title: HRI, a stress response eIF2α kinase, exhibits structural and functional stability at high temperature and alkaline conditions.
abstract: The Heme Regulated Inhibitor (HRI) is a key regulator of protein synthesis in mammalian cells. Once activated under heme-deficiency and other stress conditions, it phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α) leading to inhibition of protein synthesis. In the present study, our objective was to establish the structural and functional credentials of this kinase so as to qualify it as a stress responsive eIF2α kinase. When the catalytic kinase domain of the HRI (HRI.CKD) protein was subjected to high temperature, 45°C (above mammalian heat shock temperature), it could still phosphorylate the substrate, indicating its potential as a stress response kinase. At a temperature beyond 45°C, loss in secondary structure of the HRI.CKD is attributable to loss of its function. Furthermore, no significant structural changes were observed at the broad pH range of 3.0--10.0. The HRI.CKD incubated at any pH between 8.0-10.0, exhibited more than 60% of its kinase activity, demonstrating structural and functional stability of the kinase at an alkaline pH. These data taken together establish that the structural stability of this kinase at high temperature and alkaline conditions is due to conservation of its secondary structure and that the resulting functional activity qualifies this kinase as a stress responsive kinase.
authors.affiliation: Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra 411007, India.
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhavnani
authors.affiliation: Vaccine Formulation & Research Centre, Gennova Biopharmaceuticals Limited, Pune, Maharashtra 411057, India.
authors.forename: Kaviraj
authors.initials: K
authors.lastname: Swarnendu
authors.affiliation: Vaccine Formulation & Research Centre, Gennova Biopharmaceuticals Limited, Pune, Maharashtra 411057, India.
authors.forename: Laxman
authors.initials: L
authors.lastname: Savergave
authors.affiliation: Vaccine Formulation & Research Centre, Gennova Biopharmaceuticals Limited, Pune, Maharashtra 411057, India.
authors.forename: Arjun Singh
authors.initials: AS
authors.lastname: Raghuwanshi
authors.affiliation: Vaccine Formulation & Research Centre, Gennova Biopharmaceuticals Limited, Pune, Maharashtra 411057, India.
authors.forename: Ankit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Vaccine Formulation & Research Centre, Gennova Biopharmaceuticals Limited, Pune, Maharashtra 411057, India.
authors.forename: Avinash
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra 411007, India. Electronic address: jkpal@unipune.ac.in.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Pal
mesh_terms: D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D018869:Heat-Shock Response; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D066329:Protein Aggregates; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D019892:eIF-2 Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D066329:Protein Aggregates; C583541:EIF2AK1 protein, human; D019892:eIF-2 Kinase
keywords: Alkaline stability; CD; DSC; Heme Regulated Inhibitor (HRI); Thermal unfolding