ABSTRACT
The application of machine learning to longitudinal gene-expression profiles has demonstrated potential to decrease the assessment gap, between biochemical determination and clinical manifestation, of a patient's response to treatment. Although psoriasis is a proven testing ground for treatment-response prediction using transcriptomic data from clinically accessible skin biopsies, these biopsies are expensive, invasive, and challenging to obtain from certain body areas. Response prediction from blood biochemical measurements could be a cheaper, less invasive predictive platform. Longitudinal profiles for 92 inflammatory and 65 cardiovascular disease proteins were measured from the blood of psoriasis patients at baseline, and 4-weeks, following tofacitinib (janus kinase-signal transducer and activator of transcription-inhibitor) or etanercept (tumor necrosis factor-inhibitor) treatment, and predictive models were developed by applying machine-learning techniques such as bagging and ensembles. This data driven approach developed predictive models able to accurately predict the 12-week clinical endpoint for psoriasis following tofacitinib (area under the receiver operating characteristic curve [auROC] = 78%), or etanercept (auROC = 71%) treatment in a validation dataset, revealing a robust predictive protein signature including well-established psoriasis markers such as IL-17A and IL-17C, highlighting potential for biologically meaningful and clinically useful response predictions using blood protein data. Although most blood classifiers were outperformed by simple models trained using Psoriasis Area Severity Index scores, performance might be enhanced in future studies by measuring a wider variety of proteins.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Biomarkers, Pharmacological/metabolism , Etanercept/therapeutic use , Inflammation Mediators/metabolism , Interleukin-17/metabolism , Piperidines/therapeutic use , Psoriasis/diagnosis , Pyrimidines/therapeutic use , Adult , Aged , Cohort Studies , Computer Simulation , Double-Blind Method , Female , Humans , Interleukin-17/genetics , Machine Learning , Male , Middle Aged , Placebos , Predictive Value of Tests , Prognosis , Psoriasis/drug therapy , Psoriasis/immunology , Time Factors , Treatment Outcome , Young AdultABSTRACT
Patients with psoriasis have an increased risk of myocardial infarction, and psoriasis is now recognized as an independent risk factor for coronary heart disease and cardiovascular mortality. To understand the effects of psoriasis medications on systemic inflammation associated with cardiovascular risks, we studied blood proteins related to inflammation and cardiovascular disease archived from a phase 3 clinical trial of tofacitinib and etanercept in adults with moderate-to-severe psoriasis. A total of 157 blood proteins were quantified by a proximity extension assay from 266 patients at baseline and week 4. Protein changes in the blood after 1 month of treatment were compared between tofacitinib (10 mg two times a day) and etanercept (50 mg biweekly), and by response status at week 12. Tofacitinib and etanercept commonly reduced IL-6, CCL20, and CXCL10, but IL-17A was significantly reduced only in responders of either treatment. Compared with etanercept, tofacitinib showed a wider spectrum of cardiovascular blood protein reduction, but the protein reduction effects of tofacitinib were strictly confined to treatment responders. Tumor necrosis factor receptor 1, E-selectin, hK11, tumor necrosis factor-related activation-induced cytokine, CHI3L1, IL-16, and matrix metalloproteinase-12 were cardiovascular proteins significantly reduced only in tofacitinib responders. Our data suggest that a short-term systemic psoriasis treatment can cause reductions in circulating inflammatory and other proteins associated with cardiovascular risks.
Subject(s)
Cardiovascular Diseases/prevention & control , Cytokines/blood , Etanercept/therapeutic use , Inflammation/prevention & control , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Psoriasis/drug therapy , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Adult , Aged , Cardiovascular Diseases/blood , Cardiovascular Diseases/immunology , Cytokines/immunology , Etanercept/pharmacology , Female , Humans , Inflammation/blood , Inflammation/immunology , Male , Middle Aged , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proteomics , Psoriasis/blood , Psoriasis/complications , Psoriasis/immunology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Risk Factors , Treatment Outcome , Young AdultABSTRACT
Acidic mammalian chitinase (AMCase) is known to be induced by allergens and helminths, yet its role in immunity is unclear. Using AMCase-deficient mice, we show that AMCase deficiency reduced the number of group 2 innate lymphoid cells during allergen challenge but was not required for establishment of type 2 inflammation in the lung in response to allergens or helminths. In contrast, AMCase-deficient mice showed a profound defect in type 2 immunity following infection with the chitin-containing gastrointestinal nematodes Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. The impaired immunity was associated with reduced mucus production and decreased intestinal expression of the signature type 2 response genes Il13, Chil3, Retnlb, and Clca1. CD103(+) dendritic cells, which regulate T cell homing, were also reduced in mesenteric lymph nodes of infected AMCase-deficient mice. Thus, AMCase functions as a critical initiator of protective type 2 responses to intestinal nematodes but is largely dispensable for allergic responses in the lung.
