Unravelling the role of zinc and copper in the regulation of the co-chaperone DNAJA1 and its interactions with Hsp70. Impact on the cellular metabolome under metal stress

• Grant number: 24/20734-3
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2025
• End date: June 30, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Carlos Henrique Inacio Ramos
• Grantee: Ketolly Natanne da Silva Leal
• Host Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 17/26131-5 - The chaperome: study of the relationship of the structure of its components and the maintenance of proteostasis, AP.TEM

Abstract

This postdoctoral project seeks to examine the role of zinc and copper in the regulation of the human cochaperone DNAJA1 and its influence on the interaction with the chaperone Hsp70, with an innovative focus on modifications at the single-cell level. DNAJA1 is a zinc-binding protein essential for the proper functioning of the Hsp70 system, particularly under conditions of metal stress. The central hypothesis of this work is that the cochaperone DNAJA1 utilises zinc and copper to maintain its cellular functions, and that these metal interactions directly impact metabolic pathways. The innovation of this project lies in the use of advanced analytical techniques, such as single-cell Inductively Coupled Plasma Mass Spectrometry (scICP-MS), for the detailed characterisation of intracellular alterations in zinc and copper in yeast, enabling the acquisition of quantitative intracellular information at the individual level. Furthermore, an online multimodal coupling involving atomic and molecular mass spectrometry (LC-ICP-MS-ESI-HRMS) will be employed to identify types of inorganic, organic, and metallorganic compounds (metal-biomolecules) that correspond to metabolic pathways modulated by these metals and their interactions with the metabolome. These integrative approaches are unprecedented in Brazil, as there are no prior studies employing these techniques to explore metal interactions at the cellular level. Thus, the project not only elucidates the impact of zinc and copper speciation on the functionality of DNAJA1 and its interaction with Hsp70 but also establishes new paradigms for research in cellular homeostasis under conditions of metal stress. Additionally, an international BEPE internship grant will be requested for analytical development at the School of Medicine, University of St Andrews, Scotland, under the supervision of Dr Alan James Stewart. This collaboration aims at single-cell proteomics analysis through high-resolution mass spectrometry. The project will be divided into three main phases: (i) development of a method for quantifying metals in yeast by single-cell ICP-MS; (ii) studies of intracellular, metabolomic, and proteomic alterations under metal stress using atomic and molecular mass spectrometry; and (iii) analysis of the results and scientific communication. The generated data will address the following questions: (a) how do zinc and copper modulate the function of DNAJA1 and its interaction with Hsp70? (b) which metabolic pathways are affected by the availability of these metals? and (c) what are the consequences for cellular homeostasis and stress response? The proposed duration of the project is three years, and it promises to provide unprecedented insights into metal dynamics at the cellular level. (AU)