Proadrenomedullin N-terminal Peptide Research Articles

In silico optimization of analogs derived pro-adrenomedullin peptide to evaluate antimicrobial potential.

Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Adrenomedullin: Not Just Another Gastrointestinal Peptide.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two bioactive peptides derived from the same precursor with several biological functions including vasodilation, angiogenesis, or anti-inflammation, among others. AM and PAMP are widely expressed throughout the gastrointestinal (GI) tract where they behave as GI hormones, regulating numerous physiological processes such as gastric emptying, gastric acid release, insulin secretion, bowel movements, or intestinal barrier function. Furthermore, it has been recently demonstrated that AM/PAMP have an impact on gut microbiome composition, inhibiting the growth of bacteria related with disease and increasing the number of beneficial bacteria such as Lactobacillus or Bifidobacterium. Due to their wide functions in the GI tract, AM and PAMP are involved in several digestive pathologies such as peptic ulcer, diabetes, colon cancer, or inflammatory bowel disease (IBD). AM is a key protective factor in IBD onset and development, as it regulates cytokine production in the intestinal mucosa, improves vascular and lymphatic regeneration and function and mucosal epithelial repair, and promotes a beneficial gut microbiome composition. AM and PAMP are relevant GI hormones that can be targeted to develop novel therapeutic agents for IBD, other GI disorders, or microbiome-related pathologies.

Proadrenomedullin N-Terminal 20 Peptides (PAMPs) Are Agonists of the Chemokine Scavenger Receptor ACKR3/CXCR7.

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two peptides with vasodilative, bronchodilative, and angiogenic properties, originating from a common precursor, proADM. Previous studies proposed that the atypical chemokine receptor ACKR3 might act as a low-affinity scavenger for ADM, regulating its availability for its cognate receptor calcitonin receptor-like receptor (CLR) in complex with a receptor activity modifying protein (RAMP). In this study, we compared the activation of ACKR3 by ADM and PAMP, as well as other related members of the calcitonin gene-related peptide (CGRP) family. Irrespective of the presence of RAMPs, ADM was the only member of the CGRP family to show moderate activity toward ACKR3. Remarkably, PAMP, and especially further processed PAMP-12, had a stronger potency toward ACKR3 than ADM. Importantly, PAMP-12 induced β-arrestin recruitment and was efficiently internalized by ACKR3 without inducing G protein or ERK signaling in vitro. Our results further extend the panel of endogenous ACKR3 ligands and broaden ACKR3 functions to a regulator of PAMP-12 availability for its primary receptor Mas-related G-protein-coupled receptor member X2 (MrgX2).

Proadrenomedullin N-terminal 20 peptide (PAMP) and its C-terminal 12-residue peptide, PAMP(9–20): Cell selectivity and antimicrobial mechanism

Proadrenomedullin N-terminal 20 peptide (PAMP) is a regulatory peptide that is found in various cell types. It is involved in many biological activities and is rich in basic and hydrophobic amino acids, a common feature of antimicrobial peptides (AMPs). In this study, the cell selectivity and antimicrobial mechanism of PAMP and its C-terminal peptide, PAMP(9–20), were investigated. PAMP and PAMP(9–20) displayed potent antimicrobial activity (minimum inhibitory concentration: 4–32 μM) against standard bacterial strains, but showed no hemolytic activity even at the highest tested concentration of 256 μM. PAMP(9–20) showed 2- to 4-fold increase in antimicrobial activity against gram-negative bacteria compared to PAMP. Cytoplasmic membrane depolarization, leakage of calcein dye from membrane mimic liposomes, SYTOX Green uptake, membrane permeabilization, and flow cytometry studies indicated that the major target of PAMP and PAMP(9–20) is not the microbial cell membrane. Interestingly, laser-scanning confocal microscopy demonstrated that FITC-labeled PAMP and PAMP(9–20) enter the cytoplasm of Escherichia coli similar to buforin-2, and gel retardation assay indicated that PAMP and PAMP(9–20) effectively bind to bacterial DNA. These results suggest that the intracellular target mechanism is responsible for the antimicrobial action of PAMP and PAMP(9–20). Collectively, PAMP and PAMP(9–20) could be considered promising candidates for the development of new antimicrobial agents.

