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MolBioChem Talks I: Molecular Biology & Biochemistry: Talks I
8:30am - 9:30am
Session Chair: Prof. Torsten Ochsenreiter
Location:DCB, U113, basement Department of Chemistry and Biochemistry, basement, Freiestrasse 3, 3012 Bern
Presentations T-017 to T-020
8:30am - 8:45am
CRISPR-Trap: A Clean Approach for the Generation of Gene Knockouts and Gene Replacements in Human Cells
Stefan Reber1, Jonas Mechtersheimer1, Sofia Nasif1, Julio Aguila Benitez2, Martino Colombo1, Michal Domanski1, Daniel Jutzi1, Eva Hedlund2, Marc-David Ruepp1
1Department of Chemistry and Biochemistry, University of Bern, Switzerland; 2Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
CRISPR/Cas9-based genome editing offers the possibility to knock out (KO) almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to about 50 % of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to KO several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal KOs. Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.
8:45am - 9:00am
Superoxide Quenching Within the Membrane by E. coli Cytochrome b561
Olivier Biner1, Camilla Lundgren2, Dan Sjöstrand2, Martin Högbom2, Christoph von Ballmoos1
1Department of Chemistry and Biochemistry, University of Bern, Switzerland; 2Department of Biochemistry and Biophysics, University of Stockholm, Sweden
Superoxides are reactive oxygen species that can damage various cellular components such as lipids, proteins, and DNA and are involved in many diseases. Most superoxide is produced in the cytoplasm by flavin containing proteins. To combat this superoxide all life forms that live in an aerobic habitat contain superoxide dismutase (SOD). SOD is a soluble enzyme that eliminates superoxides produced in the bulk. However, superoxides can also be produced within the membrane by respiratory chain complexes. Here, we describe the first protein that can combat superoxides in the membrane.
In the year 1984, the laboratory of Yasuhiru Anraku discovered and purified an integral membrane protein in E. coli that contains two b-type hemes. The protein was called cytochrome b561 (CybB). They further determined its mid-point potential (20 mV) and predicted that it might reduce quinones and be part of the respiratory chain. However, the function of this protein has never been established.
We performed a functional characterisation of E. coli CybB and identified its molecular partners. The protein was reducible by superoxides and quinol and further experiments showed that it can catalyse electron transfer between superoxides and ubiquinone in detergent. This is the first report of an enzyme that catalyses this type of reaction. Experiments with reconstituted enzyme further suggest that CybB mainly reacts with superoxides formed within the membrane, because soluble SOD could not compete with reconstituted CybB. Furthermore, in collaboration with the group of Martin Högbom at Stockholm University we determined the structure of CybB at 2.0 Å resolution. The structure corroborates the biochemical data and shows a quinone binding site as well as a positively-charged pocket that might attract negatively charges superoxides.
To sum up, we describe the first enzyme that can combat superoxides produced within the membrane.
9:00am - 9:15am
Involvement of Epigenetic Factors and Metabolism in Pluripotency Maintenance in C. elegans
Francesca Coraggio, Ringo Pueschel, Alisha Marti, Peter Meister
Institute of Cell Biology, University of Bern, Switzerland
A promising therapy for degenerative diseases is the replacement of the deficient cells by injection of in vitro differentiated progenitors. Differentiation is a crucial step since incompletely differentiated cells could lead to the formation of teratomas. Understanding which signalling pathways and epigenetic determinants maintain and drive cell fate is therefore of prime importance for regenerative medicine.
Using C. elegans and an induced muscle transdifferentiation system, we test cell plasticity in fully differentiated animals. We find that the silent histone mark H3K27 methylation deposited by the MES/Polycomb complex is essential to restrict plasticity in fully differentiated animals. In the absence of this mark, animals completely arrest development upon fate challenge. As most animals arrest development, we used this system to screen for plasticity enhancers, which knock-down would suppress developmental arrest. We find that the Notch signalling pathway can rescue the arrest upon fate challenge, suggesting Notch acts to enhance plasticity in vivo. Surprisingly, we discovered that cell plasticity additionally depends on the metabolic state of the animal: starved animals are insensitive to fate challenges, while fed ones arrest development. We are currently investigating the processes which lead to larval arrest and characterizing the link between metabolism and cell fate plasticity.
9:15am - 9:30am
Non-Conservative Mutation in the Canine Distemper Virus Attachment Protein Disrupts Cell Invasion by Increasing the Intermolecular Interaction
Michael Herren, Marianne Wyss, Neeta Shrestha, Philippe Plattet
Division of Experimental Clinical Research, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Switzerland
Host cell entry by morbilliviruses (e.g. measles virus (MeV) or canine distemper virus (CDV)) is coordinated by two interacting envelope glycoproteins; a tetrameric attachment (H) protein and a trimeric fusion (F) protein. Upon receptor engagement, it is assumed that H- and F-proteins undergo series of conformational changes ultimately leading to membrane merging and fusion pore formation. More specifically, the ectodomain of H-tetramers consists of stalk, connector and head domains that adopt “F-triggering-permissive” native structures. While the central region of the stalk is proposed to interact and activate F, the heads carry the receptor binding activity. In contrast, the precise functional role of the C-terminal module of the stalk (termed “linker”) and the following connector domain, although hypothesized to assume flexible structures to support putative receptor-induced head-stalk structural rearrangements, remains largely unexplored. In this study, to gain mechanistic insights, we conducted a thorough “non-conservative” mutagenesis-scan analysis of the MeV and CDV H-linker/connector domains. Our data provided evidence that substituting the hydrophobic isoleucine residue encompassed within the linker module (H-I146) into any amino acids with charged chemical properties translated into the assembly of over-stabilized H-tetramers, which correlated with fusion promotion-deficiency. Since H-I146 mutants remained entirely competent in intracellular trafficking, F-interaction, receptor binding activity and activating a highly destabilized F-mutant, our findings suggest that the morbillivirus H-stalk C-terminal linker module requires some structural freedom to enable the generation of fully bioactive H-tetramers. Altogether, our data demonstrate that the H-stalk linker module regulates the folding of loosely-assembling functional H-tetramers. Noteworthy, stabilized H-tetramers may offer useful probes for structural determination and ensuing antiviral drug design.