IL-17 Signaling triggers degradation of the constitutive NF-κB inhibitor ABIN-1

IL-17 activates NF-κB and induces expression of proinflammatory genes. IL-17 drives disease in autoimmune conditions, and anti–IL-17 Abs have shown impressive success in the clinic. Although produced by lymphocytes, IL-17 predominantly signals in fibroblasts and epithelial cells. IL-17–driven inflammation is kept in check by negative feedback signaling molecules, including the ubiquitin editing enzyme A20, whose gene TNFAIP3 is linked to autoimmune disease susceptibility. The A20 binding inhibitor of NF-κB activation 1 (ABIN-1) is an A20-binding protein encoded by the TNIP1 gene, which is also linked to autoimmune disease susceptibility including psoriasis. Accordingly, we hypothesized that ABIN-1 might play a role in negatively regulating IL-17 signaling activity. Indeed, ABIN-1 enhanced both tonic and IL-17–dependent NF-κB signaling in IL-17–responsive fibroblast cells. Interestingly, the inhibitory activities of ABIN-1 on IL-17 signaling were independent of A20. ABIN-1 is a known NF-κB target gene, and we found that IL-17–induced activation of NF-κB led to enhanced ABIN-1 mRNA expression and promoter activity. Surprisingly, however, the ABIN-1 protein was inducibly degraded following IL-17 signaling in a proteasome-dependent manner. Thus, ABIN-1, acting independently of A20, restricts both baseline and IL-17–induced inflammatory gene expression. We conclude that IL-17–induced signals lead to degradation of ABIN-1, thereby releasing a constitutive cellular brake on NF-κB activation

Characterization of ABIN-1 in the Traumatically Injured Brain

Traumatic brain injury (TBI) is associated with chronic pain and persistent neuroinflammation. Opioids are often prescribed in order to relieve pain symptoms, but recent evidence suggests that their use negatively impacts the neuropathology of TBI leading to behavioral impairments and exacerbated neuroinflammation. The de-ubiquinating enzyme tumor necrosis factor alpha-induced protein (TNFAIP)3 or A20, inhibits the inflammatory signaling transcription factor nuclear factor (NF)-kB leading to attenuation of the inflammatory response. A20-binding inhibitor of nuclear factor kB (ABIN-1), which physically interacts with A20, also plays a role in the inhibition of NF-kB and is poorly researched in the central nervous system (CNS). Additionally, recent evidence suggests that ABIN-1 negatively regulates mu-opioid receptors. Therefore, dysregulation of ABIN-1 may contribute to the neuropathology post-TBI, especially after treatment with opioids. Phase 1 of the project involved determining the influence of neuroinflammation on ABIN-1 expression. Ten-week old male mice were injected intraperitoneally with a single dose (10mg/kg) of lipopolysaccharide (LPS), a potent immunoactivator, or saline (control). Brains were harvested 24 hours after injection for immunoblotting and co-immunoprecipitation assays. Preliminary results suggest that ABIN-1 protein is poorly expressed in the prefrontal cortex, nucleus accumbens, and ventral hippocampus, but that brain levels of ABIN-1 may be modulated by LPS treatment. Future research will investigate ABIN-1 protein expression throughout the mouse brain at 1, 7, and 10 days after a moderate level TBI in the presence or absence of morphine exposure to evaluate the role of ABIN-1 in mediating opioid action in the injured brain

A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-κB signaling in activated T cells

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation. © 2022, The Author(s)

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Hydrodynamical simulations of the tidal stripping of binary stars by massive black holes

In a galactic nucleus, a star on a low angular momentum orbit around the central massive black hole can be fully or partially disrupted by the black hole tidal field, lighting up the compact object via gas accretion. This phenomenon can repeat if the star, not fully disrupted, is on a closed orbit. Because of the multiplicity of stars in binary systems, also binary stars may experience in pairs such a fate, immediately after being tidally separated. The consumption of both the binary components by the black hole is expected to power a double-peaked flare. In this paper, we perform for the first time, with GADGET2, a suite of smoothed particle hydrodynamics simulations of binary stars around a galactic central black hole in the Newtonian regime. We show that accretion luminosity light curves from double tidal disruptions reveal a more prominent knee, rather than a double peak, when decreasing the impact parameter of the encounter and when elevating the difference between the mass of the star which leaves the system after binary separation and the mass of the companion. The detection of a knee can anticipate the onset of periodic accretion luminosity flares if one of the stars, only partially disrupted, remains bound to the black hole after binary separation. Thus knees could be precursors of periodic flares, which can then be predicted, followed up and better modelled. Analytical estimates in the black hole mass range $10^5-10^8 \rm M_{\rm \odot}$ show that the knee signature is enhanced in the case of black holes of mass $10^6-10^7 \rm M_{\rm \odot}$.Comment: 14 pages, 6 figures, 8 tables. Accepted by MNRA

