Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivative

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives

RhoA Regulates Peroxisome Association to Microtubules and the Actin Cytoskeleton

Peer reviewe

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template

Nuclear Progesterone Receptors Are Up-Regulated by Estrogens in Neurons and Radial Glial Progenitors in the Brain of Zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Degradation studies and directed evolution of the human O6-alkylguanine-DNA-alkyltransferase

A new technology for the specific and covalent labelling of fusion proteins with small organic molecules in vivo and in vitro has recently been developed in our laboratory. The method is based on the genetically encoded fusion of a target protein to O6-alkylguanine- DNA-alkyltransferase (hAGT). hAGT irreversibly and covalently transfers the benzyl group of O6-benzylguanine (BG) to its reactive cysteine and is subsequently degraded in vivo afterwards. hAGT possesses specific activity towards BG-based compounds which are otherwise chemically inert and have no other reaction partners in the cellular environment. This property allows for the labelling of hAGT fusion proteins with BG-fluorescein compounds inside the living cell. Mutants of hAGT that overcome several limitations of the wild-type protein such as binding to DNA, low activity, unfavourable folding properties under oxidising conditions and reaction with endogenous AGT have been generated. One mutant (named MAGT) was generated that combines most of these features with the exception of a high reactivity. In the course of this project the activity of this mutant was increased using directed molecular evolution: the phage display technology was applied to select for MAGT based highly reactive mutants. The final mutant (named SMAGT) exhibits a two-old higher activity against BG derivatives than the fastest so far described hAGT and relative to its predecessor MAGT a 16-fold higher activity. Furthermore, SMAGT combines all the described positive features of MAGT. The mutant was subsequently used for fluorescence labelling experiments on the cell surface of mammalian cells: a highly specific labelling was obtained after short incubation times with low substrate concentrations. A previously used hAGT mutant engineered only with respect to activity gave no fluorescence signal under the same conditions. SMAGT thus extends the range of possible applications of the hAGT technology. It is reported that hAGT is degraded by the ubiquitin proteasome pathway after reaction with BG substrates. Relatively few data are available concerning the post-alkylation fate of hAGT and no data at all about the in vivo stability of alkylated hAGT fusion proteins. Therefore in another part of this work the in vivo stability of hAGT fusion proteins in mammalian cells and yeast was examined: it was demonstrated that the degradation of hAGT after alkylation is strongly dependent on the C-terminal fusion partner of the protein. Furthermore it was shown that the addition of degradation signals to the hAGT (such as a N-end rule degron) can enhance the BG-induced degradation in vivo. These experiments are the first steps towards the generation of a hAGT with optimised degradation properties. Such a protein would be suitable to generate a system for the BG-induced knock-out of hAGT fusion proteins. Gene- and protein knock-out systems are powerful tools to study the physiological function of a protein in an organism. It would be highly desirable to have a system that allows the conditional knock-out of a target protein induced by an external stimulus such as a small molecule that can be used in higher organisms like mice