural effects of the isomerization of each Ser residue in HN. By using a thioflavin-T assay to detect fibril formation, we found that an HN derivative that contained two isomerized d-Ser residues had a greater tendency to form fibrils than did wild-type HN or HNs containing single d-Ser residues. A previous report showed that HN containing two d-Ser residues exerts neuroprotective activity. Our data, therefore, suggest that the fibril formation by HN that contains two d-Ser residues may promote HN neuroprotective activity.     

Structure of three Humanin peptides with different activities upon interaction with liposome

We have recently shown that a 24 amino acid Humanin (HN) adopts an anti-parallel β-sheet structure in the presence of a negatively charged 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and suggested a possibility that it interacts with lipid membranes and thereby exerts neuroprotective effects through the target cell surface receptors or the intracellular signaling molecules following membrane interaction events. The structures of two HN analogs, having either a S7A mutation or a S14G mutation, were examined under the identical conditions, as the S7A analog is inactive and the S14G analog is 1000-fold more active than the wild type HN. These analogs showed a secondary structure indistinguishable from the structure of HN in the presence of DOPG liposome, while unrelated peptides were disordered with and without DOPG. It thus appeared that HN and the analogs, regardless of the biological activities, have an ability to interact with DOPG liposome and form an anti-parallel β-sheet structure. While the wild type HN and the S7A and S14G analogs were largely disordered in buffer, the S14G analog showed greater stability as a disordered structure in the buffer at a physiological temperature, suggesting that it maintains the disordered structure presumably required for the interaction with the DOPG liposome and thereby greater neuroprotective activity.     

Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.     

14-3-3 binds to and mediates phosphorylation of microtubule-associated tau protein by Ser9-phosphorylated glycogen synthase kinase 3beta in the brain

In mammalian brain, tau, glycogen synthase kinase 3beta (GSK3beta), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3beta (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722-12728). The molecular mechanism by which 14-3-3 connects GSK3beta to tau within the complex is not clear. In this study, we find that GSK3beta within the tau phosphorylation complex is phosphorylated on Ser(9). From extracts of rat brain and rat primary cultured neurons, Ser(9)-phosphorylated GSK3beta precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser(9)-phosphorylated GSK3beta co-immunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3beta only when the kinase is phosphorylated on Ser(9). In transfected HEK-293 cells, 14-3-3 binds to Ser(9)-phosphorylated GSK3beta and does not bind to GSK3beta (S9A). Tau, on the other hand, binds to both GSK3beta (WT) and GSK3beta (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser(9)-phosphorylated GSK3beta by approximately 3-fold but not with GSK3beta (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser(9)-phosphorylated GSK3beta but not by GSK3beta (S9A). In transfected HEK-293 cells, Ser(9) phosphorylation suppresses GSK3beta-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser(9)-phosphorylated GSK3beta remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser(9)-phosphorylated GSK3beta to tau and Ser(9)-phosphorylated GSK3beta phosphorylates tau.     

A polymorphism in the beta1 adrenergic receptor is associated with resting heart rate

Resting heart rate is significantly associated with cardiovascular morbidity and mortality. However, the extent to which resting heart rate is genetically determined is poorly understood, and no genes have been found that contribute to variation in resting heart rate. Because signaling through the beta1 adrenergic receptor is a key determinant of cardiac function, we tested whether polymorphisms in this receptor are associated with resting heart rate. A cohort of >1,000 individuals of Chinese and Japanese descent, from nuclear families, was genotyped for two polymorphisms, resulting in a serine/glycine substitution at amino acid 49 (Ser49Gly) and an arginine/glycine substitution at residue 389 (Arg389Gly), in the beta1 adrenergic receptor. For comparison, polymorphisms in the beta2 and beta3 adrenergic receptors were also evaluated. The Ser49Gly polymorphism was significantly associated (P=.0004) with resting heart rate, independent of other variables, such as body-mass index, age, sex, ethnicity, exercise, smoking, alcohol intake, hypertension status, and treatment with beta blockers. The data support an additive model in which individuals heterozygous for the Ser49Gly polymorphism had mean heart rates intermediate to those of either type of homozygote, with Ser homozygotes having the highest mean heart rate and with Gly homozygotes having the lowest. Neither the Arg389Gly polymorphism in the beta1 adrenergic receptor nor polymorphisms in the beta2 and beta3 adrenergic receptors were associated with resting heart rate. The heritability of heart rate was 39.7% +/- 7.1% (P<10-7).     

Identification of soluble WSX-1 not as a dominant-negative but as an alternative functional subunit of a receptor for an anti-Alzheimer’s disease rescue factor Humanin

Humanin (HN) inhibits Alzheimer’s disease (AD)-relevant neuronal death and dysfunction, by interacting with a receptor (s) involving ciliary neurotrophic factor receptor α (CNTFR), WSX-1, and gp130. It remains unknown whether this complex is the sole HN receptor that mediates HN-induced anti-AD activity. We here report that an alternatively spliced WSX-1 isoform, encoding an extracellular 270-amino-acid region of WSX-1 with cytokine-binding regions (named soluble WSX-1; sWSX-1), is expressed in neuronal cells lacking function of full-length WSX-1 and enables HN to rescue AD-relevant death. This result suggests that CNTFR/soluble WSX-1/gp130 behaves as an alternative functional HN receptor.     

The predicted beta12-beta13 loop is important for inhibition of PP2Acalpha by the antitumor drug fostriecin

The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1. Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding. A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro. Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect. The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs.     

Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis

The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.     

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.     

Role of the N-terminal helix I for dimerization and stability of the calcium-binding protein S100B

Human S100B(beta beta) is a small intracellular EF-hand calcium-binding protein that consists of two noncovalently associated 91-residue beta monomers. The three-dimensional structures of S100B reveal the dimer interface consists of four alpha-helices (I, I' and IV, IV') packed in an X-type bundle. In this study, guanidine hydrochloride denaturation and dynamic light scattering were used to assess the impact of single (L3A, L3S, M7A, I11A, F14A) and double (L3A/I11A and L3A/F14A) substitution mutations in helix I on the stability and dimerization propensity of S100B. The free energy of unfolding (Delta G(u)) of wild-type apo-S100B was determined to be 72.4 +/- 4.0 kJ mol(-1), consistent with it being the most stable calcium-binding protein to date. The order of stability of the mutants in their apo form is S100B > L3A > L3S > I11A > M7A approximately L3A/I11A > F14A > L3A/F14A. Further, there is a strong correlation between the stability and the cooperativity of unfolding. Each mutation proved to be more stable in its calcium form compared to its apo form. The calcium-bound L3S substitution proved to be significantly more stable than calcium-saturated S100B, whereas the L3A, I11A, and L3A/I11A mutants are only slightly more stable than the wild-type protein. The F14A and L3A/F14A mutants are significantly reduced in stability, even in the presence of calcium.