Correlation of Aqueous, Vitreous, and Serum Protein Levels in Patients With Retinal Diseases.

Purpose: To further establish aqueous humor (AH) as a clinically suitable source of protein biomarkers in retinal diseases by evaluating the correlation of a large panel of proteins between AH, vitreous humor (VH), and serum (SE). Methods: We enrolled 60 subjects (eyes) with various non-infectious retinal diseases. AH, VH, and SE proteins were analyzed using the Olink Target 96 platform (1196 protein assays in total). We compared these three matrices in terms of quantification overlap, principal component analysis, and correlation. Results: In the AH, VH, and SE samples, 841, 917, and 1133 proteins, respectively, were consistently quantified above the limit of detection in more than 30% of patients. AH and VH shared 812 of these proteins. AH and VH samples overlapped along principal component 1, but SE samples were distinct. We identified 490 proteins with significant (false discovery rate [FDR]-adjusted P < 0.05) and relevant correlations (correlation coefficient > 0.5) between AH and VH, compared to only 33 and 40 proteins for VH and SE and for AH and SE, respectively. Conclusions: Due to a close correlation between protein concentrations in the AH and VH and a clear difference from the SE, AH has the potential to serve as a substitute for VH and may hold significance in identifying protein biomarkers and novel targets related to retinal diseases. Translational Relevance: This study further supports AH as a clinically suitable source of protein biomarkers in retinal diseases. In addition, the identified AH and VH correlations can inform the selection of protein biomarker candidates in future translational research.

Early changes in the immune microenvironment of oral potentially malignant disorders reveal an unexpected association of M2 macrophages with oral cancer free survival.

Understanding the dynamics of the immune microenvironment is critical to the development of immuno-based strategies for the prevention of oral potentially malignant disorders transformation to oral squamous cell carcinoma (OSCC). We used laser capture microdissection and RNA-sequencing to profile the expression of 13 matched pairs of epithelial versus stromal compartments from normal mucosa, hyperplasia, dysplasia, and invasive tumors in the 4-nitroquinolein (4-NQO) murine model of oral carcinogenesis. Genes differentially expressed at each step of transformation were defined. Immune cell deconvolution and enrichment scores of various biological processes including immune-related ones were computed. Immunohistochemistry was also performed to characterize the immune infiltrates by T-cells (T-cells CD3+, helper CD4+, cytotoxic CD8+, regulatory FoxP3+), B-cells (B220+), and macrophages (M1 iNOS+, M2 CD163+) at each histological step. Enrichment of three independent M2 macrophages signatures were computed in 86 oral leukoplakia with available clinical outcome. Most gene expression changes were observed in the stromal compartment and related to immune biological processes. Immune cell deconvolution identified infiltration by the macrophage population as the most important quantitatively especially at the stage of dysplasia. In 86 patients with oral leukoplakia, three M2 macrophages signatures were independently associated with improved oral cancer-free survival. This study provides a better understanding of the dynamics of the immune microenvironment during oral carcinogenesis and highlights an unexpected association of M2 macrophages gene expression signatures with oral cancer free survival in patients with oral leukoplakia.

Combined proteomic and transcriptomic approaches reveal externalized keratin 8 as a potential therapeutic target involved in invasiveness of head and neck cancers.

Keratin 8 (K8) expressed at the surface of cancer cells, referred as externalized K8 (eK8), has been observed in a variety of carcinoma cell lines. K8 has been previously reported to be expressed in poorly differentiated head and neck squamous cell carcinoma (HNSCC); however, its role during the invasive phase of upper aerodigestive tract tumorigenesis is unknown. Cohorts of HNSCC tumors for protein and mRNA expression and panel of cell lines were used for investigation. K8 was found to be externalized in a majority of HNSCC cell lines. Among the two main K8 protein isoforms only the 54 kDa was found to be present at the plasma membrane of HNSCC cells. The plasminogen-induced invasion of HNSCC cells was inhibited by the anti-eK8 D-A10 antagonist monoclonal antibody. Overexpression of K8 mRNA and protein were both correlated with tumor aggressive features and poor outcome. The effect of eK8 neutralization on invasion, its presence exclusively in cancer cells and the association of K8 expression with aggressive features and poor clinical outcome in HNSCC unravel eK8 as key player in invasion and a promising therapeutic target in HNSCC.

CDYL2 Epigenetically Regulates MIR124 to Control NF-κB/STAT3-Dependent Breast Cancer Cell Plasticity.

