John D. Wade

INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4–8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. Here, we show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8 but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.

Neurotoxic, Redox-competent Alzheimer's β-Amyloid Is Released from Lipid Membrane by Methionine Oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimers disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimers disease.

Amyloid-β Peptide (Aβ) Neurotoxicity Is Modulated by the Rate of Peptide Aggregation: Aβ Dimers and Trimers Correlate with Neurotoxicity

Alzheimers disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-β peptide (Aβ). Within the Aβ sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Aβ toxicity. We synthesized modified Aβ peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo β-sheet and fibril formation at an increased rate compared with wild-type Aβ. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Aβ in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Aβ toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Aβ toxicity, specifically inhibited Aβ dimers from binding to lipid membranes.

Copper-mediated Amyloid-β Toxicity Is Associated with an Intermolecular Histidine Bridge

Amyloid-β peptide (Aβ) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Aβ-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Aβ-Cu2+ complex to cultured primary cortical neurons was attenuated when either the π -or τ-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Aβ and Aβ-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Aβ toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Alzheimer disease β-amyloid activity mimics cholesterol oxidase

The abnormal accumulation of amyloid beta-peptide (Abeta) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2+ have been implicated in AD pathogenesis and plaque formation. Abeta binds Cu2+ with very high affinity, forming a redox-active complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that Abeta:Cu2+ complexes oxidize cholesterol selectively at the C-3 hydroxyl group, catalytically producing 4-cholesten-3-one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. Abeta toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2+ chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4-cholesten-3-one levels. Brain tissue from AD subjects had 98% more 4-cholesten-3-one than tissue from age-matched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates; the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain Abeta accumulation elevates 4-cholesten-3-one levels in AD. Cu2+-mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD.

R3(BΔ23–27)R/I5 Chimeric Peptide, a Selective Antagonist for GPCR135 and GPCR142 over Relaxin Receptor LGR7 IN VITRO AND IN VIVO CHARACTERIZATION

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BΔ23–27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BΔ23–27)R/I5 binds to human GPCR135 (IC50 = 0.67 nm) and GPCR142 (IC50 = 2.29 nm) with high affinity and is a potent functional GPCR135 antagonist (pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3(BΔ23–27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50 = 0.25 nm, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BΔ23–27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BΔ23–27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.

Methylation of the Imidazole Side Chains of the Alzheimer Disease Amyloid-β Peptide Results in Abolition of Superoxide Dismutase-like Structures and Inhibition of Neurotoxicity

The toxicity of the amyloid-β peptide (Aβ) is thought to be responsible for the neurodegeneration associated with Alzheimer disease. Generation of hydrogen peroxide has been implicated as a key step in the toxic pathway. Aβ coordinates the redox active metal ion Cu2+ to catalytically generate H2O2. Structural studies on the interaction of Aβ with Cu have suggested that the coordination sphere about the Cu2+ resembles the active site of superoxide dismutase 1. To investigate the potential role for such structures in the toxicity of Aβ, two novel Aβ40 peptides, Aβ40(HisτMe) and Aβ40(HisπMe), have been prepared, in which the histidine residues 6, 13, and 14 have been substituted with modified histidines where either the π- or τ-nitrogen of the imidazole side chain is methylated to prevent the formation of bridging histidine moieties. These modifications did not inhibit the ability of these peptides to form fibrils. However, the modified peptides were four times more effective at generating H2O2 than the native sequence. Despite the ability to generate more H2O2, these peptides were not neurotoxic. Whereas the modifications to the peptide altered the metal binding properties, they also inhibited the interaction between the peptides and cell surface membranes. This is consistent with the notion that Aβ-membrane interactions are important for neurotoxicity and that inhibiting these interactions has therapeutic potential.

Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation

Established methodology for the preparation of peptide thioesters requires the use of t‐butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc‐based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non‐nucleophilic base, 1,8‐diazabicyclo[5.4.0]undec‐7‐ene, together with 1‐hydroxybenzotriazole in dimethylformamide, were used as the Nα‐Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N‐terminal cysteine‐containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human α‐synuclein. The formation of aspartimide (cyclic imide) in a base‐sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2‐hydroxy‐4‐methoxybenzyl group. Copyright

Physiological and pathophysiological influences on thirst.

Thirst motivates animals to seek fluid and drink it. It is regulated by the central nervous system and arises from neural and chemical signals from the periphery interacting in the brain to stimulate a drive to drink. Our research has focussed on the lamina terminalis and the manner in which osmotic and hormonal stimuli from the circulation are detected by neurons in this region and how that information is integrated with other neural signals to generate thirst. Our studies of osmoregulatory drinking in the sheep and rat have produced evidence that osmoreceptors for thirst exist in the dorsal cap of the organum vasculosum of the lamina terminalis (OVLT) and in the periphery of the subfornical organ, and possibly also in the median preoptic nucleus. In the rat, the hormones angiotensin II and relaxin act on neurons in the periphery of the subfornical organ to stimulate drinking. Studies of human thirst using functional magnetic resonance imaging (fMRI) techniques show that systemic hypertonicity activates the lamina terminalis and the anterior cingulate cortex, but the neural circuitry that connects sensors in the lamina terminalis to cortical regions subserving thirst remains to be determined. Regarding pathophysiological influences on thirst mechanisms, both excessive (polydipsia) and inadequate (hypodisia) water intake may have dire consequences. One of the most common primary polydipsias is that observed in some cases of schizophrenia. The neural mechanisms causing the excessive water intake in this disorder are unknown, so too are the factors that result in impaired thirst and inadequate fluid intake in some elderly humans.

Design and development of a peptide-based adiponectin receptor agonist for cancer treatment.

BackgroundAdiponectin, a fat tissue-derived adipokine, exhibits beneficial effects against insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Circulating adiponectin levels are decreased in obese individuals, and this feature correlates with increased risk of developing several metabolic, immunological and neoplastic diseases. Thus, pharmacological replacement of adiponectin might prove clinically beneficial, especially for the obese patient population. At present, adiponectin-based therapeutics are not available, partly due to yet unclear structure/function relationships of the cytokine and difficulties in converting the full size adiponectin protein into a viable drug.ResultsWe aimed to generate adiponectin-based short peptide that can mimic adiponectin action and be suitable for preclinical and clinical development as a cancer therapeutic. Using a panel of 66 overlapping 10 amino acid-long peptides covering the entire adiponectin globular domain (residues 105-254), we identified the 149-166 region as the adiponectin active site. Three-dimensional modeling of the active site and functional screening of additional 330 peptide analogs covering this region resulted in the development of a lead peptidomimetic, ADP 355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2). In several adiponectin receptor-positive cancer cell lines, ADP 355 restricted proliferation in a dose-dependent manner at 100 nM-10 μM concentrations (exceeding the effects of 50 ng/mL globular adiponectin). Furthermore, ADP 355 modulated several key signaling pathways (AMPK, Akt, STAT3, ERK1/2) in an adiponectin-like manner. siRNA knockdown experiments suggested that ADP 355 effects can be transmitted through both adiponectin receptors, with a greater contribution of AdipoR1. In vivo, intraperitoneal administration of 1 mg/kg/day ADP 355 for 28 days suppressed the growth of orthotopic human breast cancer xenografts by ~31%. The peptide displayed excellent stability (at least 30 min) in mouse blood or serum and did not induce gross toxic effects at 5-50 mg/kg bolus doses in normal CBA/J mice.ConclusionsADP 355 is a first-in-class adiponectin receptor agonist. Its biological activity, superior stability in biological fluids as well as acceptable toxicity profile indicate that the peptidomimetic represents a true lead compound for pharmaceutical development to replace low adiponectin levels in cancer and other malignancies.