Reconstructing the TIR side of the Myddosome: a paradigm for TIR-TIR interactions?

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the alpha E-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The alpha C'-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains

TYK2-induced phosphorylation of Y640 suppresses STAT3 transcriptional activity

STAT3 is a pleiotropic transcription factor involved in homeostatic and host defense processes in the human body. It is activated by numerous cytokines and growth factors and generates a series of cellular effects. Of the STAT-mediated signal transduction pathways, STAT3 transcriptional control is best understood. Jak kinase dependent activation of STAT3 relies on Y705 phosphorylation triggering a conformational switch that is stabilized by intermolecular interactions between SH2 domains and the pY705 motif. We here show that a second tyrosine phosphorylation within the SH2 domain at position Y640, induced by Tyk2, negatively controls STAT3 activity. The Y640F mutation leads to stabilization of activated STAT3 homodimers, accelerated nuclear translocation and superior transcriptional activity following IL-6 and LIF stimulation. Moreover, it unlocks type I IFN-dependent STAT3 signalling in cells that are normally refractory to STAT3 transcriptional activation

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARa receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids

BACKGROUND: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses. RESULTS: In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids. CONCLUSION: Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

HyperISGylation of Old World Monkey ISG15 in Human Cells

BACKGROUND: ISG15 is an Ubiquitin-like protein, highly induced by Type I Interferons. Upon the cooperative activity of specific Ubiquitinating enzymes, ISG15 can be conjugated to its substrates. Increasing evidence points to a role for protein ISGylation in anti-viral and anti-tumoral defense. PRINCIPAL FINDINGS: We identified ISG15 from Old World Monkeys (OWm) as a hyper-efficient protein modifier. Western blot analysis visualized more efficient conjugation of OWmISG15 relative to HuISG15 in human (Hu), monkey and mouse (Mo) cell-lines. Moreover, the substrates of OWmISG15 identified upon Tandem Affinity Purification followed by LC-MS/MS identification largely outnumbered these of HuISG15 itself. Several Ubiquitin-Conjugating enzymes were identified as novel ISGylated substrates. Introduction of a N89D mutation in HuISG15 improved its ISGylation capacity, and additional Q31K/T33A/D133N mutations yielded a HuISG15 variant with an ISGylation efficiency comparable to OWmISG15. Homology modeling and structural superposition situate N89 in the interaction interface with the Activating enzyme. Analysis of the UbE1L residues in this interface revealed a striking homology between OWmUbE1L and HuUbE1, the Activating enzyme of Ubiquitin. In line with this observation, we found efficient activation of AgmISG15, but not HuISG15 or MoISG15, by HuUbE1, thus providing a likely explanation for OWm hyperISGylation. CONCLUSIONS: This study discloses the poor conjugation competence of HuISG15 compared to OWmISG15 and maps the critical determinants for efficient conjugation. HyperISGylation may greatly assist ISGylation studies and may enhance its function as positive regulator of Interferon-related immune responses or as anti-tumoral modulator