PAMP protects intestine from Enterohemorrhagic Escherichia coli infection through destroying cell membrane and inhibiting inflammatory response

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Genetic loss of proadrenomedullin N-terminal 20 peptide (PAMP) in mice is compatible with survival

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are small peptides derived from a common precursor, pre-proadrenomedullin. Although AM and PAMP share hypotensive effects in the cardiovascular system, the peptides also exert diverse and distinct effects on endocrine physiology, innate immunity, cytoskeletal biology and receptor signaling pathways. Tremendous knowledge has been gleaned from the study of several genetic animal models of AM deletion or overexpression, some of which also simultaneously delete the coding region for PAMP peptide. However, deletion of PAMP without concurrent deletion of AM in an animal model is not currently available for the study of PAMP function. Here, we present the generation of AdmΔPAMP/ΔPAMP and AdmΔPAMP/− mice, which lack the coding sequence for PAMP while preserving the coding sequence for AM. AdmΔPAMP/ΔPAMP mice survive to adulthood without any obvious abnormalities and are fertile, though AdmΔPAMP/− females have small litters. Interestingly, these animals express lower levels of Adm mRNA and AM peptide than wild type animals, but these levels are still compatible with survival. Importantly, despite reduced levels, the spatiotemporal expression of AM peptide within the hearts of AdmΔPAMP/− mice remains similar to wild type animals. AdmΔPAMP/ΔPAMP mice are now a publicly available tool for future investigations of PAMP function.

Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer

To investigate the contribution of adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we studied the effects of four small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. For each SM, four experimental groups of male mice were used: (i) Control group; (ii) SM group; (iii) DSS group (injected with azoxymethane [AOM] and drank dextran sulfate sodium [DSS]); and (iv) DSS + SM group (treated with AOM, DSS, and the SM). None of the mice in groups i and ii developed tumors, whereas all mice in groups iii and iv developed colon neoplasias. No significant differences were found among mice treated with PAMP modulators (87877 and 106221). Mice that received the AM negative modulator, 16311, had worse colitis symptoms than their control counterparts, whereas mice injected with the AM positive modulator, 145425, had a lower number of tumors than their controls. SM 145425 regulated the expression of proliferation marker Lgr5 and had an impact on microbiota, preventing the DSS-elicited increase of the Bacteroides/Prevotella ratio. These results suggest that treatment with AM or with positive modulator SMs may represent a novel strategy for colon cancer.

Adrenomedullin Contributes to Age-Related Memory Loss in Mice and Is Elevated in Aging Human Brains.

Memory decline is common in elderly individuals and is the hallmark of Alzheimer’s disease (AD). Memory failure follows the loss of synaptic contacts in the cerebral cortex and hippocampus, caused in part by cytoskeleton disruption. Adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), are microtubule-associated proteins (MAP) whose expression has been identified as a potential biomarker for predicting progression from predementia to clinical AD. Here we analyze the connection between AM levels and memory preservation. Mice lacking neuronal AM and PAMP (knockout, KO) and their wild type (WT) littermates were subjected, at different ages, to the novel object recognition test and the contextual fear conditioned test. Aged KO mice have significantly better retention memory than their WT counterparts. This feature was more prominent in females than in males. Prefrontal cortex and hippocampus samples from these animals were subjected to Western blotting for phospho-Tau and acetylated tubulin. Aged female KO mice had significantly less accumulation of phospho-Tau than their WT littermates. In addition, protein extracts from the frontal cortex of non-demented mature (65.10 ± 3.86 years) and aged (77.14 ± 2.77 years) human donors were analyzed by Western blotting. Aged human brains had significantly higher levels of AM and lower levels of acetylated tubulin than younger donors. These observations suggest that drugs or interventions that reduce AM/PAMP expression may constitute a new avenue to prevent memory decline during normal aging and in patients suffering moderate AD in high risk of rapid cognitive decline.

