Transmembrane zipper of polar residues modulates the activation of the protein kinase Ire1

JUAN CRUZ ALMADA; ANA BORTOLOTTI; MERCEDES ESTELA; PAULA DUNAYEVICH; JULIA CRICCO; LORENA FALCONE; PAULA CASATTI; PABLO AGUILAR; ALEJANDRO COLMAN-LERNER; ANDREA CARINA CUMINO; LARISA CYBULSKI

Paris

Conferencia; Endoplasmic Reticulum Conference ?Cell biology, development and evolution? (ITMO BCDE); 2021

TRANSMEMBRANE ZIPPER OF POLAR RESIDUES MODULATES THE ACTIVATION OF THE PROTEIN KINASE IRE1.Juan Cruz Almada1, Ana Bortolotti1, Mercedes Estela1, Paula Dunayevich2, Julia Cricco3, Lorena Falcone4, Paula Casatti4, Pablo Aguilar2, Andrea Cumino5, Alejandro Colman-Lerner2, Larisa Cybulski11Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-Argentine National Research Council-CONICET, Suipacha 531, 2000 Rosario, Argentina. 2[IFIBYNE] Instituto De Fisiología, Biología Molecular Y Neurociencias - Argentine National Research Council – CONICET. 3[IBR] Instituto De Biología Molecular Y Celular De Rosario - Argentine National Research Council – CONICET. 4[CEFOBI] Centro De Estudios Fotosintéticos Y Bioquímicos - Argentine National Research Council – CONICET. 5[IIPROSAM] Instituto De Investigaciones En Producción, Sanidad Y Ambiente - Argentine National Research Council – CONICET.Receptor kinases play a key role in transmitting signals: they are essential for detecting physicochemical stimuli in the environment and for activating adaptive cellular responses. Our research studies the molecular biophysical activation mechanism of Inositol-required enzyme 1 (Ire1), which locates in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae, plants, humans and other organisms.Ire1 is an ER transmembrane sensor with kinase and endoribonuclease activities that is activated by two different stimuli: 1) by misfolded proteins, which are detected by the luminal domain, and 2) by a decrease in the fluidity of the ER membrane, which is detected by the transmembrane helix of Ire1. The activation of Ire1 promotes dimerization and oligomerization, which stimulates its activity. Ire1 cleaves an mRNA that codes for a transcription factor that induces the expression of 381 genes involved in the biosynthesis of lipids and chaperones. If homeostasis is not recovered, chronic stimulation of this pathway initiates the apoptotic program. In mammals, the insulin deficiency associated with type 2 diabetes is due to the death of pancreatic cells, triggered by constant activation of Ire1.We focus on responding how the transmembrane helix can detect changes in membrane fluidity. The single TM helix of Ire1 has a group of polar hydrogen-bond forming amino acids (S533, Y536, E540) that are located on the same face of the TM helix, as a heptad zipper and they could form reversible inter-helix hydrogen-bonds according to environmental conditions.We used CRISPr Cas9 technology, to introduce mutations in the potential zipper to either reinforce or to weaken it. We found that introduction of additional Hydrogen-bond forming amino acids reinforcing the zipper face results in active proteins in the absence of a stimulus. On the contrary, replacing Hydrogen-bond forming amino acids from the original zipper to alanine weakens it, resulting in inactive proteins.To sum up, through genetic and biophysical studies we identified a potential hydrogen-bond zipper located in the transmembrane helix of Ire1, which could be responsible for sensing eukaryotic membrane fluidity. The activation mechanism would imply a dimerization through the hydrophilic face delimited by the zipper of polar residues forming hydrogen bonds in less fluid ER membranes, which would result in activation of the protein.