Luhua Song

Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress

C2H2‐zinc finger proteins that contain the EAR repressor domain are thought to play a key role in modulating the defense response of plants to abiotic stress. Constitutive expression of the C2H2‐EAR zinc finger protein Zat10 in Arabidopsis was found to elevate the expression of reactive oxygen‐defense transcripts and to enhance the tolerance of plants to salinity, heat and osmotic stress. Surprisingly, knockout and RNAi mutants of Zat10 were also more tolerant to osmotic and salinity stress. Our results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.

The EAR-motif of the Cys2/His2-type Zinc Finger Protein Zat7 Plays a Key Role in the Defense Response of Arabidopsis to Salinity Stress

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Thiamin Confers Enhanced Tolerance to Oxidative Stress in Arabidopsis

Thiamin and thiamin pyrophosphate (TPP) are well known for their important roles in human nutrition and enzyme catalysis. In this work, we present new evidence for an additional role of these compounds in the protection of cells against oxidative damage. Arabidopsis (Arabidopsis thaliana) plants subjected to abiotic stress conditions, such as high light, cold, osmotic, salinity, and oxidative treatments, accumulated thiamin and TPP. Moreover, the accumulation of these compounds in plants subjected to oxidative stress was accompanied by enhanced expression of transcripts encoding thiamin biosynthetic enzymes. When supplemented with exogenous thiamin, wild-type plants displayed enhanced tolerance to oxidative stress induced by paraquat. Thiamin application was also found to protect the reactive oxygen species-sensitive ascorbate peroxidase1 mutant from oxidative stress. Thiamin-induced tolerance to oxidative stress was accompanied by decreased production of reactive oxygen species in plants, as evidenced from decreased protein carbonylation and hydrogen peroxide accumulation. Because thiamin could protect the salicylic acid induction-deficient1 mutant against oxidative stress, thiamin-induced oxidative protection is likely independent of salicylic acid signaling or accumulation. Taken together, our studies suggest that thiamin and TPP function as important stress-response molecules that alleviate oxidative stress during different abiotic stress conditions.

NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.

This work describes biochemical, biophysical, structural, and genetic analyses of an Arabidopsis homolog of mammalian NEET proteins, which are involved in a wide range of cellular processes. It finds that At-NEET plays a key role in plant development, senescence, reactive oxygen species homeostasis, and iron metabolism. The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

The Fe-S cluster-containing NEET proteins mitoNEET and NAF-1 as chemotherapeutic targets in breast cancer

Significance Cancer is a leading cause of mortality worldwide, with the identification of novel drug targets and chemotherapeutic agents being a high priority in the fight against it. The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) were recently shown to be required for cancer cell proliferation. Utilizing a combination of experimental and computational techniques, we identified a derivative of the mitocan cluvenone that binds to NEET proteins at the vicinity of their 2Fe-2S clusters and facilitates their destabilization. The new drug displays a high specificity in the selective killing of human epithelial breast cancer cells, without any apparent effects on normal breast cells. Our results identify the 2Fe-2S clusters of NEET proteins as a novel target in the chemotherapeutic treatment of breast cancer. Identification of novel drug targets and chemotherapeutic agents is a high priority in the fight against cancer. Here, we report that MAD-28, a designed cluvenone (CLV) derivative, binds to and destabilizes two members of a unique class of mitochondrial and endoplasmic reticulum (ER) 2Fe-2S proteins, mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1), recently implicated in cancer cell proliferation. Docking analysis of MAD-28 to mNT/NAF-1 revealed that in contrast to CLV, which formed a hydrogen bond network that stabilized the 2Fe-2S clusters of these proteins, MAD-28 broke the coordinative bond between the His ligand and the cluster’s Fe of mNT/NAF-1. Analysis of MAD-28 performed with control (Michigan Cancer Foundation; MCF-10A) and malignant (M.D. Anderson–metastatic breast; MDA-MB-231 or MCF-7) human epithelial breast cells revealed that MAD-28 had a high specificity in the selective killing of cancer cells, without any apparent effects on normal breast cells. MAD-28 was found to target the mitochondria of cancer cells and displayed a surprising similarity in its effects to the effects of mNT/NAF-1 shRNA suppression in cancer cells, causing a decrease in respiration and mitochondrial membrane potential, as well as an increase in mitochondrial iron content and glycolysis. As expected, if the NEET proteins are targets of MAD-28, cancer cells with suppressed levels of NAF-1 or mNT were less susceptible to the drug. Taken together, our results suggest that NEET proteins are a novel class of drug targets in the chemotherapeutic treatment of breast cancer, and that MAD-28 can now be used as a template for rational drug design for NEET Fe-S cluster-destabilizing anticancer drugs.

