Aimé Vazquez

Mitochondrial dynamics regulate the RIG‐I‐like receptor antiviral pathway

The intracellular retinoic acid‐inducible gene I‐like receptors (RLRs) sense viral ribonucleic acid and signal through the mitochondrial protein mitochondrial antiviral signalling (MAVS) to trigger the production of type I interferons and proinflammatory cytokines. In this study, we report that RLR activation promotes elongation of the mitochondrial network. Mimicking this elongation enhances signalling downstream from MAVS and favours the binding of MAVS to stimulator of interferon genes, an endoplasmic reticulum (ER) protein involved in the RLR pathway. By contrast, enforced mitochondrial fragmentation dampens signalling and reduces the association between both proteins. Our finding that MAVS is associated with a pool of mitofusin 1, a protein of the mitochondrial fusion machinery, suggests that MAVS is capable of regulating mitochondrial dynamics to facilitate the mitochondria–ER association required for signal transduction. Importantly, we observed that viral mitochondria‐localized inhibitor of apoptosis, a cytomegalovirus (CMV) antiapoptotic protein that promotes mitochondrial fragmentation, inhibits signalling downstream from MAVS, suggesting a possible new immune modulation strategy of the CMV.

Casein kinase 1α governs antigen-receptor-induced NF-κB activation and human lymphoma cell survival

The transcription factor NF-κB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-κB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-κB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent ‘activated B-cell-like’ (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1α (CK1α) as a bifunctional regulator of NF-κB. CK1α dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1α kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1α has thus a dual ‘gating’ function which first promotes and then terminates receptor-induced NF-κB. ABC DLBCL cells required CK1α for constitutive NF-κB activity, indicating that CK1α functions as a conditionally essential malignancy gene—a member of a new class of potential cancer therapeutic targets.

MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors

BackgroundDuring a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear.ResultsWe report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria.ConclusionsThese results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival.

The transcription factor NF-κB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-κB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-κB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent ‘activated B-cell-like’ (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1α (CK1α) as a bifunctional regulator of NF-κB. CK1α dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1α kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1α has thus a dual ‘gating’ function which first promotes and then terminates receptor-induced NF-κB. ABC DLBCL cells required CK1α for constitutive NF-κB activity, indicating that CK1α functions as a conditionally essential malignancy gene—a member of a new class of potential cancer therapeutic targets.

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt’s lymphoma cells. Whereas soluble anti-μ Ab triggers caspase-independent mitochondrial activation, cross-linked anti-μ Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt’s lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.

Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34

BackgroundNF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated.FindingsHere, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.ConclusionsCollectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

Interplay between BCL10, MALT1 and IκBα during T-cell-receptor-mediated NFκB activation

T-cell-receptor (TCR) signalling to NFκB requires the assembly of a large multiprotein complex containing the serine/threonine kinase CK1α, the scaffold protein CARMA1, the heterodimer BCL10-MALT1 (the CBM complex) and the IκB kinase complex (IKK). Although the mechanisms regulating recruitment and activation of IKK within the CBM microenvironment have been extensively studied, there is little understanding of how IKK subsequently binds and phosphorylates IκBα, the inhibitor of NFκB, to promote IκBα ubiquitylation and proteasomal degradation. Here, we show that BCL10, MALT1 and IKK inducibly associate with IκBα in a complex that is physically distinct from the early CK1α-CBM signalosome. This IκBα-containing complex probably maturates from the CBM, because siRNA-based knockdown of CARMA1, CK1α and BCL10 hampered its assembly, leading to a reduction in NFκB activation. By contrast, CK1α normally recruited both BCL10 and ubiquitylated species of MALT1 when IκBα levels were reduced. However, knockdown of IκBα led to an altered ubiquitylation profile of BCL10-MALT1 combined with a defect in MALT1 reorganisation within large cytoplasmic structures, suggesting that, following stimulation, IκBα might also participate in MALT1 recycling. Altogether, our data suggest a two-step mechanism to connect active IKK to IκBα, and further unveil a potential role for IκBα in resetting TCR-mediated signalling.

The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation.

BackgroundThe nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.FindingsWe performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.ConclusionsThese results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers. We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment. In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen.

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) antigen-pulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.