A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Elucidating the role of WRNIP1 in the protection of stalled replication forks

DNA is the blueprint of life. It contains all the information necessary for the synthesis of proteins – the building blocks and workhorses of the cell. Maintenance of DNA in an unchanged state is therefore of utmost importance, since any mutations or loss of the information it holds may lead to catastrophic events, such as cell death or tumorigenesis. DNA needs to be faithfully copied with every cell division, which is an enormous task, given its size and importance. DNA replication is constantly challenged by endogenous and exogenous factors, which have the potential to stall or stop replication – such a circumstance is called replication stress. While cells have evolved a number of mechanisms to deal with replication stress, e.g. checkpoint signaling that halts the cell cycle, DNA repair pathways that remove obstacles or factors that stabilise stalled replication forks, persisting replication stress inevitably leads to genomic instability and tumorigenesis. Interestingly, cancer cells, due to their unrestricted proliferation, have elevated level of replication stress, which makes them more susceptible to anti-cancer therapies that exacerbate genomic instability. It is thus obvious that dissecting the events and mechanisms leading to and following replication stress is important from a basic science and clinical research point of view. Among the many aspects of replication stress, one that is gaining an increasing amount of attention is replication fork stability. Over the last years it was shown that a stalled replication fork is rapidly remodeled into a 4-way structure, an event that is believed to contribute to its stabilisation. However, such a structure needs to be carefully maintained, since lack of its protection may cause an unscheduled processing by nucleases, leading to genomic instability. Interestingly, among the factors that protect stalled replication forks are many proteins previously associated with other DNA repair pathways, such as homologous recombination or Fanconi anaemia. The most recent member of the ’protectosome’ group is WRNIP1 (Werner helicase interacting protein 1), but its mode of action is unclear. In this project, we attempted to characterise WRNIP1’s biochemical activities and investigate further its cellular function. We have found that the protein exerts its protective function downstream of replication fork reversal, challenging the current model. Our in vitro data show that WRNIP1 binds specifically to 4-way DNA junctions, a structure resembling a reversed replication fork. We have also found that WRNIP1 interacts directly with the replication fork remodeler ZRANB3 and that it is able to limit its replication fork reversal activity in vitro. Combined with published data, our data led us to propose a mechanism in which WRNIP1 binds to reversed replication forks immediately after their generation, thus protecting them against unscheduled MRE11-dependent degradation

WRNIP1 Protects Reversed DNA Replication Forks from SLX4-Dependent Nucleolytic Cleavage

During DNA replication stress, stalled replication forks need to be stabilized to prevent fork collapse and genome instability. The AAA + ATPase WRNIP1 (Werner Helicase Interacting Protein 1) has been implicated in the protection of stalled replication forks from nucleolytic degradation, but the underlying molecular mechanism has remained unclear. Here we show that WRNIP1 exerts its protective function downstream of fork reversal. Unexpectedly though, WRNIP1 is not part of the well-studied BRCA2-dependent branch of fork protection but seems to protect the junction point of reversed replication forks from SLX4-mediated endonucleolytic degradation, possibly by directly binding to reversed replication forks. This function is specific to the shorter, less abundant, and less conserved variant of WRNIP1. Overall, our data suggest that in the absence of BRCA2 and WRNIP1 different DNA substrates are generated at reversed forks but that nascent strand degradation in both cases depends on the activity of exonucleases and structure-specific endonucleases

New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens

The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline-dependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells

Prolonged estrogen deprivation triggers a broad immunosuppressive phenotype in breast cancer cells

Among others, expression levels of programmed cell death 1 ligand 1 (PD-L1) have been explored as biomarkers of the response to immune checkpoint inhibitors in cancer therapy. Here, we present the results of a chemical screen that interrogated how medically approved drugs influence PD-L1 expression. As expected, corticosteroids and inhibitors of Janus kinases were among the top PD-L1 downregulators. In addition, we identified that PD-L1 expression is induced by antiestrogenic compounds. Transcriptomic analyses indicate that chronic estrogen receptor alpha (ER alpha) inhibition triggers a broad immunosuppressive program in ER-positive breast cancer cells, which is subsequent to their growth arrest and involves the activation of multiple immune checkpoints together with the silencing of the antigen-presenting machinery. Accordingly, estrogen-deprived MCF7 cells are resistant to T-cell-mediated cell killing, in a manner that is independent of PD-L1, but which is reverted by estradiol. Our study reveals that while antiestrogen therapies efficiently limit the growth of ER-positive breast cancer cells, they concomitantly trigger a transcriptional program that favors their immune evasion.De 2 sista författarna delar sistaförfattarskapet.</p