Sérine/thréonine protéine kinase

A serine/threonine protein kinase () is a kinase enzyme, in particular a protein kinase, that phosphorylates the OH group of the amino-acid residues serine or threonine, which have similar side chains. At least 350 of the 500+ human protein kinases are serine/threonine kinases (STK). In enzymology, the term serine/threonine protein kinase describes a class of enzymes in the family of transferases, that transfer phosphates to the oxygen atom of a serine or threonine side chain in proteins. This process is called phosphorylation. Protein phosphorylation in particular plays a significant role in a wide range of cellular processes and is a very important posttranslational modification. The chemical reaction performed by these enzymes can be written as ATP + a protein ADP + a phosphoprotein Thus, the two substrates of this enzyme are ATP and a protein, whereas its two products are ADP and phosphoprotein. The systematic name of this enzyme class is ATP:protein phosphotransferase (non-specific). Serine/threonine kinases play a role in the regulation of cell proliferation, programmed cell death (apoptosis), cell differentiation, and embryonic development. While serine/threonine kinases all phosphorylate serine or threonine residues in their substrates, they select specific residues to phosphorylate on the basis of residues that flank the phosphoacceptor site, which together comprise the consensus sequence. Since the consensus sequence residues of a target substrate only make contact with several key amino acids within the catalytic cleft of the kinase (usually through hydrophobic forces and ionic bonds), a kinase is usually not specific to a single substrate, but instead can phosphorylate a whole "substrate family" which share common recognition sequences. While the catalytic domain of these kinases is highly conserved, the sequence variation that is observed in the kinome (the subset of genes in the genome that encode kinases) provides for recognition of distinct substrates.

An Oculus to Profile and Probe Target Engagement In Vivo: How T-REX Was Born and Its Evolution into G-REX

Yimon Aye, Chloé Rogg

Here we provide a personal account of innovation and design principles underpinning a method to interrogate precision electrophile signaling that has come to be known as "REX technologies". This Account is framed in the context of trying to improve methods of target mining and understanding of individual target-ligand engagement by a specific natural electrophile and the ramifications of this labeling event in cells and organisms. We start by explaining from a practical standpoint why gleaning such understanding is critical: we are constantly assailed by a battery of electrophilic molecules that exist as a consequence of diet, food preparation, ineluctable endogenous metabolic processes, and potentially disease. The resulting molecules, which are detectable in the body, appear to be able to modify function of specific proteins. Aside from potentially being biologically relevant in their own right, these labeling events are essentially identical to protein-covalent drug interactions. Thus, on what proteins and even in what ways a covalent drug will work can be understood through the eyes of natural electrophiles; extending this logic leads to the postulate that target identification of specific electrophiles can inform on drug design. However, when we entered this field, there was no way to interrogate how a specific labeling event impacted a specific protein in an unperturbed cell. Methods to evaluate stoichiometry of labeling, and even chemospecificity of a specific phenotype were limited. There were further no generally accepted ways to study electrophile signaling that did not hugely disturb physiology. We developed T-REX, a method to study single-protein-specific electrophile engagement, to interrogate how single-protein electrophile labeling shapes pathway flux. Using T-REX, we discovered that labeling of several proteins by a specific electrophile, even at low occupancy, leads to biologically relevant signaling outputs. Further experimentation using T-REX showed that in some instances, single-protein isoforms were electrophile responsive against other isoforms, such as Akt3. Selective electrophile-labeling of Akt3 elicited inhibition of Akt-pathway flux in cells and in zebrafish embryos. Using these data, we rationally designed a molecule to selectively target Akt3. This was a fusion of the naturally derived electrophile and an isoform-nonspecific, reversible Akt inhibitor in phase-II trials, MK-2206. The resulting molecule was a selective inhibitor of Akt3 and was shown to fare better than MK-2206 in breast cancer xenograft mouse models. Recently, we have also developed a means to screen electrophile sensors that is unbiased and uses a precise burst of electrophiles. Using this method, dubbed G-REX, in conjunction with T-REX, we discovered new DNA-damage response upregulation pathways orchestrated by simple natural electrophiles. We thus emphasize how deriving a quantitative understanding of electrophile signaling that is linked to thorough and precise mechanistic studies can open doors to numerous medicinally and biologically relevant insights, from gleaning better understanding of target engagement and target mining to rational design of targeted covalent medicines.

