CIISB Research Highlights Archive

Dendrite-shaped hematite microrobots have been developed as an effective GSH depletion agent for photodynamic therapy (PDT) of prostate cancer cells. These single-component microrobots with dual light/magnetic field actuation can induce GSH depletion, greatly enhancing in vitro PDT performance and accomplishing the non-contact transportation of micro-sized objects.

Martin Pumera Research Group

Significance

Photocatalytic micromotors that exhibit wireless and controllable motion by light have been extensively explored for cancer treatment by photodynamic therapy (PDT). However, overexpressed glutathione (GSH) in the tumor microenvironment can down-regulate the reactive oxygen species (ROS) level for cancer therapy. Herein, we present dendrite-shaped light-powered hematite microrobots as an effective GSH depletion agent for PDT of prostate cancer cells. These hematite microrobots can display negative phototactic motion under light irradiation and flexible actuation in a defined path controlled by an external magnetic field. Non-contact transportation of micro-sized cells can be achieved by manipulating the microrobot's motion. In addition, the biocompatible microrobots induce GSH depletion and greatly enhance PDT performance. The proposed dendrite-shaped hematite microrobots contribute to developing dual light/magnetic field-powered micromachines for the biomedical field.

Xia Peng, Urso, M., Balvan, J., Masařík, M., and Pumera, M.:

Self-Propelled Magnetic Dendrite-Shaped Microrobots for Photodynamic Prostate Cancer Therapy, Angew. Chem. Int. Ed. 2022, on-line version e202213505, https://doi.org/10.1002/anie.202213505

a Surface representation of composite cryo-EM map of virion of phage SU10 colored according to protein type. The major capsid protein (gp9) is shown in turquoise, portal protein (gp6) in magenta, adaptor protein (gp11) in yellow, long tail fibers (gp12) in violet, nozzle protein (gp17) in red, short tail fibers (gp16) in green, and tail needle (gp18) in light blue. The length of the virion is 1590 Å. For details on the construction of the composite map, please see the Materials and methods section. b The same as A, but the front half of the composite map of the SU10 head was removed to show the structure of the genome in grey. The inset shows a 2D class average of the SU10 virion. The scale bar indicates 45 nm. c Composite cryo-EM map of portal and tail complexes of SU10 virion. The length of the complex is 540 Å. d Cryo-EM reconstruction of portal and tail complexes from an SU10 genome release intermediate. e Composite cryo-EM map of genome-release intermediate of SU10. The front half of the head was removed to show the structure of the genome remaining in the capsid. The inset shows a 2D class average of the SU10 genome release intermediate. f Schematic representation of segment of SU10 genome encoding structural proteins color-coded the same as the proteins in panels a to e. Proteins shown in white are either non-structural or were not identified in the reconstructions.

Pavel Plevka Research Group

Significance

Escherichia coli phage SU10 belongs to the genus Kuravirus from the class Caudoviricetes of phages with short non-contractile tails. In contrast to other short-tailed phages, the tails of Kuraviruses elongate upon cell attachment. Here we show that the virion of SU10 has a prolate head, containing genome and ejection proteins, and a tail, which is formed of portal, adaptor, nozzle, and tail needle proteins and decorated with long and short fibers. The binding of the long tail fibers to the receptors in the outer bacterial membrane induces the straightening of nozzle proteins and rotation of short tail fibers. After the re-arrangement, the nozzle proteins and short tail fibers alternate to form a nozzle that extends the tail by 28 nm. Subsequently, the tail needle detaches from the nozzle proteins and five types of ejection proteins are released from the SU10 head. The nozzle with the putative extension formed by the ejection proteins enables the delivery of the SU10 genome into the bacterial cytoplasm. It is likely that this mechanism of genome delivery, involving the formation of the tail nozzle, is employed by all Kuraviruses.

