Biology subjects

Gebel, J.

Publications and source records attributed to Gebel, J..

5 recordsLinked to original sources

bioRxiv2024

Potent photoswitch for expression of biotherapeutics in mammalian cells by light

AbstractPrecise temporal and spatial control of gene expression is of great benefit for the study of specific cellular circuits and activities. Compared to chemical inducers, light-dependent control of gene expression by optogenetics achieves a higher spatial and temporal resolution. This could also prove decisive beyond basic research for manufacturing difficult-to-express proteins in pharmaceutical bioproduction. However, current optogenetic gene-expression systems limit this application in mammalian cells as expression levels and fold induction upon light stimulation are not sufficient. To overcome this limitation, we designed a photoswitch by fusing the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter litoralis to the three tandem transcriptional activator domains VP64, p65, and Rta. The resultant photoswitch, dubbed DEL-VPR, allows an up to 400-fold induction of target gene expression by blue light, achieving expression levels that surpass those for strong constitutive promoters. Here, we utilized DEL-VPR to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression, increasing the yield of functional protein complexes. Our approach offers temporally controlled yet strong gene expression and applies to both academic and industrial settings.

molecular biology↗

bioRxiv2022

VASH1/2 inhibition accelerates functional recovery of injured nerves

Treatments accelerating axon regeneration in the nervous system are still unavailable in the clinic. However, in culture, parthenolide markedly promotes adult sensory neurons axon growth by inhibiting microtubule detyrosination. Here, we show that overexpression of vasohibins increases microtubule detyrosination in growth cones and compromises growth in culture and in vivo. Moreover, overexpression of these proteins increases the required parthenolide concentrations to promote axon regeneration, while the knockdown of vasohibins or their enhancer SVBP abolishes parthenolides effects, verifying them as pharmacological targets for promoting axon growth. In vivo, repeated intravenous application of parthenolide or its prodrug di-methyl-amino-parthenolide (DMAPT) markedly facilitates regeneration of sensory, motor, and sympathetic axons in injured murine and rat nerves and accelerates functional re-covery. Moreover, orally applied DMAPT was similarly effective in promoting nerve regeneration. Thus, pharmacological inhibition of vasohibins facilitates axon regeneration in different species and nerves, making parthenolide and DMAPT promising drugs for curing nerve injury.

neuroscience↗

bioRxiv2021

Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein TDP-43, is hyperphosphorylated in disease on several C-terminal serine residues, which is generally believed to promote TDP-43 aggregation. Here, we show that hyperphosphorylation by Casein kinase 1{delta} or C-terminal phosphomimetic mutations surprisingly reduce TDP-43 phase separation and aggregation and render TDP-43 condensates more liquid-like and dynamic. Multi-scale simulations reveal reduced homotypic interactions of TDP-43 low complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We propose that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

biochemistry↗

bioRxiv2019

p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63 is the key regulator of genome integrity in oocytes. After DNA damage TAp63 is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kinetics in vitro with peptides and full length protein we show that TAp63 phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63 substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

biochemistry↗

bioRxiv2018

The washing machine as a reservoir for transmission of ESBL-producing Klebsiella oxytoca in newborns

During the period from April 2012 to May 2013, 13 newborns and one child in a paediatric hospital ward in Germany were found to be colonised with a distinct clinical clone of an (extended spectrum {beta}-lactamase) (ESBL)-producing Klebsiella oxytoca. This clone was specific to this hospital and had not been previously isolated in Germany.\n\nA source-tracking analysis was carried out to identify the source and transmission pathways of the ESBL-producing K. oxytoca clone. A systematic environmental survey of the ward and an audit of the procedures for cleaning and disinfecting surfaces, instruments, incubators, and washing machines was performed. Microbiological samples were obtained from environmental surfaces. Risk factors were analysed for epidemiological linkage.\n\nIsolates of an ESBL-producing K. oxytoca were found in the detergent drawer and on the rubber door seal of a washing machine and in two sinks. These strains were typed by pulsed-field-gel-electrophoresis (PFGE) and compared with the isolates from the newborns and their clothing and found to be identical. The retrospective analysis demonstrated that only newborns who had worn clothes that had been washed in the washing machine were colonised with the identical clone. After the washing machine was taken out of use, no further cases were detected over the following 4-year period.\n\nWe conclude that washing machines are potential reservoirs and vectors for transmission of Enterobacteriaceae, and likely other bacteria.\n\nImportanceWashing machines should be further investigated as possible sites for horizontal gene transfer (ESBL-/carbapenemase-genes) and cross-contamination of clinically important Gram-negative strains. Particularly in the healthcare sector, the knowledge of possible (re-) contamination of laundry (patients clothes, staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and control nosocomial infections. To date, the potential of the washing machine as a source and vector of antibiotic-resistant gram-negative bacteria causing an outbreak in a clinical setting has not been investigated. This report shows that sampling of washing machines should be included in environmental audits associated with outbreak control management, and conditions for the laundering of baby 64 clothing should be reviewed.

microbiology↗