Renaturation of thermally denatured luciferase by CacyBP/SIP.

<p>Purified, recombinant luciferase (0.04 μM) was denatured alone (control) or in the presence of 0.5 μM Hsp90, 0.8 μM CacyBP/SIP or both at 31°C for 3 min and then left to recover at 25°C for 120 min <b>(A)</b> or for 90 min <b>(B)</b>. In <b>(B)</b> 0.8 μM Hsp40 and 0.4 μM Hsp70 were added directly after denaturation. Luciferase activity was calculated as a percentage of the value before denaturation. Data from 4 independent experiments are shown as a mean ± standard error (SEM); *** p ≤ 0.001, ** p ≤ 0.0.01, * p ≤ 0.05.</p

Pattern of spots representing Hsp90 forms with different pI values.

<p><b>(A)</b> Pattern of Hsp90 spots in HEp-2 cell lysate. Upper panel: HEp-2 cells co-transfected with plasmids encoding Hsp90β-3xFLAG and 3xFLAG (control). Middle panel: HEp-2 cells co-transfected with plasmids encoding Hsp90β-3xFLAG and CacyBP/SIP-3xFLAG. Bottom panel: HEp-2 cells co-transfected with plasmids encoding Hsp90β-3xFLAG and 3xFLAG (control) but lysate was incubated with Lambda Protein Phosphatase (Lambda PP). (<b>B)</b> Pattern of spots corresponding to purified Hsp90. Upper panel: Hsp90 (control). Middle panel: Hsp90 incubated with recombinant CacyBP/SIP. Bottom panel: Hsp90 incubated with Lambda Protein Phosphatase (Lambda PP). A representative Western blot, out of 3 performed after two dimensional (2D) electrophoresis, is shown.</p

Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

<div><p>The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.</p></div

List of restriction enzymes and primers used for cloning.

<p>List of restriction enzymes and primers used for cloning.</p

Hsp90-CacyBP/SIP complex formation.

<p>Co-immunoprecipitation of CacyBP/SIP and Sgt1 with <b>(A)</b> FLAG-tagged Hsp90α and Hsp90β or <b>(B)</b> full-length Hsp90β (FL) and its deletion mutants containing N-terminal + middle domains (N+M), middle + C-terminal (M+C) or middle (M) domains. Anti-FLAG staining shows expression of Hsp90α-3xFLAG, Hsp90β-3xFLAG and FLAG-tagged Hsp90β deletion mutants. For control, HEp-2 cells were transfected with plasmid encoding 3xFLAG alone. A representative Western blot, out of 3 performed, is shown.</p

Influence of Hsp90 inhibition on CacyBP/SIP protein level.

<p>Level of CacyBP/SIP in HEp-2 cells treated with different concentrations of radicicol (<b>A</b>) or novobiocin (<b>B</b>). Representative Western blots (left panels) and densitometric analysis of data from 3 independent experiments (right panels) are shown as means ± standard errors (SEM); *** p ≤ 0.001, ** p ≤ 0.0.01, * p ≤ 0.05.</p

Comparison of human Sgt1 and CacyBP/SIP.

<p><b>(A)</b> Schematic representation of CacyBP/SIP and both isoforms of Sgt1 (Sgt1A and Sgt1B). Grey and black boxes show the CS (CHORD-containing proteins and Sgt1) and the SGS (Sgt1-specific) domains. Hatched box indicates the fragment of sequence specific for Sgt1B. <b>(B)</b> Sequence alignment of CacyBP/SIP and Sgt1A. The sequences were compared using ClustalW software. Black and grey boxes indicate identical and similar amino acid residues, respectively. The amino acid identity and similarity in the SGS domain are 29.3% and 63.4%, respectively, and in the CS domain are 36.8% and 55.2%, respectively.</p

Binding of purified Hsp90 to CacyBP/SIP.

<p>(<b>A</b>) ELISA assay with increasing concentrations of CacyBP/SIP. <b>(B)</b> Competitive ELISA assay with increasing concentrations of CacyBP/SIP in the absence (black bars) or presence (white bars) of constant concentration of Sgt1 (0.67 μM). <b>(C)</b> Competitive ELISA assay with constant concentration of CacyBP/SIP (1.33 μM) in the absence (black bar) or in the presence of increasing concentrations of Sgt1 (grey bars). The results from 3 independent experiments are presented as a mean value of absorbance ± standard error (SEM).</p

Influence of Hsp90 inhibition on Hsp90-CacyBP/SIP and Hsp90-Sgt1 complex formation.

<p>Co-immunoprecipitation of CacyBP/SIP and Sgt1 with FLAG-tagged Hsp90β from HEp-2 cell lysate obtained after 2 hrs treatment with radicicol <b>(A)</b> or novobiocin <b>(B)</b>. Anti-FLAG staining shows expression of FLAG-tagged Hsp90β. A representative Western blot, out of 3 performed, is shown.</p

Influence of CacyBP/SIP on heat-induced aggregation of citrate synthase.

<p>Aggregation of 0.15 μM citrate synthase (CS) was measured by the increase in optical density due to light scattering (at 360 nm) during incubation at 43°C for 21 min in the absence (control) or presence of GST (negative control), Hsp90, CacyBP/SIP or both Hsp90 and CacyBP/SIP at 0.15 μM final concentration each; <b>A)</b> a representative result (out of three performed) is shown as a function of time. <b>B)</b> Mean relative levels of CS aggregation in the presence of equimolar concentrations of indicated proteins at the 20-min time point of the aggregation reaction. Data from 3 independent experiments are shown as a mean ± standard error (SEM); * p<0.05, ** p< 0.01.</p