Abstract 3220: Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer

Abstract Adrenomedullin (AM) expression is elevated in colon cancer patients and a correlation between higher AM levels and lower disease-free survival has been described in Japan. Nevertheless, the influence of AM in colon cancer is somewhat controversial: on the one hand, antibodies against either the peptide or the receptor reduce the growth of xenografted tumors, whereas application of the peptide clearly reduces inflammation and clinical severity in human and mouse models of colitis, which is an important risk factor for colon cancer. To investigate the contribution of AM and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we have studied the effects of several small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. Colon tumors were induced by intraperitoneal injection of a carcinogen: 10 mg/Kg azoxymethane, followed by 3 cycles of a colitis-inducing chemical: 2.5% dextran sulfate sodium (DSS) in the drinking water for a week followed by 2 weeks of regular water. Four SM were tested: 16311 (a negative modulator of AM), 145425 (a positive modulator of AM), 87877 (a negative modulator of PAMP), and 106221 (a positive modulator of PAMP). For each SM, 4 experimental groups of male C57BL6 mice were used: i) Control group (injected with vehicle and drank regular water); ii) Control+SM (vehicle, regular water, and injected with the SM 3 times a week at a concentration of 20 nm/Kg); iii) Cancer group (injected with AOM and drank DSS); and iv) Cancer+SM (treated with AOM, DSS, and the SM). At the end of the experimental procedures, animals were sacrificed and the colon was extracted and analyzed. None of the mice belonging to groups i and ii developed any tumor or had any pathological finding, indicating that the SMs do not present overt toxicity. All mice in groups iii and iv developed colon neoplasias ranging from carcinomas in situ to adenocarcinomas. No significant differences were found among mice treated with PAMP modulators indicating that PAMP may not play a major role in colon cancer or that the PAMP-related SMs were not very effective. On the other hand, mice that received SM 16311 had a higher severity index (p<0.001) and weight loss (p<0.05) than their control counterparts, whereas mice injected with SM 145425 had a lower number of tumors than their controls (p<0.001). These results suggest that AM may have a protective role during the progression phase of colon cancer, and that treatment with AM or with positive modulator SMs may represent a novel treatment for colon cancer. This study was funded by Instituto de Salud Carlos III (PI13/02166), FEDER, and Junta Provincial de La Rioja de la Asociación Española Contra el Cáncer (AECC). Citation Format: Laura Ochoa-Callejero, Josune García-Sanmartín, Sonia Martínez-Herrero, Susana Rubio-Mediavilla, Alfredo Martínez. Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3220. doi:10.1158/1538-7445.AM2017-3220

The effect of adrenomedullin and proadrenomedullin N-terminal 20 peptide on angiotensin II induced vascular smooth muscle cell proliferation.

The study aimed to investigate the effects of adrenomedullin (ADM) and proadrenomedullin N- terminal 20 peptide (PAMP) on angiotensin (AngII)-stimulated proliferation in vascular smooth muscle cells (VSMCs). Thoracic aorta was obtained from Wistar rats and VSMCs were isolated from aorta tissues and then cultured. In vitro cultured VSMCs were stimulated with Ang II (10(-8) mol/l) followed by various doses of PAMP or ADM (10(-9), 10(-8), or 10(-7) mol/l). Cell proliferation as assessed by (3)H-TdR incorporation. Protein kinase C (PKC) activity was measured by counting γ-(32)P radioactivity with liquid scintillation. In a separate cohort, in vitro cultured rat aortic vessels were treated with different doses of Ang II or PAMP (10(-9), 10(-8), or 10(-7) mol/l). Cellular and secreted levels of PAMP, ADM and Ang II were measured using radioimmunoassay in the tissues and intubation mediums, respectively. Ang II (10(-8) mol/l) treatment significantly increased both (3)H-TdR incorporation and PKC activity in VSMCs (by 2.68 and 1.02-fold, respectively; both P<0.01 vs. the control). However, Ang II-induced elevation of (3)H-TdR incorporation, and PKC activity was significantly inhibited by various doses of ADM and PAMP (all P<0.01 vs. the Ang II group). In rat aortic vascular tissues or intubation media, Ang II treatments stimulated the expression and secretion of PAMP and ADM in a dose-dependent manner, while PAMP treatments had no significant effects on Ang II levels. ADM and PAMP inhibit Ang II-induced VSMCs proliferation. The interaction of Ang II, ADM and PAMP may regulate VSMCs and cardiovascular function.

