Probing the 14-3-3 Isoform-Specificity Profile of Protein–Protein Interactions Stabilized by Fusicoccin A

Abstract

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. Recently, FC has emerged as an important chemical probe of human 14-3-3 PPIs involved in cancer and neurobiology. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of 14-3-3 isoforms on FC activity remains underexplored. This is a relevant question for the continued development of FC variants because there are seven isoforms of 14-3-3 in humans. Despite their sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions in vivo . Herein, we interrogate the isoform-specificity profile of FC in vitro using recombinant 14-3-3 isoforms and a library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal recognition domains of client proteins that are characterized targets of FC in vivo . Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3σ. Together, these data support the feasibility of developing FC variants with enhanced isoform selectivity.

Understanding the interaction of 14‐3‐3 proteins with hDMX and hDM2: a structural and biophysical study

“…Ser166 and Ser186 on hDM2 are involved in apoptosis, signalling pathway regulation and ubiquitination. protein are clear between the isoforms [36,37]. The 14-3-3 proteins exist as homo or heterodimers where each monomer consists of nine α-helices forming an amphipathic binding groove.…”

“…protein are clear between the isoforms [36,37]. The 14-3-3 proteins exist as homo or heterodimers where each monomer consists of nine α-helices forming an amphipathic binding groove.…”

“…S1). Even though sequence conservation between the isoforms can indicate redundancy in protein function, this is not the case for 14‐3‐3 proteins as different affinities towards a target protein are clear between the isoforms [36,37]. The 14‐3‐3 proteins exist as homo or heterodimers where each monomer consists of nine α‐helices forming an amphipathic binding groove.…”

The Surprising Story of Fusicoccin: A Wilt-Inducing Phytotoxin, a Tool in Plant Physiology and a 14-3-3-Targeted Drug

“…Lentini Santo et al [148] reported that the FC derivatives called FC-H and 16-O-Me FC-H (Figure 11), lacking substitutions in the C ring, stabilized the interaction between the N-terminal non canonical mode III motif, whereas FC, FC-J, and FC-THF which carry substituents at the C ring were ineffective, due to steric hindrance with the Gln residue of the peptide within the binding groove. Finally, in a study aimed to assess 14-3-3 isoform specificity of FC association to mode III targets, Sengupta et al [149] tested the binding of different recombinant human 14-3-3 isoforms to phosphorylated hexapeptides containing known mode III motifs. Despite that isoform-dependent interactions were displayed better in the absence of FC, the stabilization by FC of the association of the 14-3-3σ isoform was apparently sensitive to the type of client peptide.…”

Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

“…Even though sequence conservation between the isoforms can indicate redundancy in protein function, this is not the case for 14-3-3 proteins as different affinities towards a target protein are clear between the isoforms. [35][36] 14-3-3 proteins exist as homo or heterodimers where each monomer consists of nine α-helices forming an amphipathic binding groove. Four α-helices are directly responsible for dimer formation, while the rest of the α-helices form the "W" shape of the protein dimer.…”