Biology subjects

Szakacs, G.

Publications and source records attributed to Szakacs, G..

5 recordsLinked to original sources

bioRxiv2026

SSB1 links stress granule regulation to cellular stress responses and renal ischemia-reperfusion injury

Mammalian single-stranded DNA binding protein 1 (SSB1) has been established as an essential component of genome stability in both human cells and mice. Moreover, SSB1 was recently implicated in cytoplasmic stress response by its involvement in Ras GTPase-activating protein-binding protein 1 (G3BP1)-containing cytoplasmic stress granules (SGs) upon various forms of stress. Here, we generated and analyzed human cellular knockout and rodent ischemia-reperfusion (I/R) models to define SSB1s roles in cytoplasmic stress response. Analysis of wild-type as well as SSB1 and G3BP1 knockout human retinal pigment epithelial (RPE-1) cells shows stress-specific incorporation of SSB1 into SGs and a negative regulator role for SSB1 in SG dynamics under sublethal stress conditions. We find that SSB1 knockout measurably increases cellular sensitivity to oxidative stress but does not alter cell proliferation following mild acute stress. Moreover, we detect SSB1 efflux from the nucleus upon stress that is dependent upon the presence of G3BP1 in a stress-specific manner. In addition, using mouse and rat models we observe significant upregulation and robust cytoplasmic granulation of SSB1 upon renal ischemia-reperfusion stress, establishing SSB1s involvement in complex organismal stress response in vivo. Together, our data demonstrate active involvement of SSB1 in cytoplasmic response to cellular stress and acute kidney injury, with implications for targeting stress response functions in cancerous versus non-cancerous contexts. HIGHLIGHTSO_LISSB1 is incorporated into cytoplasmic stress granules and negatively regulates stress granule assembly under sublethal stress conditions C_LIO_LISSB1 shows stress- and G3BP1-dependent nuclear efflux C_LIO_LISSB1 is upregulated and undergoes apical granulation in renal epithelial cells during renal ischemia-reperfusion injury C_LI

cell biology↗

bioRxiv2025

Enhanced AkaLuc Bioluminescence Imaging for Longitudinal Intravital Monitoring of Minimal Residual Disease in a Murine Model of Triple-negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive form of cancer with poor prognosis. Beyond the absence of targeted therapies, a major challenge is its high recurrence rate, driven by the outgrowth of residual tumor cells that survive chemotherapy and persist during minimal residual disease (MRD). To monitor the therapy response of TNBC by enhanced intravital imaging, we established a clinically relevant combination chemotherapy protocol for the treatment of mouse mammary tumors engrafted from K14cre;Brca1F/F;Trp53F/F (KB1P) organoids engineered to express an mCherry-AkaLuc dual reporter (mCA-KB1P). Reproducible MRD and relapse response patterns with significantly extended relapse-free survivals were achieved with the TAC protocol, consisting of docetaxel, doxorubicin and cyclophosphamide. AkaLuc bioluminescence imaging (AkaBLI) of mCA-KB1P organoids verified the single-cell sensitivity of the system in vitro, and showed a detection limit of approximately 1000 cells in the mammary gland of living mice. Unexpectedly, mCA-KB1P organoids elicited an immune response, which necessitated the use of immunodeficient hosts for the longitudinal intravital monitoring of MRD. AkaBLI and an adapted TAC protocol enabled, for the first time, the non-invasive intravital tracking and an estimation of the number of surviving tumor cells in the MRD state following intensive chemotherapy. Engineering KB1P organoids for Histon2B-mCherry reporter expression (HmC-KB1P) enabled the estimation of tumor cell survival also in syngeneic immunocompetent hosts. Flow cytometry and histological analysis revealed that immunocompetent hosts harbored only a few residual cells at MRD, which exhibited a transient loss of epithelial characteristics, whereas immunodeficient hosts had a greater number of surviving cells with a maintained epithelial phenotype. These findings are consistent with the immune systems role in shaping phenotypic changes influencing survival following chemotherapy Together, the results demonstrate the utility of the AkaBLI system for rare tumor cell tracking and highlight the immune systems role in triggering adaptive responses to chemotherapy.

cancer biology↗

bioRxiv2023

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of E. coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2s C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

biochemistry↗

bioRxiv2023

Redox-dependent condensation and cytoplasmic granulation by human ssDNA binding protein 1 delineate roles in oxidative stress response

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in the repair of DNA breaks and oxidized DNA lesions. Here we show that hSSB1 undergoes liquid-liquid phase separation (LLPS) that is redox-dependent and requires the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions, in response to treatment resulting in cellular oxidative stress. hSSB1s intrinsically disordered region (IDR) is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding (OB) fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we also detected hSSB1 condensates in the cytoplasm in response to oxidative stress in various cell lines. hSSB1 condensates colocalize with stress granules, implying unexplored extranuclear roles in cellular stress response. Our results suggest novel, condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.

biochemistry↗

bioRxiv2022

Nucleotide binding is the critical regulator of ABCG2 conformational transitions

ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

biochemistry↗