ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance.

ILDR2 is a member of the Ig superfamily, which is implicated in tricellular tight junctions, and has a putative role in pancreatic islet health and survival. We recently found a novel role for ILDR2 in delivering inhibitory signals to T cells. In this article, we show that short-term treatment with ILDR2-Fc results in long-term durable beneficial effects in the relapsing-remitting experimental autoimmune encephalomyelitis and NOD type 1 diabetes models. ILDR2-Fc also promotes transplant engraftment in a minor mismatch bone marrow transplantation model. ILDR2-Fc displays a unique mode of action, combining immunomodulation, regulation of immune homeostasis, and re-establishment of Ag-specific immune tolerance via regulatory T cell induction. These findings support the potential of ILDR-Fc to provide a promising therapeutic approach for the treatment of autoimmune diseases.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

The novel Mas agonist, CGEN-856S, attenuates isoproterenol-induced cardiac remodeling and myocardial infarction injury in rats.

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg(-1)·day(-1)) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg(-1)·day(-1)) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg(-1)·day(-1)) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson's trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.

Prevention of bleomycin-induced pulmonary fibrosis by a novel antifibrotic peptide with relaxin-like activity.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-ß1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.

Vascular relaxation, antihypertensive effect, and cardioprotection of a novel peptide agonist of the MAS receptor.

Mas stimulation with angiotensin (Ang)-(1-7) produces cardioprotective effects and vasorelaxation. Using a computational discovery platform for predicting novel naturally occurring peptides that may activate G protein-coupled receptors, we discovered a novel Mas agonist peptide, CGEN-856S. An endothelium- and NO-dependent vasodilating effect was observed for CGEN-856S in thoracic aorta rings of rats (maximal value for the relaxant effect: 39.99+/-5.034%), which was similar to that produced by Ang-(1-7) (10(-10) to 10(-6) mol/L). In addition, the vasodilator activity of this peptide depended on a functional Mas receptor, because it was abolished in aorta rings of Mas-knockout mice. CGEN-856S appears to bind the Mas receptor at the same binding domain as Ang-(1-7), as suggested by the blocking of its vasorelaxant effect with the Ang-(1-7) analogue d-Ala(7)-Ang-(1-7), and by its competitive inhibition of Ang-(1-7) binding to Mas-transfected cells. The effect of CGEN-856S on reperfusion arrhythmias and cardiac function was studied on ischemia reperfusion of isolated rat hearts. We found that picomolar concentration of CGEN-856S (0.04 nmol/L) had an antiarrhythmogenic effect, as demonstrated by a reduction in the incidence and duration of reperfusion arrhythmias. Furthermore, acute infusion of CGEN-856S produced a shallow dose-dependent decrease in mean arterial pressure of conscious spontaneously hypertensive rats. The maximum change during infusion was observed at the highest dose. Strikingly, blood pressure continued to drop in the postinfusion period. The results presented here indicate that the novel Mas agonist, CGEN-856S, might have a therapeutic value, because it induces vasorelaxing, antihypertensive, and cardioprotective effects.

A novel peptide agonist of formyl-peptide receptor-like 1 (ALX) displays anti-inflammatory and cardioprotective effects.

Activation of the formyl-peptide receptor-like (FPRL) 1 pathway has recently gained high recognition for its significance in therapy of inflammatory diseases. Agonism at FPRL1 affords a beneficial effect in animal models of acute inflammatory conditions, as well as in chronic inflammatory diseases. TIPMFVPESTSKLQKFTSWFM-amide (CGEN-855A) is a novel 21-amino acid peptide agonist for FPRL1 and also activates FPRL2. CGEN-855A was discovered using a computational platform designed to predict novel G protein-coupled receptor peptide agonists cleaved from secreted proteins by convertase proteolysis. In vivo, CGEN-855A displays anti-inflammatory activity manifested as 50% inhibition of polymorphonuclear neutrophil (PMN) recruitment to inflamed air pouch and provides protection against ischemia-reperfusion-mediated injury to the myocardium in both murine and rat models (36 and 25% reduction in infarct size, respectively). Both these activities are accompanied by inhibition of PMN recruitment to the injured organ. The secretion of inflammatory cytokines, including interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha, was not affected upon incubation of human peripheral blood mononuclear cells with CGEN-855A, whereas IL-8 secretion was elevated up to 2-fold upon treatment with the highest CGEN-855A dose only. Collectively, these new data support a potential role for CGEN-855A in the treatment of reperfusion-mediated injury and in other acute and chronic inflammatory conditions.