Šiborová, M., Füzik, T., Procházková,M., Nováček, J., Benešík, M., Nilsson, A.S., and Plevka, P.:

Tail proteins of phage SU10 reorganize into the nozzle for genome delivery, Nature Comm. (2022)13:5622,https://doi.org/10.1038/s41467-022-33305-w

a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC₅₀ = 353 nM and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPII^CTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Dalibor Blažek Research Group

Significance

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex^. Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein—is phosphorylated during spliceosome activation, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B^act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B^act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Hluchý, M., Gajdušková, P., Ruiz de los Mozos, I., Rájecký, M., Kluge, M., Berger, B-T., Slabá, Z., Potěšil, D., Weiß, E., Ule, J., Zdráhal, Z., Knapp, S., Paruch, K., Friedel, C.C., and Blažek, D.:

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, Nature. (2022) 609, 829-834. https://doi.org/10.1038/s41586-022-05204-z

a)Ribbon diagram of the NKR-P1 CTLD. Secondary structure elements are labeled in different colors: helix α1 is red, helix α2 is yellow, and β-strands and loops are cyan. b)Comparison between NKR-P1 dimers formed by the glycosylated (cyan), deglycosylated free (green), and LLT1-bound (blue) forms of NKR-P1. c) Comparison between helices α1- and α2-centered dimerization of murine dectin-1 (magenta) and human LLT1 (green), respectively; helices α1 and α2 are shown in red and yellow. Structural alignments of dectin-1 and NKR-P1 homodimers and LLT1 and NKR-P1 homodimers, prepared by aligning only one monomer from each dimer, are shown on the right-hand side. Although the CTLD fold is conserved in each pair of the aligned monomers, the helix α1- and helix α2-centered dimers show inverse arrangement.

Ondřej Vaněk Research Group

Significance

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Blaha, J., Skalova, T. Kalouskova, B., Skorepa, O., Cmunt, D., Grobarova, V.,Pazicky, S., Polachova, E., Abreu, C., Stransky, J., Koval, T., Duskova, J.,Zhao, Y.,Harlos, K., Hasek, J. Dohnalek, J., and Vanek, O.:

Structure of the human NK cell NKR-P1:LLT1 receptor: ligand complex reveals clustering in the immune synapse, Nature Comm. (2022)13:5022, https://doi.org/10.1038/s41467-022-32577-6

Model for cooperation between the intrinsically disordered and structured regions of Spt6 which regulates nucleosome and Pol II CTD binding.

Richard Štefl Research Group

Significance

Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6’s function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.

Kasiliauskaite, A., Kubicek, K., Klumpler, T., Zanova, M., Zapletal, D., Koutna, E., Novacek, J., and Stefl. R.: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly, Nucleic Acids Res. 2022, 50 (10), 5961–5973, https://doi.org/10.1093/nar/gkac451

Cryo-electron density map of CV-A6 virion (a), altered particle (b), and empty particle (c), viewed along the twofold icosahedral symmetry axis, coloured according to distance from centre of particle. The insets show central slices through the particle interior (central slice, thickness 5 Å). Virion and altered particle contain genomic RNA (a, b). Positions of icosahedral symmetry axes are indicated by dashed lines in a. Scale bar 5 nm.

Pavel Plevka Research Group

Significance

Coxsackievirus A6 (CV-A6) has recently overtaken enterovirus A71 and CV-A16 as the primary causative agent of hand, foot, and mouth disease worldwide. Virions of CV-A6 were not identified in previous structural studies, and it was speculated that the virus is unique among enteroviruses in using altered particles with expanded capsids to infect cells. In contrast, the virions of other enteroviruses are required for infection. Here we used cryo-electron microscopy (cryo-EM) to determine the structures of the CV-A6 virion, altered particle, and empty capsid. We show that the CV-A6 virion has features characteristic of virions of other enteroviruses, including a compact capsid, VP4 attached to the inner capsid surface, and fatty acid-like molecules occupying the hydrophobic pockets in VP1 subunits. Furthermore, we found that in a purified sample of CV-A6, the ratio of infectious units to virions is 1 to 500. Therefore, it is likely that virions of CV-A6 initiate infection, like those of other enteroviruses. Our results provide evidence that future vaccines against CV-A6 should target its virions instead of the antigenically distinct altered particles. Furthermore, the structure of the virion provides the basis for the rational development of capsid-binding inhibitors that block the genome release of CV-A6

Büttner, C.R., Spurný, R., Füzik, T., and Plevka, P.: Cryo-electron microscopy and image classification reveal the existence and structure of the coxsackievirus A6 virio

Commun. Biol. 2022, 5, 898, https://doi.org/10.1038/s42003-022-03863-2