Limits...
Dynamic impacts of the inhibition of the molecular chaperone Hsp90 on the T-cell proteome have implications for anti-cancer therapy.

Fierro-Monti I, Echeverria P, Racle J, Hernandez C, Picard D, Quadroni M - PLoS ONE (2013)

Bottom Line: Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects.As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients.The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.

Show MeSH

Related in: MedlinePlus

Validation of new Hsp90 clients.A) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int [21] were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. B). Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90β in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90β (mouse), and the corresponding non-immune (NI) control antibodies from rabbit and mouse were used in co-IP experiments, and then analysed by immunoblotting (WB). C) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 h were analysed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842317&req=5

pone-0080425-g005: Validation of new Hsp90 clients.A) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int [21] were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. B). Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90β in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90β (mouse), and the corresponding non-immune (NI) control antibodies from rabbit and mouse were used in co-IP experiments, and then analysed by immunoblotting (WB). C) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 h were analysed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.

Mentions: Hsp90Int assisted in the identification of proteins that exhibited the expected behaviour for a novel Hsp90 client, i.e. GA-induced depletion, and no previously reported interaction with Hsp90 at the same time of the analysis. Concurrently, these potential candidates could be selected based on the fact that they were associated with protein complexes that contained known Hsp90 interactors (2nd level Hsp90 interacting protein) (Fig. 5A). We made use of functional metadata contained in the Hsp90Int to detect interesting potentially novel clients, such as the IL2-inducible T-cell kinase (ITK), the BRCA1-associated ATM activator 1 (BRAT1), and the UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT) (Table S6). ITK is a tyrosine kinase that plays an essential role in regulation of the adaptive immune response. It is recruited to the cell membrane upon activation of the T-cell receptor following a series of phosphorylation events. BRAT1 is required for the activation of ataxia telangiectasia mutated (ATM) following ionizing radiation, and it may act by regulating the dephosphorylation of ATM [48]. OGT is an O-GlcNAc transferase, the main enzyme responsible for intracellular O-glycosylation. It is a component of the THAP1/THAP3-HCFC1-OGT complex required for the regulation of the transcriptional activity of RRM1, which catalyses the biosynthesis of deoxyribonucleotides, providing the precursors necessary for DNA synthesis. Each one of the three proteins was independently shown to co-immunoprecipitate with Hsp90, and their levels decreased post-GA-treatment as confirmed by immunoblotting (Fig. 5B).


Dynamic impacts of the inhibition of the molecular chaperone Hsp90 on the T-cell proteome have implications for anti-cancer therapy.

Fierro-Monti I, Echeverria P, Racle J, Hernandez C, Picard D, Quadroni M - PLoS ONE (2013)

Validation of new Hsp90 clients.A) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int [21] were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. B). Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90β in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90β (mouse), and the corresponding non-immune (NI) control antibodies from rabbit and mouse were used in co-IP experiments, and then analysed by immunoblotting (WB). C) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 h were analysed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842317&req=5

pone-0080425-g005: Validation of new Hsp90 clients.A) Network analysis of a selected set of potentially new Hsp90 client proteins. stSILAC data and the Hsp90 interaction network Hsp90Int [21] were combined to identify interesting candidates (OGT, ITK and BRAT1) with no reported interactions with Hsp90 at the time of the analysis. Edges connecting candidate proteins with known Hsp90 interacting proteins are highlighted in red. B). Co-immunoprecipitation (co-IP) experiment demonstrating interactions between BRAT1, OGT and ITK with Hsp90β in Jurkat cells. Equal concentrations of specific antibodies against BRAT1 (rabbit), OGT (rabbit), ITK (mouse), Hsp90β (mouse), and the corresponding non-immune (NI) control antibodies from rabbit and mouse were used in co-IP experiments, and then analysed by immunoblotting (WB). C) GA-induced degradation of BRAT1, ITK and OGT in Jurkat cells. Lysates from cells treated with GA or with the equivalent volume of the solvent DMSO (control) for 6 and 20 h were analysed by WB for these three mentioned proteins and also for Hsp70 and CDK6 as positive controls of GA action.
Mentions: Hsp90Int assisted in the identification of proteins that exhibited the expected behaviour for a novel Hsp90 client, i.e. GA-induced depletion, and no previously reported interaction with Hsp90 at the same time of the analysis. Concurrently, these potential candidates could be selected based on the fact that they were associated with protein complexes that contained known Hsp90 interactors (2nd level Hsp90 interacting protein) (Fig. 5A). We made use of functional metadata contained in the Hsp90Int to detect interesting potentially novel clients, such as the IL2-inducible T-cell kinase (ITK), the BRCA1-associated ATM activator 1 (BRAT1), and the UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT) (Table S6). ITK is a tyrosine kinase that plays an essential role in regulation of the adaptive immune response. It is recruited to the cell membrane upon activation of the T-cell receptor following a series of phosphorylation events. BRAT1 is required for the activation of ataxia telangiectasia mutated (ATM) following ionizing radiation, and it may act by regulating the dephosphorylation of ATM [48]. OGT is an O-GlcNAc transferase, the main enzyme responsible for intracellular O-glycosylation. It is a component of the THAP1/THAP3-HCFC1-OGT complex required for the regulation of the transcriptional activity of RRM1, which catalyses the biosynthesis of deoxyribonucleotides, providing the precursors necessary for DNA synthesis. Each one of the three proteins was independently shown to co-immunoprecipitate with Hsp90, and their levels decreased post-GA-treatment as confirmed by immunoblotting (Fig. 5B).

Bottom Line: Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects.As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients.The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.

Show MeSH
Related in: MedlinePlus