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

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

The surprising features of the TEAD4-Vgll1 protein-protein interaction

The Hippo signaling pathway, which controls organ size in animals, is altered in various human cancers. The TEAD transcription factors, the most downstream elements in this pathway, are regulated by different cofactors, such as the Vgll (vestigial-like) proteins. Having studied the interaction between Vgll1-derived peptides and human TEAD4, we show that, although it lacks a key secondary structure element required for tight binding by two other TEAD cofactors (YAP and TAZ), Vgll1-derived peptides bind to TEAD with nanomolar affinity. We identify a β-strand:loop:α-helix motif as the minimal Vgll binding site. Finally, we reveal an unexpected difference between mouse and human Vgll1-derived peptides. Shared interests: TEAD transcription factors are regulated by cofactors such as Vgll, YAP, and TAZ. Vgll1 (blue) binds to TEAD at a similar site to YAP/TAZ (red) but lacks the Ω-loop that confers high affinity. Synthetic peptides mimicking Vgll1 also bind to TEAD with nanomolar affinity, and we reveal an unexpected difference between mouse and human Vgll1-derived peptides. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The TEAD4-YAP/TAZ protein-protein interaction: expected similarities and unexpected differences

The Hippo pathway controls cell homeostasis, and its deregulation can lead to human diseases. Within this pathway, the YAP and TAZ transcriptional cofactors hold a key role in stimulating gene transcription via their interaction with the TEAD transcriptional factors. Our study of YAP and TAZ peptides in biochemical and biophysical assays shows that both proteins have the same affinity for TEAD. Molecular modeling and structural biology data suggest that they also bind to the same site on TEAD. However, this apparent similarity hides differences in the way both proteins interact with TEAD. The secondary structure elements of their TEAD-binding site do not contribute equally to the overall affinity, and critical interactions with TEAD are made by different residues. This convergent optimization of the YAP/TAZ TEAD-binding site suggests that binding of YAP and TAZ with similar affinity to TEAD is important for Hippo pathway functionalit

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

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary study were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbonate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in-vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using non-cleavable linkers, and the resulting conjugates (ADC-4, ADC-11 and ADC-12) led to in-vivo efficacy in a HER-2 expressing (SK-OV-3ip) mouse xenograft model in a target-dependent manner