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IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity.

ABSTRACT: A central means by which mammalian cells respond to low oxygen tension is through the activation of the transcription factor HIF-1 (hypoxia-inducible factor-1). Under normoxic conditions, HIF-1alpha (the alpha subunit of HIF-1) is targeted for rapid degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this degradation is inhibited, thereby leading to the stabilization and activation of HIF-1alpha. Here, we report the identification of IOP1 (iron-only hydrogenase-like protein 1), a protein homologous with enzymes present in anaerobic organisms that contain a distinctive iron-sulfur cluster. IOP1 is present in a broad range of cell types. Knockdown of IOP1 using siRNA (small interfering RNA) in mammalian cells increases protein levels of HIF-1alpha under both normoxic and hypoxic conditions, and augments hypoxia-induced HRE (hypoxia response element) reporter gene and endogenous HIF-1alpha target gene expressions. We find that IOP1 knockdown up-regulates HIF-1alpha mRNA levels, thereby providing a mechanism by which knockdown induces the observed effects. The results collectively provide evidence that IOP1 is a component of the protein network that regulates HIF-1alpha in mammalian cells.

SUBMITTER: Huang J

PROVIDER: S-EPMC1698691 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity.

Huang Jianhe J Song Daisheng D Flores Adrian A Zhao Quan Q Mooney Sharon M SM Shaw Leslie M LM Lee Frank S FS

The Biochemical journal 20070101 1

A central means by which mammalian cells respond to low oxygen tension is through the activation of the transcription factor HIF-1 (hypoxia-inducible factor-1). Under normoxic conditions, HIF-1alpha (the alpha subunit of HIF-1) is targeted for rapid degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this degradation is inhibited, thereby leading to the stabilization and activation of HIF-1alpha. Here, we report the identification of IOP1 (iron-only hydrogenase-like protei ...[more]

PMID: 16956324

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Project description:BACKGROUND: Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1alpha protein overexpression has been shown in breast cancer. The impact of HIF-1alpha alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. METHODS: Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1alpha and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1alpha splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. RESULTS: HIF-1alpha isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1alphaTAG) and HIF-1alpha736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1alphaTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1alphaTAG (P = 0.048), and total HIF-1alpha (P = 0.048) mRNA levels. HIF-1alphaTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1alphaTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). CONCLUSIONS: Our results show for the first time that mRNA expression of a HIF-1alphaTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45.

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

IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity.

Publications

IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity.

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