Expression of peptide fragments from proADM and involvement of mitogen-activated protein kinase signaling pathways in pulmonary remodeling induced by high pulmonary blood flow.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive pulmonary arterial remodeling and right ventricular failure. Despite recent advances in pathophysiological mechanism exploration and new therapeutic approaches, PAH remains a challenging condition. In this study, we investigated the roles of the peptide fragments from proadrenomedullin (proADM) such as adrenomedullin (ADM), adrenotensin (ADT), and proadrenomedullin N-terminal 20 peptide (PAMP) during pulmonary remodeling caused by high pulmonary blood flow, and probed the possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways. Sixteen rat models of PAH were artificially established by surgically connecting the left common carotid artery to the external jugular vein. We subcutaneously injected an extracellular signal-regulated protein kinase (ERK1/2) inhibitor, PD98059, in eight rats, treated another eight rats with an equal volume of saline. Eight rats without connections served as the control group. We observed that mRNA expression levels of ADM, stress-activated protein kinase (SAPK), and ERK1/2 were significantly elevated in the shunted rats; furthermore, ERK1/2 levels were significantly inhibited by PD98059. Protein levels of ADM, PAMP, p-SAPK, and p-ERK1/2 were significantly higher ADT was lower, and p-p38 remained unchanged in the rat models compared with the controls. However, the protein expression of both ADM and p-ERK1/2 was significantly inhibited by PD98059. Our results suggest that levels of ADM, ADT, and PAMP respond to pulmonary remodeling, and that activation of the SAPK and ERK1/2 signaling pathways is involved in pulmonary hypertension and artery remodeling caused by high pulmonary blood flow.

Peptidomics for Studying Limited Proteolysis.

Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.

Effect of vasoactive peptides in Tetrahymena: chemotactic activities of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP).

Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny.

Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents

The article deals with the design, preparation, and evaluation of a new bilayered dressing for application in the healing of compromised wounds. The system is based on the sequential release of two complementary bioactive components to enhance the activation of the regeneration of dermal tissue. The internal layer is a highly hydrophilic and biodegradable film of gelatin and hyaluronic acid (HG), crosslinked with the natural compound genipin, which reacts with the amine groups of gelatin. This film is loaded with the proangiogenic, anti-inflammatory, and antibacterial peptide, proadrenomedullin N-terminal 20 peptide (PAMP), that is released slowly in the wound site. The external layer, more stable and less hydrophilic, is constituted by a biodegradable polyurethane derived from poly(caprolactone) and pluronic L61. This layer is loaded with resorbable nanoparticles of bemiparin (a fractionated low molecular weight heparin), which promotes the activation of growth factors, FGF and VEGF, and provides a good biomechanical stability and controlled permeability of the bilayered dressing.Experiments carried out in mice demonstrate the excellent angiogenic effect of the HG film in the dermal tissue. Application of the bilayered dressing in the wound healing rabbit ear model shows an improved cicatrization of the wound in both ischemic and non-ischemic defects, favoring epithelialization and reducing noticeably the contraction and the inflammation.

The heart as an endocrine organ

The concept of the heart as an endocrine organ arises from the observation that the atrial cardiomyocytes in the mammalian heart display a phenotype that is partly that of endocrine cells. Investigations carried out between 1971 and 1983 characterised, by virtue of its natriuretic properties, a polypeptide referred to atrial natriuretic factor (ANF). Another polypeptide isolated from brain in 1988, brain natriuretic peptide (BNP), was subsequently characterised as a second hormone produced by the mammalian heart atria. These peptides were associated with the maintenance of extracellular fluid volume and blood pressure. Later work demonstrated a plethora of other properties for ANF and BNP, now designated cardiac natriuretic peptides (cNPs). In addition to the cNPs, other polypeptide hormones are expressed in the heart that likely act upon the myocardium in a paracrine or autocrine fashion. These include the C-type natriuretic peptide, adrenomedullin, proadrenomedullin N-terminal peptide and endothelin-1. Expression and secretion of ANF and BNP are increased in various cardiovascular pathologies and their levels in blood are used in the diagnosis and prognosis of cardiovascular disease. In addition, therapeutic uses for these peptides or related substances have been found. In all, the discovery of the endocrine heart provided a shift from the classical functional paradigm of the heart that regarded this organ solely as a blood pump to one that regards this organ as self-regulating its workload humorally and that also influences the function of several other organs that control cardiovascular function.