Epigenetic deregulation of gene transcription is central to cancer cell plasticity and malignant progression but remains poorly understood. We found that the uncharacterized epigenetic factor chromodomain on Y-like 2 (CDYL2) is commonly over-expressed in breast cancer, and that high CDYL2 levels correlate with poor prognosis. Supporting a functional role for CDYL2 in malignancy, it positively regulated breast cancer cell migration, invasion, stem-like phenotypes, and epithelial-to-mesenchymal transition. CDYL2 regulation of these plasticity-associated processes depended on signaling via p65/NF-κB and STAT3. This, in turn, was downstream of CDYL2 regulation of MIR124 gene transcription. CDYL2 co-immunoprecipitated with G9a/EHMT2 and GLP/EHMT1 and regulated the chromatin enrichment of G9a and EZH2 at MIR124 genes. We propose that CDYL2 contributes to poor prognosis in breast cancer by recruiting G9a and EZH2 to epigenetically repress MIR124 genes, thereby promoting NF-κB and STAT3 signaling, as well as downstream cancer cell plasticity and malignant progression.

Germline MUTYH Mutation in a Pediatric Cancer Survivor Developing a Secondary Malignancy.

Radiotherapy-induced second malignant neoplasms (SMNs) are a severe late complication in pediatric cancer survivors. Germline mutations in tumor suppressor genes contribute to SMNs; however, the most relevant germline variants mediating susceptibility are not fully defined. The authors performed matched whole-exome sequencing analyses of germline and tumor DNA from 4 pediatric solid tumor survivors who subsequently developed radiation-associated SMNs. Pathogenic and predicted deleterious germline variants were identified for each patient and validated with Sanger sequencing. These germline variants were compared with germline variants in a cohort of 59 pediatric patients diagnosed with primary sarcomas. Pathway analysis was performed to test for similarities in the germline variant profiles between individuals diagnosed with SMNs or primary sarcomas. One index patient was found to have a pathogenic germline monoallelic mutation in the MUTYH gene, which encodes the base excision repair enzyme adenine DNA glycosylase. This specific germline mutation is associated with a form of familial adenomatous polyposis, a new diagnosis in the patient. Germline-level genetic similarity exists between SMN-developing patients and patients developing primary sarcomas, with relevant genes involved in signal transduction and DNA repair mechanisms. The authors identify a germline MUTYH mutation in a pediatric cancer survivor developing an SMN. Germline mutations involving specific pathways such as base excision repair may identify individuals at risk for developing SMNs. The composition of germline variants in individual patients may enable estimates of patient-specific risk for developing SMNs. The authors anticipate that further analyses of germline genomes and epigenomes will reveal diverse genes and mechanisms influencing cancer risk.

Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, µ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.

Mutational landscape of radiation-associated angiosarcoma of the breast.

PURPOSE: Radiation-associated breast angiosarcomas are a rare complication of radiation therapy for breast carcinoma. With relatively little is known about the genetic abnormalities present in these secondary tumors, we examined genomic variation in biospecimens from radiation-associated breast angiosarcomas. EXPERIMENTAL DESIGN: Patients were identified that had a previous breast cancer diagnosis, received radiation therapy, and developed angiosarcoma in the ipsilateral breast as the earlier cancer. Tumor regions were isolated from archival blocks using subsequent laser capture microdissection. Next generation sequencing was performed using a targeted panel of 160 cancer-related genes. Genomic variants were identified for mutation and trinucleotide-based mutational signature analysis. RESULTS: 44 variants in 34 genes were found in more than two thirds of the cases; this included 12 variants identified as potentially deleterious. Of particular note, the BRCA1 DNA damage response pathway was highly enriched with genetic variation. In a comparison to local recurrences, 14 variants in 11 genes were present in both the primary and recurrent lesions including variants in genes associated with the DNA damage response machinery. Furthermore, the mutational signature analysis shows that a previously defined IR signature is present in almost all of the current samples characterized by predominantly C→T substitutions. CONCLUSIONS: While radiation-associated breast angiosarcomas are relatively uncommon, their prognosis is very poor. These data demonstrate a mutational pattern associated with genes involved in DNA repair. While important in revealing the biology behind these tumors, it may also suggest new treatment strategies that will prove successful.

Somatic and Germline TP53 Alterations in Second Malignant Neoplasms from Pediatric Cancer Survivors.