Subject(s)
Chitinases/immunology , Gastrointestinal Tract/immunology , Immunity/immunology , Strongylida Infections/immunology , Animals , Chitinases/genetics , Chitinases/metabolism , Chloride Channels/genetics , Chloride Channels/immunology , Chloride Channels/metabolism , Flow Cytometry , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/parasitology , Gene Expression/immunology , Hormones, Ectopic/genetics , Hormones, Ectopic/immunology , Hormones, Ectopic/metabolism , Host-Parasite Interactions/immunology , Hypersensitivity/genetics , Hypersensitivity/immunology , Hypersensitivity/metabolism , Immunity/genetics , Intercellular Signaling Peptides and Proteins , Interleukin-13/genetics , Interleukin-13/immunology , Interleukin-13/metabolism , Lectins/genetics , Lectins/immunology , Lectins/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Nematospiroides dubius/immunology , Nematospiroides dubius/physiology , Nippostrongylus/immunology , Nippostrongylus/physiology , Reverse Transcriptase Polymerase Chain Reaction , Strongylida Infections/metabolism , Strongylida Infections/parasitology , beta-N-Acetylhexosaminidases/genetics , beta-N-Acetylhexosaminidases/immunology , beta-N-Acetylhexosaminidases/metabolismABSTRACT
While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.
Subject(s)
Antibodies, Bispecific/genetics , Complementarity Determining Regions/genetics , Protein Engineering , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/metabolism , Animals , Bacteriophages/genetics , Enzyme-Linked Immunosorbent Assay , Female , Humans , Injections, Subcutaneous , Peptide Library , Protein Stability , Rats , Single-Chain Antibodies/genetics , TemperatureABSTRACT
Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule.
Subject(s)
Antibodies, Bispecific/genetics , Interleukin-17/immunology , Interleukins/immunology , Polysaccharides/chemistry , Pyrrolidonecarboxylic Acid/chemistry , Amino Acid Sequence , Chromatography, Liquid , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Mass Spectrometry , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Interleukin-22ABSTRACT
The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.
Subject(s)
Asthma/enzymology , Chitinases/antagonists & inhibitors , Hypersensitivity/enzymology , Allergens/immunology , Animals , Asthma/genetics , Asthma/immunology , Bronchoalveolar Lavage Fluid/immunology , Chitinases/deficiency , Chitinases/genetics , Chitinases/immunology , Female , Humans , Hypersensitivity/genetics , Hypersensitivity/immunology , Inflammation/enzymology , Inflammation/genetics , Inflammation/immunology , Interleukin-13/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Neutrophils/immunology , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
Subject(s)
Complement C3a/metabolism , DNA/metabolism , Interferon-gamma/metabolism , Oligonucleotides/metabolism , Receptor for Advanced Glycation End Products/metabolism , Animals , Complement C3a/immunology , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Interferon-gamma/immunology , Mice , Mice, Knockout , Oligonucleotides/immunology , Protein Binding , Receptor for Advanced Glycation End Products/immunology , Surface Plasmon ResonanceABSTRACT
Acidic mammalian chitinase (AMCase) is a member of the glycosyl hydrolase 18 family (EC 3.2.1.14) that has been implicated in the pathophysiology of allergic airway disease such as asthma. Small molecule inhibitors of AMCase were identified using a combination of high-throughput screening, fragment screening, and virtual screening techniques and characterized by enzyme inhibition and NMR and Biacore binding experiments. X-ray structures of the inhibitors in complex with AMCase revealed that the larger more potent HTS hits, e.g. 5-(4-(2-(4-bromophenoxy)ethyl)piperazine-1-yl)-1H-1,2,4-triazol-3-amine 1, spanned from the active site pocket to a hydrophobic pocket. Smaller fragments identified by FBS occupy both these pockets independently and suggest potential strategies for linking fragments. Compound 1 is a 200 nM AMCase inhibitor which reduced AMCase enzymatic activity in the bronchoalveolar lavage fluid in allergen-challenged mice after oral dosing.
