TheTCP1γ subunit ofLeishmania donovaniforms a biologically active homo‐oligomeric complex

Abstract

Chaperonins are a class of molecular chaperons that encapsulate nascent or stress‐denatured proteins and assist their intracellular assembly and folding in an ATP‐dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero‐oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin‐like complexes and plays a role in protein folding. LdTCP1γ formed high‐molecular‐weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back‐to‐back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double‐ring group I chaperonins found in mitochondria. The LdTCP1γ homo‐oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo‐oligomer performs chaperonin reactions without partner subunit(s). Further, co‐ immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites.

Increased Abundance of Proteins Involved in Resistance to Oxidative and Nitrosative Stress at the Last Stages of Growth and Development of Leishmania amazonensis Promastigotes Revealed by Proteome Analysis

“…amazonensis promastigotes in culture. The exact function of the TCP complex is not known in Leishmania spp., although it has been suggested that the TCP1γ subunit may participate in maintaining the structural dynamics of the cytoskeleton [ 29 ]. Changes in cell morphology throughout promastigote differentiation and flagellar movement demand continuous re-organization of the cytoskeleton.…”

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

“…In addition to the heterohexadecamer form, the TRiC subunits can function as monomers (Brackley & Grantham, ; Liou & Willison, ; Tam et al . , ), other microcomplexes with two or more subunits (Liou & Willison, ), homo‐oligomers (Bhaskar, Mitra, Kuldeep, Siddiqi, & Goyal, ), and can also form homohexadecamers (Sergeeva et al . , ).…”

“…In addition to the heterohexadecamer form, the TRiC subunits can function as monomers (Brackley & Grantham, 2010;Liou & Willison, 1997;Tam et al, 2006), other microcomplexes with two or more subunits (Liou & Willison, 1997), homo-oligomers (Bhaskar, Mitra, Kuldeep, Siddiqi, & Goyal, 2015), and can also form homohexadecamers (Sergeeva et al, 2013). In our study, we observed monomeric forms and micro-complexes for all subunits examined (Figures 3-5) and notably the higher molecular weight complexes observed for the TRiC-θ subunit were different to those of other subunits (Figure 4, 5).…”

Proteomics and Leishmaniasis: Potential Clinical Applications

“…Co‐immunoprecipitation and western blot analysis of the γ subunit of the chaperonin containing T‐complex protein (TCP) complex of L. donovani promastigotes revealed high molecular weight complexes involved in protein folding. In addition, the interaction of the TCPγ with tubulin and actin suggested a structural role in the parasite cytoskeleton …”

“…In addition, the interaction of the TCPγ with tubulin and actin suggested a structural role in the parasite cytoskeleton. [74] An important complex required for the degradation and recycling of cytosolic proteins was demonstrated in L. major by MS analysis. [75] The autophagy-related protein 5 to 12 (ATG5-ATG12) complex is a key component of autophagy, and the identification of peptides from ATG5 and ATG12 in a 70 kDa band following analysis by MS confirmed the existence of this protein complex in Leishmania.…”