RESUMO
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.
Assuntos
Antígenos B7/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Citocinas/imunologia , Humanos , Domínios de Imunoglobulina/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
MOTIVATION: Many secretory peptides are synthesized as inactive precursors that must undergo post-translational processing to become biologically active peptides. Attempts to predict natural peptides are limited by the low performance of proteolytic site predictors and by the high combinatorial complexity of pairing such sites. To overcome these limitations, we analyzed the site-wise evolutionary mutation rates of peptide hormone precursors, calculated using the Rate4Site algorithm. RESULTS: Our analysis revealed that within their precursors, peptide residues are significantly more conserved than the pro-peptide residues. This disparity enables the prediction of peptides with a precision of â¼60% at a recall of 40% [receiver-operating characteristic curve (ROC) AUC 0.79]. Subsequently, combining the Rate4Site score with additional features and training a Random Forest classifier enable the prediction of natural peptides hidden within secreted human proteins at a precision of â¼90% at a recall of 50% (ROC AUC 0.96). The high performance of our method allows it to be applied to full secretomes and to predict naturally occurring active peptides. Our prediction on Homo sapiens revealed several putative peptides in the human secretome that are currently unannotated. Furthermore, the unique expression of some of these peptides implies a potential hormone function, including peptides that are highly expressed in endocrine glands. AVAILABILITY AND IMPLEMENTATION: A pseudocode is available in the SUPPLEMENTARY INFORMATION. CONTACT: doron.gerber@biu.ac.il or kliger@cgen.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional/métodos , Evolução Molecular , Hormônios Peptídicos/química , Algoritmos , Sequência de Aminoácidos , Inteligência Artificial , Calcitonina/química , Sequência Conservada , Humanos , Dados de Sequência Molecular , Taxa de Mutação , Hormônios Peptídicos/metabolismo , Curva ROC , Análise de SequênciaRESUMO
Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.
Assuntos
Interleucina-18 , Microambiente Tumoral , Animais , Humanos , Interleucina-18/metabolismo , Camundongos , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Ativação Linfocitária/imunologia , Ativação Linfocitária/efeitos dos fármacos , Feminino , Camundongos Endogâmicos C57BL , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytotoxicity thus providing an "alternative self" mechanism for MHC class I inhibition.
Assuntos
Moléculas de Adesão Celular/metabolismo , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Animais , Moléculas de Adesão Celular/química , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Técnicas In Vitro , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ligantes , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Camundongos , Nectinas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Virais/química , Receptores Virais/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix-helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFalpha, IL-1beta, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFalpha, IL-6, and IFNgamma by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.
Assuntos
Biologia Computacional/métodos , Peptídeos/química , Animais , Antineoplásicos/farmacologia , Clusterina/química , Feminino , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/química , Camundongos , Camundongos Nus , Chaperonas Moleculares , Transplante de Neoplasias , Conformação Proteica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/prevenção & controle , Infarto do Miocárdio/prevenção & controle , Peptídeos/uso terapêutico , Receptores de Formil Peptídeo/agonistas , Receptores de Lipoxinas/agonistas , Animais , Células CHO , Cricetinae , Cricetulus , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/patologia , Peptídeos/farmacologia , RatosRESUMO
MOTIVATION: Many secretory proteins are synthesized as inactive precursors that must undergo post-translational proteolysis in order to mature and become active. In the current study, we address the challenge of sequence-based discovery of proteolytic sites in secreted proteins using machine learning. RESULTS: The results revealed that only half of the extracellular proteolytic sites are currently annotated, leaving over 3600 unannotated ones. Furthermore, we have found that only 6% of the unannotated sites are similar to known proteolytic sites, whereas the remaining 94% do not share significant similarity with any annotated proteolytic site. The computational challenges in these two cases are very different. While the precision in detecting the former group is close to perfect, only a mere 22% of the latter group were detected with a precision of 80%. The applicability of the classifier is demonstrated through members of the FGF family, in which we verified the conservation of physiologically-relevant proteolytic sites in homologous proteins.
Assuntos
Proteínas da Matriz Extracelular/química , Modelos Químicos , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Dados de Sequência Molecular , Ligação ProteicaRESUMO
CD40, a TNF receptor (TNFR) family member, is composed of four cysteine rich domains (CRDs) followed by a transmembrane domain and a signaling intracellular C-terminus. CD154 ligation to CD40 regulates major inflammatory and immune processes. A natural soluble form of CD40 was detected in uremic patient's serum which might be alternative splicing product. Our aim was to identify CD40 isoforms produced by primary human cells and to evaluate their biological activity. By RT-PCR, we isolated from primary human cells three major products differing from the expected wild type (WT) transcript which lacks exon 5 (CD40-5), exon 6 (CD40-6) and both exons 5 and 6 (CD40-5 + 6). The first two were predicted computationally to be soluble decoy receptors with various CRDs numbers. Recombinant soluble CD40 (sCD40) isoforms containing either three or four CRDs are able to bind coated surfaces or membranous CD154 while the sCD40 isoform containing 2 CRDs did not. sCD40 proteins inhibited RANTES secretion, but did not block B cell proliferation induced by soluble CD154. These data suggest that sCD40 natural isoforms encompassing either three or four CRDs might exert different antagonistic effects from known CD154 antibodies by recognition of only membranous CD154.
Assuntos
Processamento Alternativo/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Antígenos CD40/biossíntese , Antígenos CD40/genética , Ligante de CD40/genética , Ligante de CD40/metabolismo , Células Cultivadas , Éxons/imunologia , Humanos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína/fisiologia , SolubilidadeRESUMO
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.
Assuntos
Processamento Alternativo/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Mioblastos/metabolismo , Osteoblastos/metabolismo , Fator de Crescimento Transformador beta , Ativinas/genética , Ativinas/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/farmacologia , Células COS , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Sequência Conservada/genética , Cisteína/genética , Cisteína/metabolismo , Evolução Molecular , Proteínas da Matriz Extracelular , Feminino , Humanos , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/metabolismo , Insulina/farmacologia , Masculino , Camundongos , Dados de Sequência Molecular , Mioblastos/citologia , Osteoblastos/citologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/genética , RNA/metabolismo , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
In a screening effort based on algorithmic predictions for novel G-protein-coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen-like repeat. Both peptides seemed to show an apparent cAMP-related effect on CHO-K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP-generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin-3 receptor (GPR135). In a set of follow-up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 micro M forskolin in cells transfected with either LGR7 or LGR8. In a dye-free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO-K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7- and LGR8-transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin-insulin-like peptide family, are potential ligands for the relaxin-related family of receptors and as such might serve as novel candidates for relaxin-related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.
Assuntos
Colágeno/química , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Modelos Teóricos , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Relaxina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Algoritmos , Animais , Biologia Computacional/métodos , AMP Cíclico/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Inflamação , Ligantes , Camundongos , Peptídeos/química , Ligação Proteica , Engenharia de Proteínas , Proteômica/métodosRESUMO
Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes.
Assuntos
Fusão Gênica/genética , Genoma Humano/genética , Splicing de RNA/genética , Transcrição Gênica/genética , Evolução Molecular , Humanos , Células Jurkat , Células K562RESUMO
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.