Interactome of Glyceraldehyde-3-Phosphate Dehydrogenase Points to the Existence of Metabolons in Paracoccidioides lutzii

Abstract

Paracoccidioides is a dimorphic fungus, the causative agent of paracoccidioidomycosis. The disease is endemic within Latin America and prevalent in Brazil. The treatment is based on azoles, sulfonamides and amphotericin B. The seeking for new treatment approaches is a real necessity for neglected infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential glycolytic enzyme, well known for its multitude of functions within cells, therefore categorized as a moonlight protein. To our knowledge, this is the first approach performed on the Paracoccidioides genus regarding the description of PPIs having GAPDH as a target. Here, we show an overview of experimental GAPDH interactome in different phases of Paracoccidioides lutzii and an in silico analysis of 18 proteins partners. GAPDH interacted with 207 proteins in P. lutzii . Several proteins bound to GAPDH in mycelium, transition and yeast phases are common to important pathways such as glycolysis and TCA. We performed a co-immunoprecipitation assay to validate the complex formed by GAPDH with triose phosphate isomerase, enolase, isocitrate lyase and 2-methylcitrate synthase. We found GAPDH participating in complexes with proteins of specific pathways, indicating the existence of a glycolytic and a TCA metabolon in P. lutzii . GAPDH interacted with several proteins that undergoes regulation by nitrosylation. In addition, we modeled the GAPDH 3-D structure, performed molecular dynamics and molecular docking in order to identify the interacting interface between GAPDH and the interacting proteins. Despite the large number of interacting proteins, GAPDH has only four main regions of contact with interacting proteins, reflecting its ancestrality and conservation over evolution.

Immunoproteomic Approach of Extracellular Antigens From Paracoccidioides Species Reveals Exclusive B-Cell Epitopes

“…These data provide the first large-scale identification of exoantigens of Paracoccidioides species. Some of these exoantigens have already been identified and described as antigens of pathogenic fungi, such as formamidase (Borges et al, 2005(Borges et al, , 2010, dihydrolipoyl dehydrogenase (Landgraf et al, 2017), HSP60 (Izacc et al, 2001;Cunha et al, 2002), HSP70 (Bromuro et al, 1998;Bisio et al, 2005), serine protease (Parente et al, 2010;Lacerda Pigosso et al, 2017), and isocitrate lyase (Cruz et al, 2011;Silva et al, 2019). This demonstrates the robustness of method used in our immunoproteomics analyses.…”

Interaction of Isocitrate Lyase with Proteins Involved in the Energetic Metabolism in Paracoccidioides lutzii

“…MET 120 and ASN 121 are contact residues between ICL and FBA1, PGK, PK, GAPDH and ENO. The fact that those proteins belong to the glycolytic pathway and since they share similar contact residues with ICL, reinforce the idea of the existence of a glycolytic metabolon in P. lutzii [84]. GLN .…”

“…MET 120 and ASN 121 are contact residues between ICL and FBA1, PGK, PK, GAPDH and ENO. The fact that those proteins belong to the glycolytic pathway and since they share similar contact residues with ICL, reinforce the idea of the existence of a glycolytic metabolon in P. lutzii [ 84 ]. GLN 502 and LYS 503 establish contact between ICL and partners in ENO, MDH, MCS and MCD, while LYS 513 of ICL interacts with PK, MDH, ECH and ACA.…”

Effect of Nitrosative Stress on the S-Nitroso-Proteome of Paracoccidioides brasiliensis

“…We identified GAPDH S-nitrosylated (SNO-GADPH) in all conditions analyzed (Supplementary Table S1). In GAPDH interactome analyzes was observed that SNO-GAPDH can interact with several P. lutzii proteins, such as acyl CoA dehydrogenase, argininosuccinate synthase, aldehydedehydrogenase, rad24, among others (Silva et al, 2019). GAPDH is well-known for its role in cytosolic glycolysis (Tristan et al, 2011).…”