Regulation of phagosome maturation and bacterial killing by the TPL-2 complex

A fundamental role of the innate immune response involves the killing of phagocytosed pathogens, such as bacteria, by macrophages. During phagocytosis, bacteria are internalised into membrane-bound vacuoles called phagosomes. The nascent phagosome undergoes a complex maturation process, which involves sequential membrane fusion and fission events with the endosomal compartment and ultimately with lysosomes to form a phagolysosome. The mature phagolysosome is an acidic, hydrolytic and highly oxidative organelle, which efficiently degrades internalised bacteria. Toll-like receptor (TLR) activation of mitogen-activated protein (MAP) kinases in macrophages is mediated by tumour progression locus 2 (TPL-2), a MAP 3 kinase that is critical for inflammatory immune responses to bacteria, viruses, and fungi. In unstimulated macrophages, TPL-2 forms a ternary complex with NF-kB1 p105 and A20-binding inhibitor of NF-kB-2 (ABIN-2). TLR-induced activation of the IkB kinase (IKK) complex leads to p105 phosphorylation and its proteasomal degradation. This liberates TPL-2 to activate MAP kinase signalling, which results in gene expression of numerous inflammatory mediators. ABIN-2 is also released, however, its physiological function in innate immune responses has remained unclear. I discovered that the TPL-2 complex promotes bead phagosome maturation in macrophages. Genetic inactivation of TPL-2 catalytic activity or ABIN-2 ubiquitin binding substantially altered the composition of the phagosome proteome in primary mouse macrophages. Further, I found that TPL-2 catalytic activity induced phagosome proteolytic activity and phagosome acidification, while ABIN-2 ubiquitin binding promoted phagosome proteolysis without affecting phagosome acidification. My genetic and pharmacological experiments indicated that TPL-2 regulates phagosome function independently of its known ability to activate MAP kinases. I demonstrated that induction of phagosome maturation was mediated by TPL-2-dependent regulation of V-ATPase function via serine 1903 phosphorylation of DMXL1, a V-ATPase-interacting regulatory protein. Importantly, I showed that TPL-2 catalytic activity also induced phagosome maturation independently of MAP kinase signalling in primary human macrophages, establishing the clinical relevance of my findings. Furthermore, my results revealed that ubiquitin binding to ABIN-2 promotes phagosome proteolytic activity independently of TPL-2 regulation of MAP kinase signalling. Consistent with these findings analysing bead phagosomes, I discovered that TPL-2 catalytic activity and ABIN-2 ubiquitin binding are required for efficient killing of internalised Staphylococcus aureus by macrophages. Genetic inactivation of TPL-2 catalytic activity or ABIN-2 ubiquitin binding impaired maturation of S. aureus phagosomes. I also found that TPL-2 catalytic activity was required for optimal killing of phagocytosed Citrobacter rodentium by inducing phagosomal acidification. Moreover, I demonstrated that ABIN-2 ubiquitin binding was essential for efficient killing of Salmonella typhimurium. Together, these discoveries demonstrated that both TPL-2 catalytic activity and ABIN-2 ubiquitin binding are important for the killing of several bacterial species, Gram-negative and Gram-positive as well as extracellular and intracellular microbes. In conclusion, my research identified novel signalling pathways that promote phagosome maturation and pathogenic bacterial killing by macrophages. This work increases our understanding of a critical process in innate immune responses and may lead to development of novel therapeutic approaches for acute bacterial infections

Circumbinary discs: Numerical and physical behaviour

We study the evolution of circumbinary disks under the gravitational influence of the binary using two-dimensional hydrodynamical simulations to investigate the impact of disk and binary parameters on the dynamical aspects of the disk. To distinguish between physical and numerical effects we apply three hydrodynamical codes. First we analyse in detail numerical issues concerning the conditions at the boundaries and grid resolution. We then perform a series of simulations with different binary (eccentricity, mass ratio) and disk parameters (viscosity, aspect ratio) starting from a reference model with Kepler-16 parameters. Concerning the numerical aspects we find that the inner grid radius must be of the order of the binary semi-major axis, with free outflow conditions applied such that mass can flow onto the central binary. A closed inner boundary leads to unstable evolutions. We find that the inner disk turns eccentric and precesses for all investigated physical parameters. The precession rate is slow with periods ($T_\mathrm{prec}$) starting at around 500 binary orbits ($T_\mathrm{bin}$) for high viscosity and large $H/R$ where the inner hole is smaller and more circular. Reducing $\alpha$ and $H/R$ increases the gap size and $T_\mathrm{prec}$ reaches 2500 $T_\mathrm{bin}$. For varying binary mass ratios $q_\mathrm{bin}$ the gap size remains constant whereas $T_\mathrm{prec}$ decreases for increasing $q_\mathrm{bin}$. For varying binary eccentricities $e_\mathrm{bin}$ we find two separate branches in the gap size and eccentricity diagram. The bifurcation occurs at around $e_\mathrm{crit} \approx 0.18$ where the gap is smallest with the shortest $T_\mathrm{prec}$. For $e_\mathrm{bin}$ smaller and larger than $e_\mathrm{crit}$ the gap size and $T_\mathrm{prec}$ increase. Circular binaries create the most eccentric disks.Comment: 20 pages, 25 figures, published in A&A, language edited versio