Corinne Cayrol

The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1

IL-33 is a chromatin-associated cytokine of the IL-1 family that has recently been linked to many diseases, including asthma, rheumatoid arthritis, atherosclerosis, and cardiovascular diseases. IL-33 signals through the IL-1 receptor-related protein ST2 and drives production of pro-inflammatory and T helper type 2-associated cytokines in mast cells, T helper type 2 lymphocytes, basophils, eosinophils, invariant natural killer T cells, and natural killer cells. It is currently believed that IL-33, like IL-1β and IL-18, requires processing by caspase-1 to a mature form (IL-33112–270) for biological activity. Contrary to the current belief, we report here that full-length IL-331–270 is active and that processing by caspase-1 results in IL-33 inactivation, rather than activation. We show that full-length IL-331–270 binds and activates ST2, similarly to IL-33112–270, and that cleavage by caspase-1 does not occur at the site initially proposed (Ser111), but rather after residue Asp178 between the fourth and fifth predicted β-strands of the IL-1-like domain. Surprisingly, the caspase-1 cleavage site (DGVD178G) is similar to the consensus site of cleavage by caspase-3, and IL-33 is also a substrate for this apoptotic caspase. Interestingly, we found that full-length IL-33, which is constitutively expressed to high levels by endothelial cells in most normal human tissues, can be released in the extracellular space after endothelial cell damage or mechanical injury. We speculate that IL-33 may function, similarly to the prototypical alarmins HMGB1 and IL-1α, as an endogenous danger signal to alert cells of the innate immune system of tissue damage during trauma or infection.

p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells

A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases δ and ε. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracycline-regulated system, that expression of wild-type p21 in p53-deficient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar effects are observed in cells expressing p21CDK−, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA−, a mutant deficient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory effects of p21 and p21CDK− result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest.

IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1β and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-331–270 and generate mature forms IL-3395–270, IL-3399–270, and IL-33109–270. These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ∼10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.

IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy.

IL-33 is a nuclear cytokine from the IL-1 family constitutively expressed in epithelial barrier tissues and lymphoid organs, which plays important roles in type-2 innate immunity and human asthma. Recent studies indicate that IL-33 induces production of large amounts of IL-5 and IL-13 by group 2 innate lymphoid cells (ILC2s), for initiation of allergic inflammation shortly after exposure to allergens or infection with parasites or viruses. IL-33 appears to function as an alarmin (alarm signal) rapidly released from producing cells upon cellular damage or cellular stress. In this review, we discuss the cellular sources, mode of action and regulation of IL-33, and we highlight its crucial roles in vivo with particular emphasis on results obtained using IL33-deficient mice.

Molecular mimicry between IL‐33 and KSHV for attachment to chromatin through the H2A–H2B acidic pocket

Interleukin‐33 (IL‐33) is an IL‐1‐like ligand for the ST2 receptor that stimulates the production of Th2‐associated cytokines. Recently, we showed that IL‐33 is a chromatin‐associated factor in the nucleus of endothelial cells in vivo. Here, we report the identification of a short IL‐33 chromatin‐binding peptide that shares striking similarities with a motif found in Kaposi sarcoma herpesvirus LANA (latency‐associated nuclear antigen), which is responsible for the attachment of viral genomes to mitotic chromosomes. Similar to LANA, the IL‐33 peptide docks into the acidic pocket formed by the H2A–H2B dimer at the nucleosomal surface and regulates chromatin compaction by promoting nucleosome–nucleosome interactions. Taken together, our data provide important new insights into the nuclear roles of IL‐33, and show a unique example of molecular mimicry of a chromatin‐associated cytokine by a DNA tumour virus. In addition, the data provide, to the best of our knowledge, the first demonstration of the existence of non‐histone cellular factors that bind to the acidic pocket of the nucleosome.

Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21

The cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 plays an essential role in the control of cell proliferation by modulating the activity of cyclin/CDK complexes in response to various intracellular or extracellular signals. Small variations in p21 expression levels may determine whether it acts as an inhibitor or an assembly factor for cyclin/CDK complexes. It is therefore critical to better characterize the mechanisms regulating p21 abundance. Here, we show, using a tetracycline-regulated system in p53-deficient DLD-1 human colon cancer cells, that p21 protein levels and stability are regulated by the proteasome-dependent degradation pathway and by association with its partners, CDKs and PCNA. A p21 mutant deficient for interaction with CDKs, p21CDK−, displayed an enhanced stability and greatly reduced sensitivity to proteasome-mediated proteolysis, indicating that association with cyclin/CDK complexes may trigger p21 degradation. In contrast, a p21 mutant impaired in the interaction with PCNA, p21PCNA−, exhibited a decreased stability, suggesting that association with PCNA protects p21 from proteasome-dependent degradation. Furthermore, the abundance of p21 itself, in addition to protein-protein interactions, may also modulate p21 stability since we found that high levels of p21 expression overcome proteasome-dependent regulation of p21 accumulation.

THAP1 is a nuclear proapoptotic factor that links prostate-apoptosis-response-4 (Par-4) to PML nuclear bodies

Promyelocytic leukemia (PML) nuclear bodies (PML NBs) are discrete subnuclear domains organized by the promyelocytic leukemia protein PML, a tumor suppressor essential for multiple apoptotic pathways. We have recently described a novel family of cellular factors, the THAP proteins, characterized by the presence at their amino-terminus of an evolutionary conserved putative DNA-binding motif, designated THAP domain. Here, we report that THAP1 is a novel nuclear proapoptotic factor associated with PML NBs, which potentiates both serum withdrawal- and TNFα-induced apoptosis, and interacts with prostate-apoptosis-response-4 (Par-4), a well characterized proapoptotic factor, previously linked to prostate cancer and neurodegenerative diseases. We show that endogenous Par-4 colocalizes with ectopic THAP1 within PML NBs in primary endothelial cells and fibroblasts. In addition, we found that Par-4 is a component of PML NBs in blood vessels, a major site of PML expression in vivo. Finally, we investigated the role of the THAP domain in THAP1 activities and found that this putative DNA-binding domain is not required for Par-4 binding and localization within PML NBs, but is essential for THAP1 proapoptotic activity. Together, our results provide an unexpected link between a nuclear factor of the THAP family, the proapoptotic protein Par-4 and PML nuclear bodies.

Mascot File Parsing and Quantification (MFPaQ), a New Software to Parse, Validate, and Quantify Proteomics Data Generated by ICAT and SILAC Mass Spectrometric Analyses Application To the Proteomics Study of Membrane Proteins from Primary Human Endothelial Cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-α, interferon-γ, and lymphotoxin α/β) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.

Direct interaction between causative genes of DYT1 and DYT6 primary dystonia

Primary dystonia is a movement disorder characterized by sustained muscle contractions and in which dystonia is the only or predominant clinical feature. TOR1A(DYT1) and the transcription factor THAP1(DYT6) are the only genes identified thus far for primary dystonia. Using electromobility shift assays and chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR), we demonstrate a physical interaction between THAP1 and the TOR1A promoter that is abolished by pathophysiologic mutations. Our findings provide the first evidence that causative genes for primary dystonia intersect in a common pathway and raise the possibility of developing novel therapies targeting this pathway. Ann Neurol 2010

The THAP-Zinc Finger Protein THAP1 Associates with Coactivator HCF-1 and O-GlcNAc Transferase A LINK BETWEEN DYT6 AND DYT3 DYSTONIAS

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.