Analysis of the Isomerase and Chaperone-Like Activities of an Amebic PDI (EhPDI)

Abstract

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. The EhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation in E. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant of E. coli, to test whether EhPDI exhibits isomerase activity in vivo. Our preliminary results showed that EhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed that EhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization of EhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding in E. histolytica.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress

“…The differentially regulated genes in NAT and the control trophozoites were classified according to the protein class which they encode (Fig. 2) using the Protein ANalysis THrough Evolutionary Relationship (PANTHER) sequence classification tool1819. The upregulated genes in NAT included those which encode chaperone proteins, such as HSP710 (EHI_130160) and DnaJ domain-containing proteins (EHI_170090), cytoskeletal proteins, such as actin (EHI_126190) and filopodin (EHI_167130), proteins associated with signal transduction, such as RAP/RAN GTPase activating protein (EHI_079910) and serine/threonine kinase 3 (EHI_096560), hydrolases, such as 26S protease regulatory subunit (EHI_052050), CP 17 (EHI_080890) and acetylornithine deacetylase (EHI_114340), membrane vesicle trafficking proteins, such as adaptor proteins (EHI_023600 and EHI_058450), and nucleic acid binding proteins, such as 40S ribosomal protein S4 (EHI_057850) and translation initiation factor (EHI_189410)).…”

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

“…We identified 154 proteins that met this condition ( S1 – S3 Tables). These 154 proteins were then classified ( Fig 1D ) using PANTHER sequence classification tool [ 27 , 28 ]. The protein classes were phosphatases (exemplified by phosphoinositide phosphatase (EHI_141860); transporters (exemplified by plasma membrane calcium-transporting ATPase, EHI_030830); membrane traffic proteins (exemplified by putative vacuolar sorting protein, EHI_025270); chaperones (exemplified by Hsc70-interacting protein, EHI_158050); hydrolases (exemplified by arginase, EHI_152330); oxidoreductases (exemplified by superoxide dismutase, EHI_159160); enzyme modulators (exemplified by Ras family GTPase, EHI_058090); lyases (exemplified by tRNA pseudouridine synthase, EHI_151650); transferases (exemplified by histone acetyltransferase, EHI_152010); nucleic acid binding proteins (exemplified by 13 kDa ribonucleoprotein-associated protein, EHI_104600); ligases (exemplified by ubiquitin-conjugating enzyme family protein, EHI_070750); kinases (exemplified by galactokinase, putative, EHI_094100); isomerases (exemplified by cysteine synthase A, EHI_024230); cytoskeletal proteins (exemplified by actin-binding protein, cofilin/tropomyosin family, EHI_168340) and proteases (exemplified by methionine aminopeptidase, EHI_126880).…”

The K-segments of wheat dehydrin WZY2 are essential for its protective functions under temperature stress

“…LEA proteins are not classical chaperones but are more likely unstructured proteins, such as α-synuclein (Kumar et al, 2014; Manda et al, 2014) and Eh PDI (Mares et al, 2014). Chaperones will translocate to specific locations to carry out their functions (Vaseva et al, 2012), and DHNs behave in the same way.…”