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(B⌬23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly 23 to Arg, a truncation at the C terminus (Gly 24 -Trp 27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(B⌬23-27)R/I5 binds to human GPCR135 (IC 50 ‫؍‬ 0.67 nM) and GPCR142 (IC 50 ‫؍‬ 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(B⌬23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC 50 ‫؍‬ 0.25 nM, pA2 ‫؍‬ 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(B⌬23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(B⌬23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.Relaxin-3 (R3) 2 (1) is the most recently identified member of the insulin-relaxin peptide family. Both relaxin-3 and its receptor, GPCR135 (2), are predominantly expressed in the brain (2, 3). GPCR135, an inhibitory receptor, is expressed in many regions of the rodent brain such as the superior colliculus, sensory cortex, olfactory bulb, amygdale, and paraventricular nucleus (4 -6), suggesting potential physiological involvement in neuroendocrine and sensory processing. Recent in vivo studies have further shown that relaxin-3 and GPCR135 are involved in the stress response and in regulation of feeding. More specifically, water restraint stress or intracerebroventricular corticotrophin-releasing factor (CRF) infusion induces relaxin-3 expression in cells of the nucleus incertus, a region where CRF receptor-1 is also expressed (7), and central administration of relaxin-3 induces feeding in rat (8, 9). These findings suggest that GPCR135 and relaxin-3 may be involved in multiple physiological processes, some of which might be as yet unknown.In vitro relaxin-3 activates GPCR135 (2), GPCR142 (10), and LGR7 (11) receptors. The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135 (2). Phar...

The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.

Hippocampal theta rhythm is thought to underlie learning and memory, and it is well established that ''pacemaker'' neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippocampal theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippocampus and promote hippocampal theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippocampus. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippocampal theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.

Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased selfadministration of alcohol in a dose-related manner and attenuated cue-and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/ RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of rewardseeking. addiction | dependenceA lcohol abuse is a major cause of morbidity and mortality worldwide, accounting for an estimated 3.8% of all global deaths and 4.6% of the global burden of disease and injury (1). Excessive alcohol use may also lead to alcohol dependence (also termed "alcohol addiction") (2, 3), which has a lifetime prevalence of ∼12.5% (4). Economic costs due to alcohol abuse were in the order of $235 billion in the United States in 2007, or ∼2.7% of GDP (1, 5). Despite the huge impact of alcohol use disorders on society, current first-line therapeutic agents, such as naltrexone and acamprosate, are far from adequate, with high relapse rates during treatment and problems with compliance (6-8). New therapeutic agents are clearly required, particularly for the reduction of hazardous drinking and prevention of relapse (9). To this end, a major goal in addiction neuroscience is to understand the neurobiology and neurocircuitry affected by alcohol use disorders and to identify factors implicated in these conditions, which may lead to improved and more targeted therapies (7-10). Here we investigate the neuropeptide relaxin-3 for its involvement in rodent models of alcohol-seeking and consumption.Relaxin-3 is the highly conserved, ancestral neuropeptide of the relaxin/insulin superfamily, and its cognate G-protein-coupled receptor is relaxin family peptide 3 receptor (RXFP3) (11-16). Relaxin-3 is predominantly expressed in gamma-aminobutyric acid (GABA) neurons in the hindbrain nucleus incertus, which projects widely to forebrain areas, including the amygdala, bed nucleus of the stria terminalis (BNST), hippocampus, and lateral hypothalamus, which also express high levels ...

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.

In a previous study, we determined that HP(2-20) (residues 2-20 of parental HP derived from the N-terminus of Helicobacter pylori Ribosomal Protein L1) and its analogue, HPA3, exhibit broad-spectrum antimicrobial activity. The primary objective of the present study was to gain insight into the relevant mechanisms of action using analogues of HP(2-20) together with model liposomes of various lipid compositions and electron microscopy. We determined that these analogues, HPA3 and HPA3NT3, exert potent antibacterial effects in low-salt buffer and antifungal activity against chitin-containing fungi, while having little or no hemolytic activity or cytotoxicity against mammalian cell lines. Our examination of the interaction of HP(2-20) and its analogues with liposomes showed that the peptides disturb both neutral and negatively-charged membranes, as demonstrated by the release of encapsulated fluorescent markers. The release of fluorescent markers induced by HP(2-20) and its analogues was inversely related to marker size. The pore created by HP(2-20) shows that the radius is approximately 1.8 nm, whereas HPA3, HPA3NT3, and melittin have apparent radii between 3.3 and 4.8 nm. Finally, as shown by electron microscopy, the liposomes and various microbial cells treated with HPA3 and HPA3NT3 showed oligomerization and blebbing similar to that seen with melittin, while HP(2-20) exhibited flabbiness. These results suggest that HP(2-20) may exert its antibiotic effects through a small pore (about 1.8 nm), whereas HPA3 and HPA3NT3 formed pores of a size consistent with those formed by melittin.

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide-and receptor-dependent.Relaxin was first identified more than 90 years ago and subsequently shown to be a peptide hormone having a two-chain structure similar to insulin ( Fig. 1) (1). It has since been established that relaxin is a member of the relaxin peptide family, comprising a total of seven members in the human (2). These are the H1, 3 H2, and H3 relaxin peptides that are encoded by the three relaxin genes RLN1 to -3 and the insulin-like peptides INSL3 to -6 (insulin-like peptides 3-6). Phylogenetic analyses indicate that all of these relaxin family peptides evolved from a relaxin-3 (H3 relaxin equivalent) ancestral gene prior to the emergence of fish (3). In most mammals other than humans and higher primates, there are only two relaxin genes that encode relaxin and relaxin-3. The RLN1 gene in these species is equivalent to the RLN2 gene in humans (encoding H2 relaxin) and higher primates and encodes the relaxin peptide that is expressed by the corpus luteum and/or placenta (2). The function of the RLN1 gene in higher primates is unknown, and an H1 relaxin peptide has not been isolated.In contrast to the receptors for insulin and insulin-like growth factors I and II, which are tyrosine kinases, the receptors for relaxin family peptides are members of two unrelated branches of the G-protein-coupled recepto...