Is the Cytoskeleton an Intracellular Receptor for Adrenomedullin and PAMP?

Classical transmembrane receptors have been described for both adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Through interactions with these membrane receptors, AM and PAMP exert a variety of endocrine, paracrine, and autocrine functions. In addition to these better known activities, recent publications have shown that both peptides can bind directly to the cytoskeleton resulting in important cellular physiological responses. In vitro and in vivo experiments show that the peptides bind to major components of the cytoskeleton: tubulin and kinesin for PAMP and a number of microtubule-associated proteins (MAPs) in the case of AM. Physiological experiments show that PAMP contributes to microtubule fluidity and increases kinesin speed. Lack of AM and PAMP results in hyperpolymerization of the cytoskeleton and a reduced motility of intracellular organelles. These data suggest that the cytoskeleton may have a novel function as an intracellular receptor, acting as the binding site and the signal transducer for specific peptide hormones such as PAMP.

Adrenomedullin Haploinsufficiency Predisposes to Secondary Lymphedema

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor–like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (AdmAM+/Δ animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both AdmAM+/+ and AdmAM+/Δ mice. However, only AdmAM+/Δ animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in AdmAM+/Δ mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

Identification of first proadrenomedullin N-terminal 20 peptide (PAMP) modulator by means of virtual screening and NMR interaction experiments

PAMP (proadrenomedullin N-terminal 20 peptide) is a regulatory peptide that is detected in a large variety of cell types and exerts important biological activities. PAMP acts as a potent angiogenic factor and decorates microtubules in cells from different origins, controlling tubulin polymerization. A high-throughput docking-based virtual screening was performed, followed by a competitive monoclonal antibody assay on selected compounds, and a detailed 1H, 15N NMR spectroscopy study. This procedure has allowed us to describe the first small molecule capable of interacting with PAMP and potentially modulate its biological activity. Molecular modeling methods such as docking and molecular dynamics were carried out to obtain a theoretical model of binding mode. Finally a directed in vivo angiogenesis assay (DIVAA) showed that the small molecule by itself has pro-angiogenic properties.

Proadrenomedullin N-Terminal 20 Peptide Increases Kinesin's Velocity Both in Vitro and in Vivo

Intracellular cargo transport relies on microtubules and motor proteins such as kinesins and dyneins. Currently we have ample knowledge of the mechanisms by which motor proteins propel themselves along the microtubules, but little is known about intracellular factors that regulate motor speed. Here we show that proadrenomedullin N-terminal 20 peptide (PAMP) increases kinesin velocity and ATP consumption in a dose-dependent manner, using a variety of human kinesins. Structure-activity studies found that the terminal amide of PAMP is required for modulating kinesin activity and that the smallest peptide fragment retaining this role is PAMP₁₂₋₂₀. On the other hand, peptide fragments as small as PAMP₁₈₋₂₀ maintained the ability of delaying tubulin polymerization, another function previously described for PAMP, indicating that these two activities depend on different regions of the molecule. To demonstrate that these observations are also relevant in vivo, hippocampal neurons were isolated from mice lacking the gene coding for PAMP and from wild type littermates. Intravital stains followed by time-lapse microscopy analysis revealed that mitochondrial speed inside neurons lacking PAMP was significantly slower than in cells expressing the peptide. External addition of synthetic PAMP reversed this phenotype in PAMP-null neurons. Besides the obvious implications for better understanding cell biology, these results may be also relevant for the rapidly evolving discipline of nanotechnology because PAMP may be used as an accelerator of nanodevices based on microtubules and motor proteins.