Haopeng Wang

Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-κB signaling

The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-κB (NF-κB) signaling in resting cells by targeting NF-κB–inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-κB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-κB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-κB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IκB kinase regulatory subunit IKKγ (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKγ were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2–induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.

ZAP-70: An Essential Kinase in T-cell Signaling

ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.

Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

Scalable signaling mediated by T cell antigen receptor–CD3 ITAMs ensures effective negative selection and prevents autoimmunity

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.

Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

Expression of the T‐cell receptor (TCR):CD3 complex is tightly regulated during T‐cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ε proline‐rich sequence, Lck, c‐Cbl, and SLAP, which collectively trigger the dynamin‐dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ‐monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T‐cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T‐cell development.

Combined Immunodeficiency Due to MALT1 Mutations, Treated by Hematopoietic Cell Transplantation

PurposeA male infant developed generalized rash, intestinal inflammation and severe infections including persistent cytomegalovirus. Family history was negative, T cell receptor excision circles were normal, and engraftment of maternal cells was absent. No defects were found in multiple genes associated with severe combined immunodeficiency. A 9/10 HLA matched unrelated hematopoietic cell transplant (HCT) led to mixed chimerism with clinical resolution. We sought an underlying cause for this patient’s immune deficiency and dysregulation.MethodsClinical and laboratory features were reviewed. Whole exome sequencing and analysis of genomic DNA from the patient, parents and 2 unaffected siblings was performed, revealing 2 MALT1 variants. With a host-specific HLA-C antibody, we assessed MALT1 expression and function in the patient’s post-HCT autologous and donor lymphocytes. Wild type MALT1 cDNA was added to transformed autologous patient B cells to assess functional correction.ResultsThe patient had compound heterozygous DNA variants affecting exon 10 of MALT1 (isoform a, NM_006785.3), a maternally inherited splice acceptor c.1019-2A > G, and a de novo deletion of c.1059C leading to a frameshift and premature termination. Autologous lymphocytes failed to express MALT1 and lacked NF-κB signaling dependent upon the CARMA1, BCL-10 and MALT1 signalosome. Transduction with wild type MALT1 cDNA corrected the observed defects.ConclusionsOur nonconsanguineous patient with early onset profound combined immunodeficiency and immune dysregulation due to compound heterozygous MALT1 mutations extends the clinical and immunologic phenotype reported in 2 prior families. Clinical cure was achieved with mixed chimerism after nonmyeloablative conditioning and HCT.

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor

Significance Intracellular signaling during complex cell–cell interactions, such as between immune cells, provides essential cues leading to cell responses. Global characterization of these signaling events is critical for systematically exploring and understanding how they eventually control cell fate. However, proteome-wide characterization of intercellular signaling under physiologically relevant conditions involving multiple interacting receptors during cell–cell interactions remains challenging. We developed an integrated proteomic strategy for quantitatively profiling intercellular-signaling events mediated by protein phosphorylation and protein–protein interaction. We applied this approach to determine the influence of a single receptor-ligand pair during T-cell stimulation by blocking the interaction of the CD28 costimulatory receptor with its ligand. This approach is generally applicable to other transmembrane receptors involved in signaling during complex cell interactions. Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell–cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell–cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails.