Functional Analysis of Hsp70 Inhibitors

Schlecht, Rainer; Scholz, Sebastian; Dahmen, Heike; Wegener, Ansgar; Sirrenberg, Christian; Müsil, Djordje; Bomke, Joerg; Eggenweiler, Hans-Michael; Mayer, Mathias; Bukau, Bernd

doi:10.1371/journal.pone.0078443

Cited by 166 publications

(189 citation statements)

References 58 publications

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“…8, eЈ and eЉ). To provide a functional test for involvement of Hsc70 in imatinib-induced TfR1 degradation, we utilized a small molecule ATP-derivative inhibitor of Hsc/Hsp70 chaperones (VER155008) that is well described to have antitumor properties (32). Importantly, we found that this inhibitor minimizes the imatinib-dependent TfR1 degradation by lysosomes.…”

Section: C-abl Regulates Tfr1 Endocytosismentioning

confidence: 99%

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

Cao

Schroeder

Chen

et al. 2016

Journal of Biological Chemistry

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Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.

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Section: C-abl Regulates Tfr1 Endocytosismentioning

confidence: 99%

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

Cao

Schroeder

Chen

et al. 2016

Journal of Biological Chemistry

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“…To test this, first we applied to tumor cells before irradiation the small-molecule inhibitor VER155008, which inhibits the activity of both the induced and constitutive forms of Hsp70 (21,22). Tumor cell numbers after 72 hours were only slightly lower when VER155008 was applied to irradiated cells, presumably because cell proliferation was already slowed down by the irradiation.…”

Section: Hsp70 Inhibition Blocks Antigen Cross-presentationmentioning

confidence: 99%

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

Salimu

Spary

Al-Taei

et al. 2015

Cancer Immunology Research

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Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8 þ T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8 þ T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G 2 -M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8 þ 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNg. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8 þ T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process. Cancer Immunol Res; 3(6); 678-88. Ó2015 AACR.

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“…Chaperone inhibitors are intended to block oncogenic or disease protein folding and function to sensitize cells to cellular stress generated by conventional chemotherapies (Neckers and Workman, 2012). HSP/C 70/90 are both attractive as therapeutic targets because they contain multiple sites amenable to drug targeting, including ATP cofactor-binding sites, client protein substrate sites, and protein-protein interfaces (PPIs), with cochaperones that facilitate client protein folding activity (Rérole et al, 2011;Assimon et al, 2013;Balaburski et al, 2013;Schlecht et al, 2013). Many HSP/C 90/70 inhibitors are indeed small molecule adenosine analogs that target the nucleotidebinding site (Chiosis et al, 2002;Dymock et al, 2004;Donnelly and Blagg, 2008;Williamson et al, 2009;Day et al, 2010;Budina-Kolomets et al, 2014).…”

Section: Therapeutic Chaperone Inhibition Is An Established Paradigmmentioning

confidence: 99%

The G ProteinαChaperone Ric-8 as a Potential Therapeutic Target

2014

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Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein a subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Ga-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein a subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Ga-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Ga unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein-coupled receptors (GPCRs). G protein bg is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Ga protein-protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies.

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Functional Analysis of Hsp70 Inhibitors

Cited by 166 publications

References 58 publications

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

The G ProteinαChaperone Ric-8 as a Potential Therapeutic Target

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