Group 5 (PI : Robert WEIL)

 
Optineurin, a multi-function protein
Antigen-induced NF-kB activation in T cells
 
The group headed by Robert Weil develops since 2003 a project centered on the mechanisms of lymphocytes activation upon antigen presentation and on the various functions of the protein Optineurin in different cellular processes.
 
 
The immune system includes two arms: the innate and the adaptive immunity. The former is the first line of defense against invading organisms and is referred to as a series of nonspecific defense mechanisms that are rapidly triggered upon viral or bacterial infection. The initiation of the adaptive immune response depends on the recognition of microbial or tumor-specific molecules by antigen receptors on the cell surface of lymphocytes. Antigen recognition leads to a variety of signaling events that result in dramatic changes in the expression of genes that control the activation, clonal proliferation and survival of the stimulated lymphocytes.
 
The nuclear factor (NF)- kB transcription factor participates in both innate and adaptive immunity through regulation of target genes encoding anti-apoptotic molecules, cell cycle regulators, cytokines and surface receptors. In normal unactivated cells, NF-kB proteins are sequestered in the cytoplasm through their interaction with the IkB inhibitors. Triggering of the T-cell antigen receptor or recognition of bacterial/viral products by Toll-like receptors present on the cell surface or in the endosomes leads to the activation of a cytoplasmic kinase complex formed of two catalytic (IKKa and IKKb) and one regulatory (NEMO) subunits, that induces phosphorylation, ubiquitination and subsequent degradation of IκB proteins. NF-kB dimers then translocate to the nucleus and activate their target genes.
Ubiquitination plays a crucial role in the NF-kB activation pathway. In particular, the role of K63-linked and linear polyubiquitin chains in NF-kB activation has been extensively studied.
NF-kB transcription factors can be uncoupled from their normal regulation and can promote tumorigenesis in different ways. For example, the Tax oncoprotein of human T cell leukemia virus (HTLV)-1 directly interacts with and constitutively activates the IKK complex, resulting in activation of NF-kB signaling pathway to promote tumorigenesis. A complex of proteins comprising Carma1, Bcl10 and Malt1 (CBM complex, Figure 1) plays key roles in the initiation of the adaptive immune response and mutations of their genes lead to dysregulation of the NF-kB activity. These mutations are associated with constitutive CBM-dependent signaling, and the development of particular subtypes of human B-cell lymphomas, such as mucosa-associated lymphoid tissue (MALT) and diffuse large B-cell (DLBCL) lymphomas (Lobry and Weil Med Sci 2007).
The NEMO/IkappaB kinase (IKK) complex is the central integrator of most stimuli leading to NF-κB activation, but the mechamisms leading to its regulation is not fully understood. Optineurin (Optn), a protein that presents structural homology with NEMO, can modulate NF-kB activation following the activation of NF-kB by TNFa or by the viral oncoprotein Tax. In addition to its NF-kB regulatory role, Optn has been suggested to be involved in a variety of functions (reviewed in Kachaner et al. Cell cycle 2012). Thus, a series of publications have focused on the role of Optn in Golgi organisation, membrane-trafficking events and protein secretion. Optn has also been involved in other pathways including protection against oxidative stress-induced apoptosis, regulation of agonist-stimulated Group I metabotropic glutamate receptors (mGluR1) signaling, cyst formation, regulation of endocytic trafficking of transferrin receptor and more recently in antiviral innate immune response and in selective autophagy of Salmonella enterica (Kachaner et al. Cell cycle 2012). Mutations within the Optn gene have been associated with primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis, and genetic variants of Optn are also considered as genetic risk factors for Paget’s disease of bone. However, the molecular mechanism by which Optn mutations lead to these pathologies has not been established so far.
 
 
- We have shown that degradation of the inhibitory protein IkBb is controlled by mechanisms that differ from those involved in the degradation of IkBa. In particular, the phosphorylation of IkBb, unlike that of IkBa, seems to be constitutive and independent of external stimuli (Weil et al. J Biol Chem 1997).
 
- Most known NF-κB activating stimuli seem to work through degradation of the IkB molecules, following phosphorylation by the IKK complex. We have uncovered a possible exception with the X protein of Hepatitis B Virus, since X could induce a sustained NF-κB activation by preventing re-association of newly synthesized nuclear IkBa with DNA-bound NF-κB complexes (Weil et al. Mol Cell Biol 1999).
 
- X is not the only viral protein involved in evading host defence systems and we have shown that viral proteins such as Tax and Rex regulate the expression of protein Tyrosine kinases in HTLV-1-infected T cells. We demonstrated that FynB, a Fyn isoform principally expressed in brain is strongly upregulated by Rex and that Zap-70 is downregulated by Tax (Weil et al. J Virol 1999).
 
 
 
We also determined the mechanisms that control the activation of T-lymphocytes, initiated upon triggering of the T-cell antigen receptor by MHC-bound antigen.
 
