In vitro studies on the recognition of NF-κB p65 subunit by the deubiquitinase enzyme USP7

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family plays a key role in the regulation of the immune response and the transcriptional response to infection through transcriptional activation of genes involved in those processes. The NF-κB response is regulated in the nucleus by the balance between ubiquitination and deubiquitination processes. Ubiquitination of the p65 subunit of NF-κB terminates the NF-κB response by targeting p65 for proteasomal degradation. Nevertheless, the ubiquitin molecules can be removed from targeted proteins by the action of deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme from the ubiquitin-specific protease (USP) family which deubiquitinates p65. Besides p65 deubiquitination, USP7 is involved in a huge variety of biological processes due to stabilisation or localisation of proteins involved in those processes. USP7 is a multidomain protein formed by an N-terminal Meprin and tumour necrosis factor receptor-associated factor homology (MATH) / tumour necrosis factor receptor-associated factor (TRAF) domain, a catalytic domain (CD) and five ubiquitin-like domains (UBLs) in the C-terminal region. p65 recognition by USP7 takes place through the C-terminal region, but the molecular determinants involved in the interaction are still unknown. New therapeutic compound design strategies are based on interrupting the interaction interface between both proteins involved in the interaction. Therefore, in order to design a specific inhibitor of the deubiquitinase activity of USP7 on p65 we performed a peptide array and subsequent alanine scan followed by site directed mutagenesis experiments. We concluded that UBL2 of USP7 is necessary for the interaction with p65. UBL2 deletion completely abolishes the interaction and the deubiquitinase activity of USP7 on p65. Specificity of this mutant was tested by immunoprecipitation assays with different USP7 substrates. In silico modelling revealed a putative binding pocket in USP7 UBL2 that may be targeted to inhibit the interaction with p65. Together our data suggest that a binding pocket present on UBL2 composed by amino-acids 627-ARSNGTK-633, 736-EEVKPNLTER-745 and 757-LDELMDGD-764 directs the interaction with p65, besides UBL2 when deleted inhibits the interaction with p65 and subsequently its deubiquitination in a substrate specific manner

Kupfer-type immunological synapses in vivo : Raison D'être of SMAC

T cells engage with antigen-presenting cells to form immunological synapses. These intimate contacts are characterized by the complex arrangement of molecules at the intercellular interface, which has been described as the supramolecular activation cluster (SMAC). However, due to T cells functioning without SMAC formation and the difficulties of studying these complex arrangements in vivo, its biological importance has been questioned. In light of recent data, we focus this review on the putative functionality of SMACs in T-cell synaptic contacts in vivo and emphasize the therapeutic potential of SMAC manipulation in immune-driven diseases

Regulation of NF-κB by PML and PML-RARα

Promyelocytic Leukemia (PML) is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. PML is a tumour suppressor and regulator of cell differentiation. We demonstrate that PML promotes TNFα-induced transcriptional responses by promoting NF-κB activity. TNFα-treated PML−/− cells show normal IκBα degradation and NF-κB nuclear translocation but significantly reduced NF-κB DNA binding and phosphorylation of NF-κB p65. We also demonstrate that the PML retinoic acid receptor-α (PML-RARα) oncofusion protein, which causes acute promyelocytic leukemia, inhibits TNFα induced gene expression and phosphorylation of NF-κB. This study establishes PML as an important regulator of NF-κB and demonstrates that PML-RARα dysregulates NF-κB

Kupfer-type immunological synapses in vivo : Raison D'être of SMAC

T cells engage with antigen-presenting cells to form immunological synapses. These intimate contacts are characterized by the complex arrangement of molecules at the intercellular interface, which has been described as the supramolecular activation cluster (SMAC). However, due to T cells functioning without SMAC formation and the difficulties of studying these complex arrangements in vivo, its biological importance has been questioned. In light of recent data, we focus this review on the putative functionality of SMACs in T-cell synaptic contacts in vivo and emphasize the therapeutic potential of SMAC manipulation in immune-driven diseases

The Ubiquitination of NF-κB Subunits in the Control of Transcription

Nuclear factor (NF)-κB has evolved as a latent, inducible family of transcription factors fundamental in the control of the inflammatory response. The transcription of hundreds of genes involved in inflammation and immune homeostasis require NF-κB, necessitating the need for its strict control. The inducible ubiquitination and proteasomal degradation of the cytoplasmic inhibitor of κB (IκB) proteins promotes the nuclear translocation and transcriptional activity of NF-κB. More recently, an additional role for ubiquitination in the regulation of NF-κB activity has been identified. In this case, the ubiquitination and degradation of the NF-κB subunits themselves plays a critical role in the termination of NF-κB activity and the associated transcriptional response. While there is still much to discover, a number of NF-κB ubiquitin ligases and deubiquitinases have now been identified which coordinate to regulate the NF-κB transcriptional response. This review will focus the regulation of NF-κB subunits by ubiquitination, the key regulatory components and their impact on NF-κB directed transcription

Three-dimensional vascular microenvironment landscape in human glioblastoma

The cellular complexity of glioblastoma microenvironments is still poorly understood. In-depth, cell-resolution tissue analyses of human material are rare but highly necessary to understand the biology of this deadly tumor. Here we present a unique 3D visualization revealing the cellular composition of human GBM in detail and considering its critical association with the neo-vascular niche. Our images show a complex vascular map of human 3D biopsies with increased vascular heterogeneity and altered spatial relationship with astrocytes or glioma-cell counterparts. High-resolution analysis of the structural layers of the blood brain barrier showed a multilayered fenestration of endothelium and basement membrane. Careful examination of T cell position and migration relative to vascular walls revealed increased infiltration corresponding with tumor proliferation. In addition, the analysis of the myeloid landscape not only showed a volumetric increase in glioma-associated microglia and macrophages relative to GBM proliferation but also revealed distinct phenotypes in tumor nest and stroma. Images and data sets are available on demand as a resource for public access

The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-kB subunits

The transcription factor NF-kB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines.Ubiquitination-triggered proteasomal degradation of DNA-bound NF-kB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase7) opposes the activities of E3ligases,stabilizes DNAbound NF-kB, and thereby promotes NF-kB–mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-kB ubiquitination and degradation, prevents Toll-like receptor–induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti inflammatory agents. To this end,we identified nNF-kB–binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-kB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ŒB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-kB activity