Discovery and validation of novel peptide agonists for G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

PURPOSE: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. EXPERIMENTAL DESIGN: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. RESULTS: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. CONCLUSIONS: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.

hCHL2, a novel chordin-related gene, displays differential expression and complex alternative splicing in human tissues and during myoblast and osteoblast maturation.

Chordin-like cysteine-rich repeats (CRs) are conserved domains present in an expanding family of secreted proteins that associate with members of the TGF beta superfamily. In this study, we report the molecular cloning and characterization of CHL2 (chordin-like 2), a novel protein closely related to CHL (chordin-like). Both are members of the chordin family of proteins, and contain a signal peptide and three CR domains. We found that recombinant human CHL2 (hCHL2) protein is secreted and binds activin A, but not BMP-2, -4, or -6. Expression of hCHL2 mRNA and protein was detected in a variety of human tissues and is particularly abundant in the uterus. Extensive and complex alternative splicing of hCHL2 was observed in different tissues, resulting in several distinct protein isoforms that vary substantially in the presence of a signal peptide and their content of CR domains. Differential expression of CHL2 variants was observed during myoblast and osteoblast differentiation, implying a role for this gene in these physiological processes.

Evolution of multicellularity in Metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis.

A comparison of the subcellular assignments of proteins between the unicellular Saccharomyces cerevisiae and the multicellular Drosophila melanogaster and Caenorhabditis elegans was performed using a computational tool for the prediction of subcellular localization. Nine subcellular compartments were studied: (1) extracellular domain, (2) cell membrane, (3) cytoplasm, (4) endoplasmic reticulum, (5) Golgi apparatus, (6) lysosome, (7) peroxisome, (8) mitochondria, and (9) nucleus. The transition to multicellularity was found to be characterized by an increase in the total number of proteins encoded by the genome. Interestingly, this increase is distributed unevenly among the subcellular compartments. That is, a disproportionate increase in the number of proteins in the extracellular domain, the cell membrane, and the cytoplasm is observed in multicellular organisms, while no such increase is seen in other subcellular compartments. A possible explanation involves signal transduction. In terms of protein numbers, signal transduction pathways may be roughly described as a pyramid with an expansive base in the extracellular domain (the numerous extracellular signal proteins), progressively narrowing at the cell membrane and cytoplasmic levels, and ending in a narrow tip consisting of only a handful of transcription modulators in the nucleus. Our observations suggest that extracellular signaling interactions among metazoan cells account for the uneven increase in the numbers of proteins among subcellular compartments during the transition to multicellularity.

Widespread occurrence of antisense transcription in the human genome.

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that > or =60% of this data set, or approximately 1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.

Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins.

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and human kallikrein 2 are produced and secreted in the prostate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3 genes that result from inclusion of intronic sequences adjacent to the first exon. The novel proteins encoded by these transcripts, named PSA-linked molecule (PSA-LM) and hK2-linked molecule (K-LM), share only the signal peptide with the original protein product of the respective gene. The mature proteins are entirely different and bear no similarity to the kallikrein family or to other proteins in the databases. As is the case with PSA, PSA-LM is expressed in the secretory epithelial cells of the prostate and is up-regulated in response to androgenic stimulation. A similar pattern of expression is suggested for K-LM.

Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.

ATM deficiency and oxidative stress: a new dimension of defective response to DNA damage.

ATM is one of the sentries at the gate of genome stability. This multifunctional protein kinase orchestrates the intricate array of cellular responses to DNA double-strand breaks. Absence or inactivation of ATM leads to the pleiotropic genetic disorder ataxia-telangiectasia (A-T), whose hallmarks are neuronal degeneration, immunodeficiency, genomic instability, premature aging and cancer predisposition. Several features of the complex clinical and cellular phenotype of A-T are reminiscent of other syndromes involving neurodegeneration, premature aging or genomic instability. A common denominator of many of these conditions is the perturbation of the cellular balance of reactive oxygen species, which leads to constant oxidative stress. Of these disorders, ATM deficiency is one of the most extensively studied with regard to the genome instability-oxidative stress connection. This connection may provide new insights into the phenotypes associated with genetic deficiencies of DNA damage responses, and point to new strategies to alleviate some of their clinical symptoms.