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Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis.

ABSTRACT: Leishmania parasites primarily infect cells of macrophage lineage and can cause leishmaniasis in the skin, mucosal, and visceral organs, depending on both host- and parasite-derived factors. The protein disulfide isomerases (PDIs) are thiol-disulfide oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds of proteins in cells. Although four Leishmania PDI genes are functionally inferred from homology in the genome sequences, only two of them have been expressed as active proteins to date. The functional relationship among various PDI enzymes remains largely unclear. In this study, we expressed and partially characterized all four L. amazonensis PDIs encoding 52-, 47-, 40-, and 15-kDa proteins. Homology analysis showed that the sequence identity between L. amazonensis (New World) PDIs and their counterpart PDI sequences from L. major (Old World) ranged from 76% to 99%. Kinetic characterization indicated that while the 15-, 40-, and 47- kDa PDI proteins displayed both insulin isomerase and reductase activities, the 52-kDa protein had only isomerase activity with no detectable reductase activity. All four PDI proteins were recognized by sera from L. amazonensis-infected mice and were sensitive to inhibition by standard PDI inhibitors. This study describes the enzymatic activities of recombinant L. amazonensis PDIs and suggests a role for these proteins in parasite development.

SUBMITTER: Hong BX

PROVIDER: S-EPMC2745823 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis.

Hong B X BX Soong L L

Parasitology research 20071206 3

Leishmania parasites primarily infect cells of macrophage lineage and can cause leishmaniasis in the skin, mucosal, and visceral organs, depending on both host- and parasite-derived factors. The protein disulfide isomerases (PDIs) are thiol-disulfide oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds of proteins in cells. Although four Leishmania PDI genes are functionally inferred from homology in the genome sequences, only two of them have been express ...[more]

PMID: 18058133

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Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1-245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.

Dataset Information

Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis.

Publications

Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis.

Similar Datasets

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