Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH.

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Functional maturation of cytochromes P450 3A4 and 2D6 relies on GAPDH- and Hsp90-Dependent heme allocation.

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease.

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO.

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution.

A natural heme deficiency that exists in cells outside of the circulation broadly compromises the heme contents and functions of heme proteins in cells and tissues. Recently, we found that the signaling molecule, nitric oxide (NO), can trigger or repress the deployment of intracellular heme in a concentration-dependent hormetic manner. This uncovers a new role for NO and sets the stage for it to shape numerous biological processes by controlling heme deployment and consequent heme protein functions in biology.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through control of cell heme allocation by nitric oxide.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.

New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals.

The intracellular trafficking of mitochondrial heme presents a fundamental challenge to animal cells. This article provides some background on heme allocation, discusses some of the concepts, and then reviews research done over the last decade, much in the author's laboratory, that is uncovering unexpected and important roles for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (hsp90), and nitric oxide (NO) in enabling and regulating the allocation of mitochondrial heme to hemeproteins that mature and function outside of the mitochondria. A model for how hemeprotein functions can be regulated in cells through the coordinate participation of GAPDH, hsp90, and NO in allocating cellular heme is presented.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through GAPDH- and Hsp90-dependent control of their heme levels.

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygense (TDO) are heme-containing dioxygenases that catalyze the conversion of tryptophan to N-formyl-kynurenine and thus enable generation of l-kynurenine and related metabolites that govern the immune response and broadly impact human biology. Given that TDO and IDO1 activities are directly proportional to their heme contents, it is important to understand their heme delivery and insertion processes. Early studies established that TDO and IDO1 heme levels are sub-saturating in vivo and subject to change but did not identify the cellular mechanisms that provide their heme or enable dynamic changes in their heme contents. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into IDO1 and TDO expressed in both normal and heme-deficient HEK293T cells and into IDO1 naturally expressed in HeLa cells in response to IFN-γ, and also investigated the interactions of TDO and IDO1 with GAPDH and Hsp90 in cells and among their purified forms. We found that GAPDH delivered both mitochondrially-generated and exogenous heme to apo-IDO1 and apo-TDO in cells, potentially through a direct interaction with either enzyme. In contrast, we found Hsp90 interacted with apo-IDO1 but not with apo-TDO, and was only needed to drive heme insertion into apo-IDO1. By uncovering the cellular processes that allocate heme to IDO1 and TDO, our study provides new insight on how their activities and l-kynurenine production may be controlled in health and disease.

Targeting Histone Chaperone FACT Complex Overcomes 5-Fluorouracil Resistance in Colon Cancer.

Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (∼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.

Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase.

Myoglobin (Mb) maturation involves heme incorporation as a final step. We investigated a role for heat shock protein (hsp) 90 in Mb maturation in C2C12 skeletal muscle myoblasts and cell lines. We found the following: 1) Hsp90 directly interacts preferentially with heme-free Mb both in purified form and in cells. 2) Hsp90 drives heme insertion into apoprotein-Mb in an ATP-dependent process. 3) During differentiation of C2C12 myoblasts into myotubes, the apo-Mb-hsp90 complex associates with 5 cell cochaperons, Hsp70, activator of hsp90 ATPase protein 1 (Aha1), alanyl-tRNA synthetase domain containing 1 (Aarsd1), cell division cycle 37 (Cdc37), and stress induced phosphoprotein 1 (STIP1) in a pattern that is consistent with their enabling Mb maturation. 4) Mb heme insertion was significantly increased in cells that had a functional soluble guanylyl cyclase (sGC)-cGMP signaling pathway and was diminished upon small interfering RNA knockdown of sGCß1 or upon overexpression of a phosphodiesterase to prevent cGMP buildup. Together, our findings suggest that hsp90 works in concert with cochaperons (Hsp70, Aha1, Aarsd1, STIP1, and Cdc37) and an active sGC-cGMP signaling pathway to promote heme insertion into immature apo-Mb, and thus generate functional Mb during muscle myotube formation. This fills gaps in our understanding and suggests new ways to potentially control these processes.-Ghosh, A., Dai, Y., Biswas, P., Stuehr, D. J. Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase.

Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis.

The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives." Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.

Transcription factors Atf1 and Sty1 promote stress tolerance under nitrosative stress in Schizosaccharomyces pombe.

Nitric Oxide (NO) and its associated reactive nitrogen species (RNS) produce nitrosative stress under various pathophysiological conditions in eukaryotes. The fission yeast Schizosaccharomyces pombe regulates stress response mainly through the Sty1-Atf1 MAP Kinase pathway. The present study deals with the role of transcription factor Atf1 and Sty1 in S. pombe under nitrosative stress. In this study, exposure to an NO donor resulted in S-phase slowdown with associated mitotic block in S. pombe. Deletion of sty1 and atf1 in S. pombe had differential growth sensitivity towards NO donor. Both Sty1 and Atf1 were involved in regulating mitotic slowdown in S. pombe under nitrosative stress. Experimental data obtained in this study reveals a novel role of Atf1 in initiating the replication slowdown in S. pombe under nitrosative stress. Both Sty1 and Atf1 were accumulated in the nucleus in S. pombe under nitrosative stress in a concentration and time dependent manner. Atf1 is also found to be nuclear delocalized under longer nitrosative stress.

Nitric oxide sensing by chlorophyll a.

Nitric oxide (NO) acts as a signalling molecule that has direct and indirect regulatory roles in various functional processes in biology, though in plant kingdom its role is relatively unexplored. One reason for this is the fact that sensing of NO is always challenging. There are very few probes that can classify the different NO species. The present paper proposes a simple but straightforward way for sensing different NO species using chlorophyll, the source of inspiration being hemoglobin that serves as NO sink in mammalian systems. The proposed method is able to classify NO from DETA-NONOate or (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate, nitrite, nitrate and S-nitrosothiol or SNO. This discrimination is carried out by chlorophyll a (chl a) at nano molar (nM) order of sensitivity and at 293 K-310 K. Molecular docking reveals the differential binding effects of NO and SNO with chlorophyll, the predicted binding affinity matching with the experimental observation. Additional experiments with a diverse range of cyanobacteria reveal that apart from the spectroscopic approach the proposed sensing module can be used in microscopic inspection of NO species. Binding of NO is sensitive to temperature and static magnetic field. This provides additional support for the involvement of the porphyrin ring structures to the NO sensing process. This also, broadens the scope of the sensing methods as hinted in the text.

The extracellular role of DNA damage repair protein APE1 in regulation of IL-6 expression.

The human apurinic/apyrimidinic endonuclease 1 (APE1) is a pleiotropic nuclear protein with roles in DNA base excision repair pathway as well as in regulation of transcription. Recently, the presence of extracellular plasma APE1 was reported in endotoxemic rats. However, the biological significance and the extracellular function of APE1 remain unclear. In this study, we found that monocytes secrete APE1 upon inflammatory challenges. Challenging the monocytic cells with extracellular APE1 resulted in the increased expression and secretion of the pro-inflammatory cytokine IL-6. Additionally, the extracellular APE1 treatment activated the transcription factor NF-κB, followed by its increased occupancy at the IL-6 promoter, resulting in the induction of IL-6 expression. APE1-induced IL-6 further served to elicit autocrine and paracrine cellular responses. Moreover, the extracellular IL-6 promoted the secretion of APE1, thus indicating a functional feedforward loop in this pathway. Furthermore, we show that APE1 is secreted through extracellular vesicles formation via endosomal sorting complex required for transport (ESCRT)-dependent pathway. Together, our study demonstrates a novel role of extracellular APE1 in IL-6-dependent cellular responses.

Multimodal control of transcription factor Pap1 in Schizosaccharomyces pombe under nitrosative stress.

