Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.

Danish Cancer Society Research Center Copenhagen Denmark

Division of Genome Biology Department of Medical Biochemistry and Biophysics Science for Life Laboratory Karolinska Institute Stockholm Sweden

Laboratory of Genome Integrity Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacky University Olomouc Czech Republic

Zobrazit více v PubMed

Hartl FU. Protein misfolding diseases. Annu. Rev. Biochem. 2017;86:21–26. doi: 10.1146/annurev-biochem-061516-044518. PubMed DOI

Deshaies RJ. Proteotoxic crisis, the ubiquitin-proteasome system, and cancer therapy. BMC Biol. 2014;12:94. doi: 10.1186/s12915-014-0094-0. PubMed DOI PMC

Guin D, Gelman H, Wang Y, Gruebele M. Heat shock-induced chaperoning by Hsp70 is enabled in-cell. PLoS ONE. 2019;14:e0222990. doi: 10.1371/journal.pone.0222990. PubMed DOI PMC

Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Ulrich Hartl F. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 2013;82:323–355. doi: 10.1146/annurev-biochem-060208-092442. PubMed DOI

Kim M, Lee J, Nam J. Plasmonic photothermal nanoparticles for biomedical applications. Adv. Sci. 2019;6:1900471. doi: 10.1002/advs.201900471. PubMed DOI PMC

Mayer KM, Hafner JH. Localized surface plasmon resonance sensors. Chem. Rev. 2011;111:3828–3857. doi: 10.1021/cr100313v. PubMed DOI

Xue C, Mirkin CA. pH-switchable silver nanoprism growth pathways. Angew. Chem. - Int. Ed. 2007;46:2036–2038. doi: 10.1002/anie.200604637. PubMed DOI

Haes AJ, Stuart DA, Nie S, Van Duyne RP. Using solution-phase nanoparticles, surface-confined nanoparticle arrays and single nanoparticles as biological sensing platforms. J. Fluoresc. 2004;14:355–367. doi: 10.1023/B:JOFL.0000031817.35049.1f. PubMed DOI

Jiang K, Smith DA, Pinchuk A. Size-dependent photothermal conversion efficiencies of plasmonically heated gold nanoparticles. J. Phys. Chem. C. 2013;117:27073–27080. doi: 10.1021/jp409067h. DOI

Plech A, Kotaidis V, Grésillon S, Dahmen C, von Plessen G. Laser-induced heating and melting of gold nanoparticles studied by time-resolved x-ray scattering. Phys. Rev. B. 2004;70:195423. doi: 10.1103/PhysRevB.70.195423. DOI

Govorov AO, Richardson HH. Generating heat with metal nanoparticles. Nano Today. 2007;2:30–38. doi: 10.1016/S1748-0132(07)70017-8. DOI

Zhao S, Zhang K, An J, Sun Y, Sun C. Synthesis and layer-by-layer self-assembly of silver nanoparticles capped by mercaptosulfonic acid. Mater. Lett. 2006;60:1215–1218. doi: 10.1016/j.matlet.2005.11.007. DOI

Mistrik M, et al. Cells and stripes: a novel quantitative photo-manipulation technique. Sci. Rep. 2016;6:19567. doi: 10.1038/srep19567. PubMed DOI PMC

Lukas C, Falck J, Bartkova J, Bartek J, Lukas J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 2003;5:255–260. doi: 10.1038/ncb945. PubMed DOI

Morgner N, et al. Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90. Cell Rep. 2015;11:759–769. doi: 10.1016/j.celrep.2015.03.063. PubMed DOI PMC

Mayer MP, Gierasch LM. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. J. Biol. Chem. 2019;294:2085–2097. doi: 10.1074/jbc.REV118.002810. PubMed DOI PMC

Trcka F, et al. Human stress-inducible Hsp70 has a high propensity to form ATP-dependent antiparallel dimers that are differentially regulated by cochaperone binding. Mol. Cell. Proteom. 2019;18:320–337. doi: 10.1074/mcp.RA118.001044. PubMed DOI PMC

Vandova V, et al. HSPA1A conformational mutants reveal a conserved structural unit in Hsp70 proteins. Biochim. Biophys. Acta - Gen. Subj. 2020;1864:129458. doi: 10.1016/j.bbagen.2019.129458. PubMed DOI

Komander D, Rape M. The ubiquitin code. Annu. Rev. Biochem. 2012;81:203–229. doi: 10.1146/annurev-biochem-060310-170328. PubMed DOI

Audas TE, et al. Adaptation to stressors by systemic protein amyloidogenesis. Dev. Cell. 2016;39:155–168. doi: 10.1016/j.devcel.2016.09.002. PubMed DOI PMC

Frottin F, et al. The nucleolus functions as a phase-separated protein quality control compartment. Science. 2019;365:342–347. doi: 10.1126/science.aaw9157. PubMed DOI

Meyer H, Bug M, Bremer S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 2012;14:117–123. doi: 10.1038/ncb2407. PubMed DOI

Hyer ML, et al. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment. Nat. Med. 2018;24:186–193. doi: 10.1038/nm.4474. PubMed DOI

Anderson DJ, et al. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell. 2015;28:653–665. doi: 10.1016/j.ccell.2015.10.002. PubMed DOI PMC

Schlecht R, et al. Functional analysis of Hsp70 inhibitors. PLoS ONE. 2013;8:e78443. doi: 10.1371/journal.pone.0078443. PubMed DOI PMC

Mogk A, Bukau B, Kampinga HH. Cellular handling of protein aggregates by disaggregation machines. Mol. Cell. 2018;69:214–226. doi: 10.1016/j.molcel.2018.01.004. PubMed DOI

Tang, W. K. & Xia, D. Mutations in the human AAA+ chaperone p97 and related diseases. Front. Mol. Biosci. 3, 79 (2016). PubMed PMC

Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461:1071–1078. doi: 10.1038/nature08467. PubMed DOI PMC

Sies H, Berndt C, Jones DP. Oxidative Stress. Annu. Rev. Biochem. 2017;86:715–748. doi: 10.1146/annurev-biochem-061516-045037. PubMed DOI

Mistrik, M. et al. Protocol for subcellular targeted microthermal protein damage in cells cultivated on plasmonic nanosilver-modified surfaces. Protoc. Exch.10.21203/rs.3.pex-1314/v1 (2021). PubMed PMC

Skrott Z, et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature. 2017;552:194–199. doi: 10.1038/nature25016. PubMed DOI PMC

Heterogeneous protein dynamics links to mitochondrial activity, glucose transporter, and ALDH cancer stem cell properties

Microthermal-induced subcellular-targeted protein damage in cells on plasmonic nanosilver-modified surfaces evokes a two-phase HSP-p97/VCP response