Biology subjects

Peter, M.

Publications and source records attributed to Peter, M..

6 recordsLinked to original sources

bioRxiv2018

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and non-enzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, decreased viability, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

biochemistry↗

bioRxiv2018

6mer Seed Toxicity Determines Strand Selection in miRNAs

Many siRNAs and shRNAs are toxic to cancer cells through a 6mer seed sequence (position 2-7 of the guide strand). A siRNA screen with all 4096 possible 6mer seed sequences in a neutral RNA backbone revealed a preference for guanine in positions 1-3 and a GC content of >80% of the 6mer seed in the most toxic siRNAs. These 6mer seed containing siRNAs exert their toxicity by targeting survival genes which contain GC-rich 3UTRs. The master tumor suppressor miRNA miR-34a was found to be toxic through such a G-rich 6mer seed suggesting that certain tumor suppressive miRNAs use a toxic 6mer seed to kill cancer cells. An analysis of all mature miRNAs suggests that most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the predominantly expressed arm. In contrast, for many tumor suppressive miRNAs the predominant arm contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.

cell biology↗

bioRxiv2017

Induction of DISE in ovarian cancer cells in vivo

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nano particles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

cancer biology↗

bioRxiv2017

Identification of DISE-inducing shRNAs by monitoring cellular responses

Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si-or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.

cancer biology↗

bioRxiv2017

CD95L mRNA And CD95L Derived si- and shRNAs Kill Cancer Cells Through An RNAi Mechanism By Targeting Survival Genes

Over 80% of multiple tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that using specific toxic RNAi-active sequences present in the genome can kill cancer cells.

cancer biology↗

bioRxiv2017

Filopodyan: An Open-Source Pipeline For The Analysis Of Filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.\n\neTOC summary: Urban[c]i[c] et al. developed an open-source platform called Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia, at their tips, and bases, concurrently with their morphological and dynamic properties. This customizable tool therefore enables researchers to determine the relationship between protein localization and filopodium behavior.

cell biology↗