Study of the Interaction between Hsp90 and Aha1 in Leishmania braziliensis by Biophysical Approaches

• Grant number: 25/13886-4
• Support Opportunities: Regular Research Grants
• Start date: October 01, 2025
• End date: September 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Julio Cesar Borges
• Grantee: Julio Cesar Borges
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• City of the host institution: São Carlos
• Associated research grant: 24/02293-0 - The Role of Phosphorylation in Modulating Hsp90 Chaperone Activity and its Co-chaperones SGT and Aha1, AP.R

Abstract

Molecular chaperones are essential for maintaining cellular proteostasis, assisting in the folding, stabilization, and maturation of client proteins. Among them, Hsp90 stands out as a highly conserved protein that plays a central role in key biological processes, including signal transduction, cell cycle control, intracellular protein trafficking, and the response to physiological and environmental stress. In protozoa of the genus Leishmania, the causative agents of leishmaniasis, these functions become particularly critical due to the hostile conditions encountered throughout their life cycle, such as drastic changes in temperature and pH, as well as the pressures imposed by the host immune system.Hsp90's functional activity is regulated by a complex cycle of ATP binding and hydrolysis, which is finely tuned by co-chaperones. A key co-chaperone is Aha1 (Activator of the Hsp90-ATPase activity 1), which stimulates Hsp90's ATPase activity and promotes conformational transitions essential for its functional cycle. The interaction between Hsp90 and Aha1 involves specific structural domains in both proteins, although significant gaps remain in our understanding of the molecular mechanisms governing this interaction.This study aims to investigate, using various biophysical approaches, the importance of the structural domains involved in the interaction between recombinant Leishmania braziliensis Hsp90 (LbHsp90) and its co-chaperone LbAha1. Full-length forms of LbHsp90 and LbAha1 will be expressed in a bacterial system, along with variants containing specific domains: LbHsp90M (middle domain only), LbHsp90NM (N-terminal and middle domains), and the LbAha1 variants LbAha1N-¿L, LbAha1NL, and LbAha1NLG4S (the latter two comprising the N-terminal domain of LbAha1 with or without the conserved linker or a generic linker). These recombinant proteins will be purified and analyzed through limited proteolysis with different proteases, cross-linking assays, covalent labeling, and mass spectrometry. These methods will allow a more detailed investigation of the interaction interfaces between LbHsp90 and LbAha1, as well as the conformational changes that occur during their functional association.The data generated in this study will be instrumental in building a more comprehensive model of the structural dynamics and regulatory mechanisms of Hsp90 in L. braziliensis. Furthermore, it will provide a solid foundation for future studies employing advanced techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS), which enables high-resolution mapping of interaction regions and conformational mobility. These findings may offer novel insights into chaperone regulation in protozoa, contributing to a broader understanding of protein regulation in lower eukaryotes.From an academic standpoint, this project represents a significant contribution to the molecular biology of parasites, especially considering that Hsp90 has emerged as a promising target for antiparasitic drug development. Functional characterization of its interactions with co-chaperones may uncover new therapeutic possibilities. Leishmaniasis remains a major public health issue in several regions of Brazil and worldwide, particularly affecting socially vulnerable populations. Thus, the advances achieved in this study have the potential to generate positive social impact in the medium and long term.In addition to its scientific and societal impacts, the project will play a valuable educational role, particularly for the students involved in its various stages. Hands-on participation in protein expression, purification, assay optimization, and data analysis will provide a rich, multidisciplinary scientific experience, involving molecular biology, protein biochemistry, and mass spectrometry techniques. At the same time, this dynamic training environment will contribute meaningfully to the students' academic development, fostering critical thinking, autonomy, and a deeper (AU)