- We have shown that NEMO is normally recruited to the activated TCR at the immunological synapse, which is located at the junction between the antigen-presenting cell and the T lymphocyte, and that artificial targeting of NEMO to the TCR or to the plasma membrane is sufficient to specifically induce NF-κB activation (Weil et al., Immunity 2003).
 
- We also provided evidence that the NEMO/IKK complex phosphorylates Bcl10 and causes its proteolysis to negatively regulate NF-κB activity. Blocking this degradation results in Bcl10 nuclear accumulation, which has been shown to be a determinant in the pathogenesis of MALT lymphomas (Lobry et al. Proc Natl Acad Sci USA 2007; Thome and Weil Trends Immunol 2007).
 
 
 
 
 
A second major aspect of our current research aims at uncovering the roles played by the protein Optn and to propose an integrated view of its functions (Kachaner et al., Cell cycle 2012)
- We observed that Optn does not associate with the IKK kinases and unlike NEMO, which is cytosolic, is associated with the Golgi apparatus. We further demonstrated that its de novo expression is induced by interferon and TNF-a (Schwamborn, Weil et al. J Biol Chem 2000).
 
- Several independant studies have shown that NF-κB activation mediated by the oncoprotein Tax is dependent upon Tax ubiquitination. In collaboration with the laboratory of Renaud Mahieux (ENS, Lyon), we identified Optn as a binding partner for Tax and showed that this interaction requires the ubiquitin-binding activity of Optn and modulates Tax1 ubiquitination and NF-kB activation (Journo et al., Plos Pathog 2009).
 
- We also uncovered an unexpected role for Optn during mitosis: Optn specifically regulates the activity of a mitotic kinase (Plk1) that plays a key role in cell division (Figure 2). The precise mechanism centers on the reciprocal activity of both Optn and Plk1. Consistently, Plk1 activity is no longer regulated when Optn is depleted, leading to major mitotic anomalies (such as chromosome separation defects and abscission failure) and eventually to multi-nucleated cells (Kachaner et al., Mol Cell 2012).
 
 
 
 


Robert Weil team includes 6 members :
 
- 3 CNRS researchers :
 
                      Robert Weil (DR2, HDR), Emmanuel Laplantine (CR1) and Pierre Génin (CR1)
 
- 1 PhD student: Nassima Messali
 
- 1 Master student: Tirsta Ackermann
 
- 1 technician: Frédérique Cuvelier (IP)
 
 
Main current research projects
Our present work focuses on three axes:
 
1     The mechanisms underlying the permanent activation of NF-kB in cancer cells
 
2     The mechanisms regulating the antiviral innate immune responses
 
3     Targeting of Optn for cancer therapy
 
 
Publications (since 1997)
R. Weil, C. Laurent Winter, and A. Israël. (1997) Regulation of IkBb degradation: similarities and differences with IkBa. J. Biol. Chem. 272: 9942-9949.

R. Weil, S. T. Whiteside, and A. Israël. (1997) Control of NF-κB activity by the IkBb inhibitor. Immunobiol. 197: 229-238.

H. Sirma, R. Weil, O. Rosmorduc, S. Urban, A. Israël, D. Kremsdorf, and C. Bréchot. (1998) Cytosol is the prime compartment of hepatitis B virus X protein where it associates with the proteasome. Oncogene. 16: 2051-2063.
 
S. Yamaoka, G. Courtois, C. Bessia, S. T. Whiteside, R. Weil, F. Agout, H. E. Kirk, R. J. Kay, and A. Israël. (1998) Complementation cloning of NEMO, a component of the IkB kinase complex essential for NF-κB activation. Cell. 93: 1231-1240.
 
R. Weil, J. P. Levraud, M. Duc Dodon, C. Bessia, U. Hazan, P. Kourilsky, and A. Israël. (1999) Altered expression and activity of tyrosine kinases of the Src and Syk families in HTLV-1-infected T cell lines. J. Virol. 73, 3709-3717.

R. Weil, H. Sirma, C. Giannini, D. Kremsdorf, C. Dargemont, C. Bréchot and A. Israël. (1999) Direct association and nuclear import of the hepatitis virus X protein with the NF-κB inhibitory protein IkBa. Mol. Cell. Biol..19, 6345-6354.

R. Eckenberg, T. Rose, J. L. Moreau, R. Weil, F. Gesbert, S. Dubois, D. Tello, M. Bossus, H. Gras, A. Tartar, J. Bertoglio, S. Chouaib, M. Goldberg, Y. Jacques, P. M. Alzari and J. These. (2000) The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells. J. Exp Med. 191, 529-540.
 
M. C. Bonnet, R. Weil, E. Dam, A. G. Hovanessian and E. F. Meurs. (2000) PKR stimulates NF-κB irrespective of its kinase function by interacting with the IKK complex. Mol. Cell. Biol. 20, 4532-4542.
 
K. Schwamborn, R. Weil (co-1er auteur), G. Courtois, S. T. Whiteside, and A. Israël. (2000) Phorbol esters and cytokines regulate the expression of the NEMO-Related Protein (NRP), a molecule involved in a NF-κB-independent pathway. J. Biol. Chem. 275, 22780-22789.
 