Ultra-fast alterations in mRNA levels uncover multiple players in light stress acclimation in plants

Summary The acclimation of plants to changes in light intensity requires rapid responses at several different levels. These include biochemical and biophysical responses as well as alterations in the steady‐state level of different transcripts and proteins. Recent studies utilizing promoter::reporter constructs suggested that transcriptional responses to changes in light intensity could occur within seconds, rates for which changes in mRNA expression are not routinely measured or functionally studied. To identify and characterize rapid changes in the steady‐state level of different transcripts in response to light stress we performed RNA sequencing analysis of Arabidopsis thaliana plants subjected to light stress. Here we report that mRNA accumulation of 731 transcripts occurs as early as 20–60 sec following light stress application, and that at least five of these early response transcripts play an important biological role in the acclimation of plants to light stress. More than 20% of transcripts accumulating in plants within 20–60 sec of initiation of light stress are H2O2‐ and ABA‐response transcripts, and the accumulation of several of these transcripts is inhibited by transcriptional inhibitors. In accordance with the association of rapid response transcripts with H2O2 and ABA signaling, a mutant impaired in ABA sensing (abi‐1) was found to be more tolerant to light stress, and the response of several of the rapid response transcripts was altered in mutants impaired in reactive oxygen metabolism. Our findings reveal that transcriptome reprogramming in plants could occur within seconds of initiation of abiotic stress and that this response could invoke known as well as unknown proteins and pathways.

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters.

Significance Elevated expression of the iron–sulfur (Fe-S) protein nutrient-deprivation autophagy factor-1 (NAF-1) is associated with the progression of multiple cancer types. Here we demonstrate that the lability of the Fe-S cluster of NAF-1 plays a key role in promoting breast cancer cell proliferation, tumor growth, and resistance of cancer cells to oxidative stress. Our study establishes an important role for the unique 3Cys-1His Fe-S cluster coordination structure of NAF-1 in promoting the development of breast cancer tumors and suggests the potential use of drugs that suppress NAF-1 accumulation or stabilize its cluster in the treatment of cancers that display high expression levels of NAF-1. Iron–sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression.

Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells.

ABSTRACT Maintaining iron (Fe) ion and reactive oxygen species homeostasis is essential for cellular function, mitochondrial integrity and the regulation of cell death pathways, and is recognized as a key process underlying the molecular basis of aging and various diseases, such as diabetes, neurodegenerative diseases and cancer. Nutrient-deprivation autophagy factor 1 (NAF-1; also known as CISD2) belongs to a newly discovered class of Fe-sulfur proteins that are localized to the outer mitochondrial membrane and the endoplasmic reticulum. It has been implicated in regulating homeostasis of Fe ions, as well as the activation of autophagy through interaction with BCL-2. Here we show that small hairpin (sh)RNA-mediated suppression of NAF-1 results in the activation of apoptosis in epithelial breast cancer cells and xenograft tumors. Suppression of NAF-1 resulted in increased uptake of Fe ions into cells, a metabolic shift that rendered cells more susceptible to a glycolysis inhibitor, and the activation of cellular stress pathways that are associated with HIF1α. Our studies suggest that NAF-1 is a major player in the metabolic regulation of breast cancer cells through its effects on cellular Fe ion distribution, mitochondrial metabolism and the induction of apoptosis. Summary: NAF-1 is a major player in the metabolic regulation of breast cancer cells through its effects on cellular Fe ion distribution, mitochondrial metabolism and the induction of apoptosis.

Interactions between mitoNEET and NAF-1 in cells

The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are required for cancer cell proliferation and resistance to oxidative stress. NAF-1 and mNT are also implicated in a number of other human pathologies including diabetes, neurodegeneration and cardiovascular disease, as well as in development, differentiation and aging. Previous studies suggested that mNT and NAF-1 could function in the same pathway in mammalian cells, preventing the over-accumulation of iron and reactive oxygen species (ROS) in mitochondria. Nevertheless, it is unknown whether these two proteins directly interact in cells, and how they mediate their function. Here we demonstrate, using yeast two-hybrid, in vivo bimolecular fluorescence complementation (BiFC), direct coupling analysis (DCA), RNA-sequencing, ROS and iron imaging, and single and double shRNA lines with suppressed mNT, NAF-1 and mNT/NAF-1 expression, that mNT and NAF-1 directly interact in mammalian cells and could function in the same cellular pathway. We further show using an in vitro cluster transfer assay that mNT can transfer its clusters to NAF-1. Our study highlights the possibility that mNT and NAF-1 function as part of an iron-sulfur (2Fe-2S) cluster relay to maintain the levels of iron and Fe-S clusters under control in the mitochondria of mammalian cells, thereby preventing the activation of apoptosis and/or autophagy and supporting cellular proliferation.