Isoforme | EPFL Graph Search

Dans le domaine de la biologie cellulaire, les isoformes d'une protéine sont les différentes formes qu'elle prend lorsqu'elle est issue d'un même gène. Le processus impliqué à la formation des isoformes s'appelle épissage alternatif. Principes Les protéines isoformes issues de la traduction d'un même gène ont des séquences peptidiques proches, mais se différencient par l'ajout ou la perte d'une partie de leurs séquences en acides aminés. De même, une modification de la séquence peut même avoir lieu dans le cas d'un décalage du cadre de lecture (frameshift). Ces phénomènes sont la plupart du temps dû à un épissage alternatif qui supprime ou ajoute la traduction d'un ou plusieurs exon(s) du gène considéré. Elles peuvent aussi résulter de l'expression de plusieurs gènes homologues dont leurs séquences ont divergé (allèle). De ce fait, la fonction biologique de ces différentes isoformes peut être légèrement différente en fonction de la modification de séquence. Les

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

Deciphering the astrocyte-neuron metabolic cooperation in vivo

Manuel Raphael Zenger

Brain energy consumption is high and a very tightly regulated energy metabolism ensures correct performance. Neurons, the cells responsible for transmitting information via synapses, interact very closely with star shaped glial cells called astrocytes. Astrocytes play a pivotal role in the regulation of brain energy metabolism and for neurotransmission. Especially, in the case of glutamate, the main excitatory neurotransmitter in the brain, astrocytes are contributing to the clearing of otherwise potentially toxic levels of glutamate in the synaptic cleft. In addition, astrocytes are capable of providing neurons with lactate, which can be used as an additional source of energy in periods of increased demand, such as during neurotransmission. Lactate released by astrocytes is also a signal for neuronal plasticity. In this work, we addressed different aspects of the astrocyte-neuron metabolic coupling. Uncoupling proteins (UCPs) are proteins of the inner mitochondrial membrane that are capable of dissipating a proton gradient (uncoupling ATP production from respiration). Five different UCP isoforms have been described. UCP1 is essentially found in brown adipose tissue and is implicated in non-shivering thermogenesis, UCP2 is widely expressed and protecting against reactive oxygen species and UCP3 is mainly expressed in skeletal muscle tissue where a possible role in fatty acid metabolism has been described. UCP4 and 5 are essentially expressed in the brain, but their precise function still needs to be determined. In the present study, we showed that in neurons, UCP4 and UCP5 are predominantly expressed, as compared to the other UCPs. In astrocytes, UCP5 is the most abundant isoform, but also UCP2 and 4 are significantly expressed indicating an important role for these three UCPs in astrocytes. The role of UCP4 was further elucidated. For this purpose, a lentiviral overexpressing UCP4 construct was produced and characterized. This construct overexpressing UCP4 at the mitochondria allowed to demonstrate a neuro-protective role of UCP4 expression in astrocytes. In a complementary approach to study neuron-astrocyte metabolic coupling, the role of different astrocytic and neuronal genes in memory and learning was explored in a spatial learning paradigm. Mice were trained 1 to 9 days in a radial maze. The gene analysis showed that genes involved in lactate production and utilization such as lactate dehydrogenases were highly increased. A gene involved in the shuttling of lactate from astrocytes to neurons, such as monocarboxylate transporter 1 was also upregulated. An unexpected decrease in glucose transporter 1 and decrease in hexokinase 2 expression were observed. Finally, genes involved in glycogen metabolism were upregulated. Na+/K+-ATPase alpha2 isoform (ATP1A2), which plays a key role in re-establishing Na+ homeostasis disrupted by the co-transport of Na+ along with glutamate during glutamate uptake, was upregulated following spatial learning. Using a lentiviral transgenesis method, an attempt was made to downregulate the expression of the ATP1A2 in vivo. However, this technique did not allow us to produce sufficient knock down to study the role of ATP1A2 in vivo. Preliminary data for an alternative method are shown. Overall, the set of results presented here provides additional support for the existence of a dynamically regulated neuron-astrocyte metabolic coupling.

EPFL2015