TNF-α synergises with IFN-γ to induce caspase-8-JAK1/2-STAT1-dependent death of intestinal epithelial cells.

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Inhibitory action of NoxA1 on dual oxidase activity in airway cells.

Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.

Biochemical and structural characterization of the gem GTPase.

RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg.GDP at 2.1 A resolution. It reveals that the G-domain fold and Mg.GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an alpha-helical conformation that extends along the alpha5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.

Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3).

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.

Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression.

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1.

Human/chicken urokinase chimeras demonstrate sequences outside the serine protease domain that dictate autoactivation.

Mammalian urokinase-type plasminogen activator (uPA) is produced as a stable single polypeptide chain zymogen and requires a distinct proteolytic cleavage to become an active, two-chain enzyme. In contrast, chicken uPA, both native and recombinant, is found predominantly as a two-chain, active enzyme even in the absence of plasmin, a physiological activator. Here we show that the proclivity to autoactivate is not a unique property of the chicken uPA catalytic domain but requires sequences distinct from and independent of the serine protease domain. Human/chicken chimeric uPA molecules and point mutants were used to determine the structural requirements for uPA autoactivation versus zymogen stability. The amino terminal fragment of chicken uPA engineered onto the human uPA molecule can induce the autoactivation of the human uPA. In fact, the first twenty residues of the chicken uPA are necessary and sufficient to induce the autoactivation of chicken and human uPA. These results indicate that sequence motifs, distal to the active site, control the substrate specificity and catalytic efficiency of uPA activity in autolytic activation.