Characterization of Hsp90 Co-Chaperone p23 Cleavage by Caspase‑7 Uncovers a Peptidase–Substrate Interaction Involving Intrinsically Disordered Regions

Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly­(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure–activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36–45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD₁₄₂↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase–substrate pair to understand the modalities of substrate recognition by caspases

Alternative splicing of WT Max leads to the expression of ΔMax.

<p>ΔMax is generated by exon 5 inclusion. This leads to a shorter LZ and a change in the primary structure of the last heptad.</p

The c-Myc*/ΔMax* heterodimer binds E-Box and non-specific DNA sequences with similar apparent affinities.

<p>(<b>A</b>) Titration of fluorescently labeled E-box (black circles) and non-specific (open circles) sequences with 1:1 mixtures of c-Myc*/ΔMax*. (<b>B</b>) Populations of ΔMax*/Max* (blue), ΔMax*/c-Myc* (red) and ΔMax*/ΔMax* (pale blue) with the E-box (full lines) and the non-specific (dotted lines) sequences as a function of the total dimeric concentrations. (<b>C</b>) Titration of fluorescently labeled E-box (black circles) and non-specific (open circles) sequences with a 1:1 mixture of c-Myc*/Max. The lines were obtained from the simulation of the binding of 3 species: (<b>D</b>) c-Myc*/Max* (purple), Max*/Max* (blue) and c-Myc*/c-Myc* (red) for the E-box (full lines) and non-specific (dotted lines) sequences.</p

Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers

<div><p>It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max.</p></div

Models of the last heptad of ΔMax and WT Max.

<p>Models of the last heptad of the LZ of ΔMax (<b>A</b>) and WT Max (<b>B</b>) in a homodimeric state. Models of the last heptad of the LZs of ΔMax/c-Myc (<b>C</b>), WT Max/c-Myc (<b>D</b>) and ΔMax/WT Max (<b>E</b>) in a heterodimeric state. Although various structures of the Max homodimeric and c-Myc/Max b-HLH-LZ have been reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174413#pone.0174413.ref016" target="_blank">16</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174413#pone.0174413.ref017" target="_blank">17</a>], the last heptad is incomplete or ill-defined in all of them. They were therefore remodeled along with the LZ of ΔMax using the program Pymol (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>) and the backbone of the structure of the heterodimeric c-Myc/Max (1A93) as a template [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174413#pone.0174413.ref018" target="_blank">18</a>].</p

Results of the analysis of the titration of the E-box and non-specific DNA with the b-HLH-LZs of Max, ΔMax and c-Myc.

<p>Results of the analysis of the titration of the E-box and non-specific DNA with the b-HLH-LZs of Max, ΔMax and c-Myc.</p

c-Myc* binds both specific and non-specific DNA sequences as a homodimer.

<p>Titration of fluorescently labeled E-box (black circles) and non-specific (open circles) sequences with c-Myc*.</p

ΔMax* binds E-box and non-specific DNA sequences as a heterodimer with Max*.

<p>(<b>A</b>) Far-UV CD spectra of ΔMax* (open triangles), Max* (open diamonds), ΔMax*/Max* (black diamonds) and the arithmetic sum of the individual spectra (gray diamonds). (<b>B</b>) Thermal denaturations of ΔMax* (open triangles), Max* (open diamonds) and ΔMax*/Max* (black diamonds) recorded by monitoring the CD signal at 222 nm and the arithmetic sum of the individual denaturations (gray diamonds). The melting temperatures (T°) are indicated by the dotted lines. (<b>C</b>) Titration of fluorescently labeled E-box (black circles) and non-specific (open circles) sequences with ΔMax*/Max*. (<b>D</b>) Populations of Max*/Max* (blue) and ΔMax*/Max* (red) with the E-box (full line) and the non-specific (dotted line) sequences as a function of the total dimeric concentrations.</p

The b-HLH-LZ of Max has a putative NLS sequence and PTD in its basic region.

<p>Primary structure of the b-HLH-LZ of Max (Max*). Also shown is the domain of NeuroD responsible for its transduction and nuclear localization and the Max NLS. Note the high content in basic side-chains and the similarity between the sequences.</p

In solution with c-Myc*, Max* forms both homo- and heterodimers while ΔMax* exclusively heterodimerizes.

<p>(<b>A</b>) Far-UV CD spectra of c-Myc* (open circles), Max* (open diamonds), c-Myc*/Max* (black circles) and the arithmetic sum of the individual spectra (gray circles). (<b>B</b>) Thermal denaturation of c-Myc*/Max* (black circles) recorded by monitoring the CD signal at 222 nm. The melting temperatures (T°) of the complexes of the mixture are indicated by the dotted lines. (<b>C</b>) Far-UV CD spectra of c-Myc* (open circles), ΔMax* (open triangles), c-Myc*/ΔMax* (black triangles) and the arithmetic sum of the individual spectra (gray triangles). (<b>D</b>) Thermal denaturation of c-Myc*/ΔMax* (black triangles) recorded by monitoring the CD signal at 222 nm and simulation of the thermal denaturation of an 8 μM dimer into two unfolded monomers (open circles). The melting temperature (T°) is indicated by the dotted line.</p