The role of lysine palmitoylation/myristoylation in the function of the TEAD transcription factors

The TEAD transcription factors are the most downstream elements of the Hippo pathway. Their transcriptional activity is modulated by different regulator proteins and by the palmitoylation/myristoylation of a specific cysteine residue. In this report, we show that a conserved lysine present in these transcription factors can also be acylated, probably following the intramolecular transfer of the acyl moiety from the cysteine. Using Scalloped (Sd), the Drosophila homolog of human TEAD, as a model, we designed a mutant protein (Glu352Gln Sd ) that is predominantly acylated on the lysine (Lys350 Sd ). This protein binds in vitro to the three Sd regulators-Yki, Vg and Tgi-with a similar affinity as the wild type Sd, but it has a significantly higher thermal stability than Sd acylated on the cysteine. This mutant was also introduced in the endogenous locus of the sd gene in Drosophila using CRISPR/Cas9. Homozygous mutants reach adulthood, do not present obvious morphological defects and the mutant protein has both the same level of expression and localization as wild type Sd. This reveals that this mutant protein is both functional and able to control cell growth in a similar fashion as wild type Sd. Therefore, enhancing the lysine acylation of Sd has no detrimental effect on the Hippo pathway. However, we did observe a slight but significant increase of wing size in flies homozygous for the mutant protein suggesting that a higher acylation of the lysine affects the activity of the Hippo pathway. Altogether, our findings indicate that TEAD/Sd can be acylated either on a cysteine or on a lysine, and suggest that these two different forms may have similar properties in cells

The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells

Different recognition of TEAD transcription factor by the conserved β-strand:loop:α-helix motif of the TEAD binding site of YAP and VGLL1

The TEAD (TEA/ATTS domain) transcription factors are regulated by various coactivator proteins. A β-strand:loop:α-helix motif is present at the TEAD binding site of all the coactivators crystallized so far. These motifs interact with the same area of TEAD, suggesting that the coactivators compete with each other in vivo to gain access to TEAD. The α-helix, which shows marked interactions with TEAD, is the key element of the β-strand:loop:α-helix motif. A very large difference in potency (>40 fold) has been measured between the isolated mouse VGLL1 (vestigial-like 1, mVGLL1) α-helix and its human YAP (Yes-associated protein, hYAP) equivalent. Elucidating the mechanisms at the origin of this difference should help in better understanding how these coactivators interact with TEAD. In this report, we show that the β-strand:loop:α-helix motif of hYAP and mVGLL1 are optimized in a very different manner suggesting a convergent evolution of these coactivators for binding to the TEAD transcription factors

Development of an Image Based Assay to Detect C5a Receptor Occupancy

Stimulation of the C5a receptor is the final common step in the three different pathways leading to activation of the complement cascade. Activation of the complement pathway has been implicated in a number of different diseases including; rheumatoid arthritis (RA), psoriasis, ischemia/reperfusion (I/R) injury, glomular nephritis and possibly even sepsis. There is strong evidence that activation of the complement cascade, by the alternative complement pathway, is involved in Age-related Macular Degeneration (AMD). Variants in two key regulators of the complement cascade, factor H and factor B, lead to an over activation of the alternative complement pathway, accounting for up to 75% of AMD cases. In addition, leukocytes, mainly monocytes and macrophages, cell types known to express C5aR, are seen by histology in neovascularization, Retinal Pigmented Epithelium degeneration, and breakdown of Bruch’s membrane leading to AMD. The C5a receptor is a GPCR that once activated leads to changes in intracellular signaling which can be detected by a number of conventional assay formats. However, while these assays can detect intracellular signaling events they give no indication as to the location of the receptor, nor do they allow monitoring of receptor binding only the subsequent signaling events. This poster describes an image based assay which is capable of monitoring the binding of fluorescently labeled C5a to its receptor. This assay not only allows measurement of binding events by monitoring competition binding of the fluorescently labeled C5a but also localization of the ligand bound receptor. The ability to track the location of the ligand bound receptor allows one to visualize receptor internalization over time. In addition, the ability to direct binding of the labeled ligand makes the requirements for antibody staining and washing steps obsolete resulting in an easily automatable protocol

Structural determinants of MALT1 protease activity

The formation of the Carma1-BCL10-MALT1 complex is pivotal for T-cell receptor mediated activation of the transcription-factor NF-κB. Recently it was shown that in this complex MALT1 not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. Here we show that, as described for caspases which play an essential role in apoptosis, proteolytic activity of MALT1 can be reconstituted by induced proximity. However, unlike caspases which cleave specifically C-terminal to an aspartate, MALT1 has an absolute specificity for arginine. We further determined the structure of the MALT1 domains responsible for proteolytic activity in the absence of ligand and in complex with a covalently bound substrate like inhibitor. The structures explain the specificity for cleavage after arginine, and show that the protease domain undergoes large conformational changes upon substrate binding that also affect the structure of the C-terminal Ig-like domain. This C-terminal Ig-like domain is required for MALT1 activity and likely important for the interaction of MALT1 with its binding partners

A new perspective on the evolution of the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors