Gonzalez-Leal, C., and Ladurner, A.G.
Mol Cell, 2021, 81, 1367-1369.
A triskelion of nucleic acids drives protein aggregation in A-T
Mutations in ataxia telangiectasia mutated (ATM) kinase lead to cerebellar neurodegeneration. In this issue of Molecular Cell, Lee et al. (2021) revealed how transcription-induced reactive oxygen species and DNA-RNA hybrids activate PARP enzymes, generating the nucleic acid poly-ADP-ribose, which promotes the accumulation of protein aggregates in A-T-like disorders.
Bauernfried, S., and Hornung, V.
Nat Struct Mol Biol, 2021, 28, 333-336.
DPP9 restrains NLRP1 activation
NLRP1 was the first inflammasome-forming sensor to be identified, but only recently has its mode of action been in the spotlight. Two groups now report cryo-EM structures demonstrating how NLRP1 is kept in check by the dipeptidyl peptidase DPP9, and they illuminate how DPP9 inhibition leads to NLRP1 inflammasome activation.
Researchers at the Max Planck Institute (MPI) of Biochemistry show that increased salt consumption has no negative effect on disease progression but is rather beneficial in a mouse model of multiple sclerosis.
Multiple sclerosis (MS) is a chronic inflammatory disease of the nervous system. In this autoimmune disease, the myelin sheath of the nerve cells is attacked by the patient's own immune system. Several animal models are available to study the disease. Researchers at the Max Planck Institute of Biochemistry have now been able to show, contrary to the results of other studies, that moderately increased salt consumption in mice has no negative effect on the course of the disease. In transgenic mice that develop spontaneous MS-like disease, increased salt consumption led to a suppression of the disease. This study was published in the journal PNAS.
Sodium chloride, table salt, is an essential mineral that we must consume for a healthy life. However, excessive salt consumption is one of the known health risks, as it has been linked to cardiovascular and kidney diseases. Researchers are also interested in understanding the effect of excessive salt consumption in autoimmune and inflammatory diseases such as MS. Therefore, an animal model of multiple sclerosis called Experimental Autoimmune Encephalomyelitis (EAE) has been used in the past to study the effect of excessive salt consumption. It has been reported that it leads to exacerbation of the disease.
Bartoschek, M.D., Ugur, E., Nguyen, T.A., Rodschinka, G., Wierer, M., Lang, K., and Bultmann, S.
(IMPRS-LS students are in bold)
Nucleic Acids Res, 2021, online ahead of print.
Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells
The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
Peritore, M., Reusswig, K.U., Bantele, S.C.S., Straub, T., and Pfander, B.
Mol Cell, 2021, online ahead of print.
Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes
In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.
Zeitler, L., Fiore, A., Meyer, C., Russier, M., Zanella, G., Suppmann, S., Gagaro, M., Sidhu, S.S., Seshagiri, S., Ohnmacht, C., Köcher, T., Fallarino, F., Linkermann, A., and Murray, P.J.
Elife, 2021, 10, online ahead of print.
Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism
Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologues in snake venoms (LAAO, L-amino acid oxidases), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell productive gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.
Mengoli, V., Jonak, K., Lyzak, O., Lamb, M., Lister, L.M., Lodge, C., Rojas, J., Zagoriy, I., Herbert, M., and Zachariae, W.
(IMPRS-LS students are in bold)
EMBO J, 2021, e106812, online ahead of print.
Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension
Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.
Researchers at the Max Planck Institute of Biochemistry have established a new technique based on Next Generation Sequencing that determines whether a protein binds to single-stranded or double-stranded DNA.
DNA in our cells is constantly exposed to damaging agents, such as UV light or byproducts of cellular metabolism like reactive oxygen species. The most severe form of DNA damage are DNA double stranded breaks. In order to keep the genetic information intact, cells need to repair these double stranded breaks. The most faithful repair process is homologous recombination, which involves DNA end resection, the degradation of one DNA strand at each side of the break. Two different domains are therefore created around the double strand breaks: one containing single-stranded DNA and one containing double-stranded. Researchers from the team of Boris Pfander, head of the research group "DNA Replication and Genome Integrity" at the Max Planck Institute of Biochemistry have now developed a ChIP-seq-technique, based on chromatin-immunoprecipitation followed by next-generation sequencing.