AMER CHEMICAL SOC2021

Fluorogenic probes for live-cell imaging

Aleksandar Salim

The development of novel fluorogenic probes and their use in live-cell imaging lead to a plethora of discoveries in biological research. Herein I report on novel red fluorogenic probes for live-cell super-resolution microscopy of various cellular organelles. First, I describe a new silicon rhodamine fluorophore termed SiR595. As most other SiR derivatives, SiR595 exists in an equilibrium between two chemical forms, a fluorescent zwitterion and a non-fluorescent spirolactone. Compared to regular SiR, the equilibrium of SiR595 is shifted towards the non-fluorescent, uncharged spirolactone. When coupled to appropriate targeting ligands, the resulting SiR595-based probes show high cell permeability. Furthermore, SiR595-based probes are fluorogenic as binding to their targets shifts the equilibrium towards the fluorescent zwitterion. I took advantage of these properties to develop novel red fluorogenic probes for live-cell imaging of Halo-tagged proteins, microtubules, F-actin and DNA. I was furthermore able to show that all of these probes are compatible with live-cell STED nanoscopy. Furthermore, I developed fluorogenic probes for live cell imaging of centrosomes. The centrosome is the principal microtubule-organizing center in animal cells. We targeted polo-like kinase 4 (Plk4), a serine/threonine-protein kinase, which localizes to centrioles throughout the cell cycle. To label Plk4 in live cells, we used Centrinone, a potent inhibitor of Plk4. Conjugating Centrinone to SiR595, I obtained and characterized several fluorogenic SiR595-Centrinone probes. SiR595-Centrinone probe labels overexpressed GFP-Plk4(K41M) during live-cell imaging and could be used to characterize the spatial distribution of GFP-Plk4(K41M) using live-cell STED nanoscopy. While the here introduced probes for Plk4 are not bright and fluorogenic enough to detect endogenous Plk4, the work provides the foundation for the development of such probes.

EPFL2020

Physiological regulation of the CDK16/PCTAIRE-1 protein kinase and its proposed role in the brain

Saifeldin Nasser Mohamed Ibrahim Shehata

Cell signalling, mediated to a large extent by protein kinase phosphorylation, plays a vital role in regulation of cellular function. PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK)16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, apoptosis, insulin secretion, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been found in patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only few substrates have been proposed with no clear functional significance and physiological role. To understand the function of PCTAIRE-1, we previously utilised a peptide library screen to determine the consensus phosphorylation motif. We showed that PCTAIRE-1 preferentially phosphorylated peptide motifs that differed from the classical CDK family substrate preference, suggesting a more distinct role for the kinase. Furthermore, we showed that cyclin Y, a novel cyclin, robustly binds and activates PCTAIRE-1 > 100-fold. In this thesis, we have identified two phosphorylation sites on cyclin Y that are essential for binding the well-known adaptor protein 14-3-3, which we propose stabilizes cyclin Y in a favourable PCTAIRE-1-binding conformation. Mutation of these sites to non-phosphorylatable Ser residues abolished PCTAIRE-1 binding and activation. Furthermore, we have cloned human PCTAIRE-1 mutants identified in XLID patients, and confirmed their failure to bind the cyclin Y-14-3-3 activating complex. In order to understand the physiological relevance of PCTAIRE-1 activity, we have utilised a chemical genetics approach that exploits the ability of an engineered PCTAIRE-1 mutant to selectively modify its substrates, allowing them to be uniquely purified. We have identified three PCTAIRE-1 substrates (AAK1, dynamin 1 and synaptojanin 1) in mouse brain that regulate crucial steps of receptor endocytosis, namely receptor binding, vesicle scission and vesicle uncoating, which are all involved in the regulation of neuronal synaptic transmission. Collectively, this thesis work has provided key molecular regulatory mechanisms and potential downstream targets of PCTAIRE-1, which has laid the foundations for future studies of the role that PCTAIRE-1 plays in specific cellular functions and physiological and pathological settings.

EPFL2017