Subject(s)
Chitinases/antagonists & inhibitors , Models, Molecular , Piperazines/chemical synthesis , Triazoles/chemical synthesis , Allergens/immunology , Animals , Bronchoalveolar Lavage Fluid , Catalytic Domain , Crystallography, X-Ray , Female , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred C57BL , Piperazines/chemistry , Piperazines/pharmacology , Protein Binding , Respiratory Hypersensitivity/drug therapy , Respiratory Hypersensitivity/enzymology , Respiratory Hypersensitivity/immunology , Structure-Activity Relationship , Surface Plasmon Resonance , Triazoles/chemistry , Triazoles/pharmacologyABSTRACT
Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.
Subject(s)
Chitinases/genetics , Evolution, Molecular , Macaca fascicularis/genetics , Macaca mulatta/genetics , Amino Acid Sequence , Animals , Base Sequence , Chitin/metabolism , Chitinases/chemistry , Chitinases/metabolism , Conserved Sequence , Databases, Genetic , Gene Expression Regulation, Enzymologic , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Organ Specificity , Sequence AlignmentABSTRACT
Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K(m) of 28 microM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.
Subject(s)
Amidohydrolases/metabolism , Chromatography, High Pressure Liquid/methods , Enzyme Inhibitors/chemistry , Mass Spectrometry/methods , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/genetics , Amino Acid Sequence , Animals , Cell Line , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/chemistry , GPI-Linked Proteins , High-Throughput Screening Assays , Humans , Kidney/enzymology , Mice , Molecular Sequence Data , Pantothenic Acid/chemistry , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry, UltravioletABSTRACT
IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.
Subject(s)
Interleukin-17/physiology , Receptors, Interleukin-17/physiology , Receptors, Interleukin/physiology , Signal Transduction/immunology , Cell Line , Dimerization , Dose-Response Relationship, Immunologic , Humans , Interleukin-17/antagonists & inhibitors , Interleukin-17/chemistry , Interleukin-17/metabolism , Protein Binding/immunology , Receptors, Interleukin/metabolism , Receptors, Interleukin-17/metabolismABSTRACT
Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Interleukin-13/metabolism , Humans , Interleukin-13/chemistry , Interleukin-13 Receptor alpha1 Subunit/chemistry , Interleukin-13 Receptor alpha1 Subunit/metabolism , Protein Binding , Receptors, Interleukin-4/chemistry , Receptors, Interleukin-4/metabolism , Reproducibility of Results , Surface Plasmon ResonanceABSTRACT
Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.
Subject(s)
Cartilage, Articular/metabolism , Glycoproteins/metabolism , Animals , Cattle , Glycoproteins/chemistry , Glycoproteins/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Engagement of the immunoinhibitory receptor, programmed death-1 (PD-1) attenuates T-cell receptor (TCR)-mediated activation of IL-2 production and T-cell proliferation. Here, we demonstrate that PD-1 modulation of T-cell function involves inhibition of TCR-mediated phosphorylation of ZAP70 and association with CD3zeta. In addition, PD-1 signaling attenuates PKCtheta activation loop phosphorylation in a cognate TCR signal. PKCtheta has been shown to be required for T-cell IL-2 production. A phosphorylated PD-1 peptide, corresponding to the C-terminal immunoreceptor tyrosine-switch motif (ITSM), acts as a docking site in vitro for both SHP-2 and SHP-1, while the phosphorylated peptide containing the N-terminal PD-1 immunoreceptor tyrosine based inhibitory motif (ITIM) associates only with SHP-2.