R. Eckenberg, T. Rose, J. L. Moreau, R. Weil, F. Gesbert, S. Dubois, D. Tello, M. Bossus, H. Gras, A. Tartar, J. Bertoglio, S. Chouaib, Y. Jacques, P. M. Alzari, J. Theze. (2001) Characterization of an IL-2 mimetic with therapeutic potential. Cell. Mol. Biol. 47, 703-707
 
R. Weil, K. Schwamborn, A. Alcover, C. Bessia, V. Di Bartolo, and A. Israël. (2003) Induction of the NF-κB cascade by recruitment of the scaffold molecule NEMO to the T cell receptor. Immunity. 18, 1-14.
 
L. Meertens, S. Chevalier, R. Weil, A. Gessain and R. Mahieux. (2004) A 10 amino acid domain within Human T-cell Leukemia Virus type 1 and type 2 Tax protein sequences is responsible for their divergent subcellular distribution. J. Biol. Chem. 279 : 43307-43320.
 
C. Lobry, T. Lopez, A. Israël and R. Weil. Negative feed back loop in T-cell activation through IkB kinase-induced phosphorylation and degradation of Bcl10. (2007) Proc. Natl. Acad. Sci. 104 : 908-913. (Citations : N. R. Gough, Applying the Brakes to the Immune Response. Sci. STKE 2007, 370 : tw28. M. Hinz and C. Scheidereit. Striking back at the activator : how IkB kinase terminates antigen receptor responses. Sci. STKE 384 : pe19)

G. Despouy, M. Joiner, E. Le Toriellec, R. Weil and M. H. Stern. The Tcl1 oncoprotein inhibits activation-induced cell death by impairing PKC-q and Erk pathways (2007) Blood. 13 : 4406-4416.
 
C. Journo, J. Filipe, F. About, S. A. Chevalier, J. N. Brady, P. V. Afonso, D. Flynn, F. Tanguy, A. Israël, P.-O. Vidalain, R. Mahieux and R. Weil. NRP/Optineurin cooperates with TAX1BP1 to potentiate the activation of NF-κB by human T-lymphotropic virus type 1 Tax protein (2009) PLoS Pathog, 5 : e100521.
 
D. Kachaner, J. Filipe, E. Laplantine, A. Bauch, K. L. Bennett, G. Superti-Furga, A. Israël, and R. Weil. Plk1-dependent phosphorylation of Optineurin provides a negative feedback mechanism for mitotic progression (2012) Mol. Cell, 45: 553-566.
 
S. Luco, O. Delmas, P-O. Vidalain, F. Tangy, R. Weil and H. Bourhy. Characterization of RelAp43, a member of the NF-kB family involved in interferon b production during lyssavirus infection. PLoS Pathog, in press.
 
 
Reviews

R. Weil, and A. Israël. (2004) T-cell-receptor and B-cell-receptor-mediated NF-kappaB activation in lymphocytes. Current opinion in Immunology. 16 : 374-381.
 
R. Weil, and A. Israël. Deciphering the pathway from the TCR to NF-κB. (2006) Review in Cell Death and differentiation. 1-8.
C. Lobry and R. Weil. New Bcl10 regulation mechanisms : a step in the comprehension of which has occured in MALT lymphomas ? (2007) Med. Sci. 23 : 11-13.
 
M. Thome and R. Weil. Postranslational modifications regulate distinct functions of Carma1 and Bcl10. (2007) Trends in Immunol. 28 : 281-288.
 
C. Lobry and R. Weil. Regulation of NF-κB pathway in T lymphocytes (2007) Med. Sci. 10 : 857-861.
 
D. Kachaner, E. Laplantine, P. Genin and R. Weil. Optineurin: a new vision on the regulation of cell division (2012) Cell cycle, 11:1481-1482.
 
D. Kachaner, P. Genin, E. Laplantine and R. Weil. Towards an integrative view of Optineurin functions. (2012) Cell cycle, 11:2808-2818.
 
 
 
Team members
 
 
Robert WEIL
MD, PhD
 
Directeur de recherché CNRS
 
Phone: 01 40 61 30 38
 
Email: rweil@pasteur.fr
 


Pierre GÉNIN
 
Chargé de recherche CNRS
 
Tél: 01 40 61 30 38
 
Email: pierre.genin@pasteur.fr
 


Emmanuel LAPLANTINE
 
Chargé de recherche CNRS
 
Tél: 01 40 61 30 38
 
Email: emmanuel.laplantine@pasteur.fr
 


Frédérique CUVELIER
 
Technicienne IP
 
Tél: 01 45 68 84 98
 
Email: frédérique.cuvelier@pasteur.fr
 


Nassima MESSALI
Etudiante en 3e année de thèse
Tél: 01 45 68 84 98


Tirtsa ACKERMANN
 
Etudiante en M2, élève à l’ENS Cachan
 
Tél: 01 45 68 84 98
 
Email: tirtsa.ackermann@pasteur.fr