In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.

• MeSH
• buněčné linie MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny metabolismus   genetika   MeSH
• histony metabolismus   MeSH
• jaderné proteiny metabolismus   genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• oprava DNA * MeSH
• poly-ADP-ribosylace MeSH
• poly(ADP-ribosa)polymerasa 1 * metabolismus   genetika   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   genetika   MeSH
• protein XRCC1 metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• DNA vazebné proteiny MeSH
• histony MeSH
• HPF1 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• PARP1 protein, human MeSH Prohlížeč
• poly(ADP-ribosa)polymerasa 1 * MeSH
• poly(ADP-ribosa)polymerasy MeSH
• protein XRCC1 MeSH
• XRCC1 protein, human MeSH Prohlížeč

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.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• povrchová plasmonová rezonance MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• protein obsahující valosin genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• reakce na tepelný šok * MeSH
• stříbro chemie   MeSH
• ubikvitin metabolismus   MeSH
• vysoká teplota škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteasomový endopeptidasový komplex MeSH
• protein obsahující valosin MeSH
• proteiny tepelného šoku HSP70 MeSH
• stříbro MeSH
• ubikvitin MeSH
• VCP protein, human MeSH Prohlížeč

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.

• Klíčová slova
• ATR pathway, BRCA1, BRCA2, DNA damage, NPL4, disulfiram, replication stress, targeted cancer therapy,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• ATM protein antagonisté a inhibitory   metabolismus   MeSH
• checkpoint kinasa 1 metabolismus   MeSH
• disulfiram farmakologie   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• jaderné proteiny antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   patologie   MeSH
• odvykací prostředky alkoholu farmakologie   MeSH
• patologická konformace proteinů chemicky indukované   MeSH
• poškození DNA účinky léků   MeSH
• protein obsahující valosin metabolismus   MeSH
• proteinové agregáty účinky léků   MeSH
• replikace DNA účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• ATM protein MeSH
• ATR protein, human MeSH Prohlížeč
• ATRIP protein, human MeSH Prohlížeč
• checkpoint kinasa 1 MeSH
• CHEK1 protein, human MeSH Prohlížeč
• disulfiram MeSH
• DNA vazebné proteiny MeSH
• jaderné proteiny MeSH
• NPLOC4 protein, human MeSH Prohlížeč
• odvykací prostředky alkoholu MeSH
• protein obsahující valosin MeSH
• proteinové agregáty MeSH
• VCP protein, human MeSH Prohlížeč