Schizosaccharomyces pombe Pap1, a bZIP transcription factor, is highly homologous to the mammalian c-Jun protein that belongs to the AP1 family of transcriptional regulators. The role of transcription factor Pap1 has been extensively studied under oxidative stress. Two cysteine residues in Pap1p namely, C278 and C501 form disulfide linkage under oxidative stress resulting in nuclear accumulation. We first time showed the involvement of Pap1 in the protection against nitrosative stress. In the present study we show that pap1 deletion makes growth of S. pombe sensitive to nitrosative stress. pap1 deletion also causes delayed recovery in terms of mitotic index under nitrosative stress. Our flow cytometry data shows that pap1 deletion causes slower recovery from the slowdown of DNA replication under nitrosative stress. This is the first report where we show that Pap1 transcription factor is localized in the nucleus under nitrosative stress. From our study it is evident that nuclear localization of Pap1 under nitrosative stress was not due to reactive oxygen species formation.

Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display.

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways.

Nitrosative stress induces a novel intra-S checkpoint pathway in Schizosaccharomyces pombe involving phosphorylation of Cdc2 by Wee1.

Excess production of nitric oxide and reactive nitrogen intermediates causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study the cell cycle regulation under nitrosative stress response. We discovered a novel intra-S-phase checkpoint that is activated in S. pombe under nitrosative stress. The mechanism for this intra-S-phase checkpoint activation is distinctly different than previously reported for genotoxic stress in S. pombe by methyl methane sulfonate. Our flow cytometry data established the fact that Wee1 phosphorylates Cdc2 Tyr15 which leads to replication slowdown in the fission yeast under nitrosative stress. We checked the roles of Rad3, Rad17, Rad26, Swi1, Swi3, Cds1, and Chk1 under nitrosative stress but those were not involved in the activation of the DNA replication checkpoint. Rad24 was found to be involved in intra-S-phase checkpoint activation in S. pombe under nitrosative stress but that was independent of Cdc25.

Global transcriptomic profiling of Schizosaccharomyces pombe in response to nitrosative stress.

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) cause nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. This is the first report on the global gene expression profile in response to NO in S. pombe using microarray. Among the 4824 genes reported for S. pombe, 818 were differentially expressed by at least 2-fold upon NO donor treatment. We previously showed that Pap1, the Activator Protein 1 transcription factor is required to combat nitrosative stress. In this study, the transcriptional response to NO in a null mutant for pap1 identified 45 genes that seem to be controlled by Pap1. Surprisingly, Pap1 regulated genes in S. pombe were distinctly different under nitrosative stress than those reported under oxidative stress. Genes of the pathway meiosis, cell cycle, spliceosome and oxidative phosphorylation were mostly affected under nitrosative stress in the fission yeast.

Nitric oxide production by necrotrophic pathogen Macrophomina phaseolina and the host plant in charcoal rot disease of jute: complexity of the interplay between necrotroph-host plant interactions.

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.

Regulation of cell cycle and stress responses under nitrosative stress in Schizosaccharomyces pombe.

Nitric oxide (NO) acts as a signaling molecule in numerous physiological processes but excess production generates nitrosative stress in cells. The exact protective mechanism used by cells to combat nitrosative stress is unclear. In this study, the fission yeast Schizosaccharomyces pombe has been used as a model system to explore cell cycle regulation and stress responses under nitrosative stress. Exposure to an NO donor results in mitotic delay in cells through G2/M checkpoint activation and initiates rereplication. Western blot analysis of phosphorylated Cdc2 revealed that the G2/M block in the cell cycle was due to retention of its inactive phosphorylated form. Interestingly, nitrosative stress results in inactivation of Cdc25 through S-nitrosylation that actually leads to cell cycle delay. From differential display analysis, we identified plo1, spn4, and rga5, three cell cycle-related genes found to be differentially expressed under nitrosative stress. Exposure to nitrosative stress also results in abnormal septation and cytokinesis in S. pombe. In summary we propose a novel molecular mechanism of cell cycle control under nitrosative stress based on our experimental results and bioinformatics analysis.