Purpose: Second malignant neoplasms (SMNs) are severe late complications that occur in pediatric cancer survivors exposed to radiotherapy and other genotoxic treatments. To characterize the mutational landscape of treatment-induced sarcomas and to identify candidate SMN-predisposing variants, we analyzed germline and SMN samples from pediatric cancer survivors.Experimental Design: We performed whole-exome sequencing (WES) and RNA sequencing on radiation-induced sarcomas arising from two pediatric cancer survivors. To assess the frequency of germline TP53 variants in SMNs, Sanger sequencing was performed to analyze germline TP53 in 37 pediatric cancer survivors from the Childhood Cancer Survivor Study (CCSS) without any history of a familial cancer predisposition syndrome but known to have developed SMNs.Results: WES revealed TP53 mutations involving p53's DNA-binding domain in both index cases, one of which was also present in the germline. The germline and somatic TP53-mutant variants were enriched in the transcriptomes for both sarcomas. Analysis of TP53-coding exons in germline specimens from the CCSS survivor cohort identified a G215C variant encoding an R72P amino acid substitution in 6 patients and a synonymous SNP A639G in 4 others, resulting in 10 of 37 evaluable patients (27%) harboring a germline TP53 variant.Conclusions: Currently, germline TP53 is not routinely assessed in patients with pediatric cancer. These data support the concept that identifying germline TP53 variants at the time a primary cancer is diagnosed may identify patients at high risk for SMN development, who could benefit from modified therapeutic strategies and/or intensive posttreatment monitoring. Clin Cancer Res; 23(7); 1852-61. ©2016 AACR.

Deep venomics of the Pseudonaja genus reveals inter- and intra-specific variation.

Australian elapid venom remains an under-investigated resource of novel bioactive peptides. In this study, the venom gland transcriptomes and proteomes of the Australian western brown snakes, Pseudonaja aspidorhyncha and Pseudonaja nuchalis, were compared to Pseudonaja textilis. A deep venomics strategy incorporating high throughput 454 pyrosequencing gave a total of 200,911 raw reads for the three venoms. Subsequent annotation identified 5716 transcripts from 20 different toxin families with inter-specific variation between species observed in eight of the less abundant families. Integration of each venom proteome with the corresponding annotated reads identified 65 isoforms from six toxin families; high sequence coverage highlighted subtle differences between sequences and intra and inter-specific variation between species. High quality MS/MS data identified unusual glycoforms with natriuretic peptides from P. aspidorhyncha and P. nuchaliscontaining O-linked trisaccharides with high homology to the glycosylated region of TNPc. Molecular evolutionary assessments indicated the accelerated evolution of all toxin families with the exception of both natriuretic peptides and P. aspidorhyncha PLA2s that were found to be evolutionarily constrained under purifying selection pressures. This study has revealed a wide range of novel peptide sequences from six bioactive peptide families and highlights the subtle differences between toxins in these closely related species. BIOLOGICAL SIGNIFICANCE: Mining Australia's vastly untapped source of toxins from its venomous creatures has been significantly advanced by employing deep venomics methodology. Technological advances in transcriptome analysis using next generation sequencing platforms and proteome analysis by highly sensitive tandem mass spectrometry allowed a more comprehensive interrogation of three underinvestigated brown snake (Pseudonaja) venoms uncovering many novel peptide sequences that are unique to these closely related species. This generic strategy will provide invaluable information when applied to other venomous snakes for a deeper understanding of venom composition, envenomation, venom evolution, as well as identifying research tools and drug leads.

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks.

Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii).

Fish venoms remain almost completely unstudied despite the large number of species. In part this is due to the inherent nature of fish venoms, in that they are highly sensitive to heat, pH, lyophilisation, storage and repeated freeze-thawing. They are also heavily contaminated with mucus, which makes proteomic study difficult. Here we describe a novel protein-handling protocol to remove mucus contamination, utilising ammonium sulphate and acetone precipitation. We validated this approach using barb venom gland tissue protein extract from the blue-spotted stingray Neotrygon kuhlii. We analysed the protein extract using 1D and 2D gels with LC-MS/MS sequencing. Protein annotation was underpinned by a venom gland transcriptome. The composition of our N. kuhlii venom sample revealed a variety of protein types that are completely novel to animal venom systems. Notably, none of the detected proteins exhibited similarity to the few toxin components previously characterised from fish venoms, including those found in other stingrays. Putative venom toxins identified here included cystatin, peroxiredoxin and galectin. Our study represents the first combined survey of gene and protein composition from the venom apparatus of any fish and our novel protein handling method will aid the future characterisation of toxins from other unstudied venomous fish lineages. BIOLOGICAL SIGNIFICANCE: These results show an efficient manner for removing mucus from fish venoms. These results are the first insights into the evolution of proteins present on stingrayvenom barbs.

Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.

Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies.

BACKGROUND: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining. RESULTS: Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies. CONCLUSIONS: Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.

Transcriptomic messiness in the venom duct of Conus miles contributes to conotoxin diversity.

Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly.

Membrane microdomains and cytoskeleton organization shape and regulate the IL-7 receptor signalosome in human CD4 T-cells.

Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.

Cysteine-rich mini-proteins in human biology.

Understanding the relationship between structure and function underpins both biochemistry and chemical biology, and has enabled the discovery of numerous agricultural and therapeutic agents. Small cysteine-rich proteins, which form a unique set of protein frameworks and folds, are found in all living organisms and often play crucial roles as hormones, growth factors, ion channel modulators and enzyme inhibitors in various biological pathways. Here we review secreted human cysteine-rich mini-proteins, classify them into broad families and briefly describe their structure and function. To systematically investigate this protein sub-class we designed a step-wise high throughput algorithm that is able to isolate the mature and active forms of human secreted cysteine-rich proteins (up to 200 amino acids in length) and extract their cysteine scaffolds. We limited our search to frameworks that contain an even number of cysteine residues (< 20), all of which are engaged in intra-molecular disulfide bonds. We found 53 different cysteine-rich frameworks spread over 378 secreted cysteine-rich mini-proteins. Restricting our search to those that contain >5% cysteine residues led to the identification of 22 cysteine-rich frameworks representing 21 protein families. Analysis of their molecular targets showed that these mini-proteins are frequently ligands for G protein- and enzyme-coupled receptors, transporters, extracellular enzyme inhibitors, and antimicrobial peptides. It is clear that these human secreted mini-proteins possess a wide diversity of frameworks and folds, some of which are conserved across the phylogenetic spectrum. Further study of these proteins will undoubtedly lead to insights into unresolved questions of basic biology, and the development of system-specific human therapeutics.

IL-2 induces conformational changes in its preassembled receptor core, which then migrates in lipid raft and binds to the cytoskeleton meshwork.

While interleukin (IL)-2 clearly initiates the sequential assembly of its soluble receptor fragments (sIL-2R) in vitro (with sIL-2Rα first, sIL-2Rß second, and sγc last), the assembly mechanism of full-length subunits (IL-2R) at the surface of living lymphocytes remains to be elucidated. Here we demonstrate by fluorescence cross-correlated spectroscopy that native IL-2Rß and γc assemble spontaneously at the surface of living human leukemia T cells (Kit-225 cell line) in the absence of IL-2 and with 1:1 stoichiometry. The dissociation constant of the membrane-embedded IL-2Rß/γc complex is measured in situ. Förster fluorescence resonance energy transfer analyzed by confocal microscopy of transfected COS-7 cells between combination pairs of various-length receptor chain constructions, using green fluorescent protein derivatives as cytoplasmic carboxy-terminal extensions, showed that IL-2Rß:ECFP and γc:EYFP bind each other through their extracellular domains, and that IL-2 binding brings their transmembrane domains 30 Å closer together. These observations demonstrate that IL-2Rß/γc heterodimers are preformed and that their cytoplasmic domains, carrying Janus kinase (Jak) 1 and Jak3, are pulled and tethered together on cytokine binding, triggering signaling transduction. IL-2 binding stabilizes IL-2/IL-2R complexes in membrane nanodomains that promote Jak1/Jak3 phosphorylation. The complexes then interact with the cytoskeleton, which slows receptor diffusion (as measured by fluorescence cross-correlated spectroscopy) and promotes STAT (signal transducer and activator of transcription) 5 phosphorylation. Separation of IL-2-activated receptors from Triton-lysed cells in detergent-resistant membrane nanodomains by ultracentrifugation on a sucrose gradient confirmed their presence in lipid rafts. The release of the IL-2-activated receptor from cytochalasin-treated cells and the IL-2-induced recruitment of actin and tubulin, analyzed by immunoprecipitation, confirmed that the activated receptor interacts with the cytoskeleton. Although IL-2Rα (the third chain that gives the IL-2Rß/γc receptor core its high affinity for IL-2) is highly expressed at the cell surface and mainly clustered in membrane microdomains at the surface of Kit-225 cells, the few free IL-2Rα present bind last to the IL-2/IL-2Rß/γc complex and lock IL-2 to its binding site for prolonged action, promoting signal amplification.