Quantitative Proteomics of Presynaptic Mitochondria Reveal an Overexpression and Biological Relevance of Neuronal MitoNEET in Postnatal Brain Development.
ABSTRACT: Although it has been recognized that energy metabolism and mitochondrial structure and functional activity in the immature brain differs from that of the adult, few studies have examined mitochondria specifically at the neuronal synapse during postnatal brain development. In this study, we examined the presynaptic mitochondrial proteome in mice at postnatal day 7 and 42, a period that involves the formation and maturation of synapses. Application of two independent quantitative proteomics approaches - SWATH-MS and super-SILAC - revealed a total of 40 proteins as significantly differentially expressed in the presynaptic mitochondria. In addition to elevated levels of proteins known to be involved in ATP metabolic processes, our results identified increased levels of mitoNEET (Cisd1), an iron-sulfur containing protein that regulates mitochondrial bioenergetics. We found that mitoNEET overexpression plays a cell-type specific role in ATP synthesis and in neuronal cells promotes ATP generation. The elevated ATP levels in SH-SY5Y neuroblastoma cells were associated with increased mitochondrial membrane potential and a fragmented mitochondrial network, further supporting a role for mitoNEET as a key regulator of mitochondrial function.
Project description:Heart failure (HF) occurs frequently among older individuals, and dysfunction of cardiac mitochondria is often observed. We here show the cardiac-specific downregulation of a certain mitochondrial component during the chronological aging of mice, which is detrimental to the heart. MitoNEET is a mitochondrial outer membrane protein, encoded by CDGSH iron sulfur domain 1 (CISD1). Expression of mitoNEET was specifically downregulated in the heart and kidney of chronologically aged mice. Mice with a constitutive cardiac-specific deletion of CISD1 on the C57BL/6J background showed cardiac dysfunction only after 12 months of age and developed HF after 16 months; whereas irregular morphology and higher levels of reactive oxygen species in their cardiac mitochondria were observed at earlier time points. Our results suggest a possible mechanism by which cardiac mitochondria may gradually lose their integrity during natural aging, and shed light on an uncharted molecular basis closely related to age-associated HF.
Project description:Fructose and ethanol are metabolized principally in the liver and are both known to contribute to the development of hepatic steatosis that can progress to hepatic steatohepatitis. The present study indentifies a synergistic interaction between fructose and ethanol in promoting hepatocyte sensitivity to TNF?-induced necroptosis. Concurrent exposure to fructose and ethanol induces the overexpression of the CDGSH iron-sulfur domain-containing protein 1 (CISD1 or mitoneet), which is localized to the outer mitochondrial membrane. The increased expression of mitoneet primes the hepatocyte for TNF?-induced cytotoxicity. Treatment with TNF? induces the translocation of a Stat3-Grim-19 complex to the mitochondria, which binds to mitoneet and promotes the rapid release of its 2Fe-2S cluster, causing an accumulation of mitochondrial iron. The dramatic increase of mitochondrial iron provokes a surge in formation of reactive oxygen species, resulting in mitochondrial injury and cell death. Additionally, mitoneet is constitutively expressed at high levels in L929 fibrosarcoma cells and is required for L929 cells to undergo TNF?-induced necroptosis in the presence of caspase inhibition, indicating the importance of mitoneet to the necroptotic form of cell death.
Project description:MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
Project description: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.
Project description:MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites.
Project description:The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1-3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.
Project description:The mitochondrial protein mitoNEET is a type of iron-sulfur protein localized to the outer membrane of mitochondria and is involved in a variety of human pathologies including cystic fibrosis, diabetes, muscle atrophy, and neurodegeneration. In the current study, we found that isoliquiritigenin (ISL), one of the components of the root of Glycyrrhiza glabra L., could decrease the expression of mitoNEET in A375 melanoma cells. We also demonstrated that mitoNEET could regulate the content of reactive oxygen species (ROS), by showing that the ISL-mediated increase in the cellular ROS content could be mitigated by the mitoNEET overexpression. We also confirmed the important role of ROS in ISL-treated A375 cells. The increased apoptosis rate and the decreased mitochondrial membrane potential were mitigated by the overexpression of mitoNEET in A375 cells. These findings indicated that ISL could decrease the expression of mitoNEET, which regulated ROS content and subsequently induced mitochondrial dysfunction and apoptosis in A375 cells. Our findings also highlight mitoNEET as a promising mitochondrial target for cancer therapy.
Project description:Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal ? helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH<sub>2</sub>) and that FMNH<sub>2</sub> can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH<sub>2</sub> using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH<sub>2</sub> The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET.
Project description:Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252-E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X(2)-(S/T)-X(3)-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates.
Project description:Neuroregeneration and apoptosis are two important pathophysiologic changes after spinal cord injury (SCI), but their underlying mechanisms remain unclear. MicroRNAs (miRNAs) play a crucial role in the regulation of neuroregeneration and neuronal apoptosis, research areas that have been greatly expanded in recent years. Here, using miRNA arrays to profile miRNA transcriptomes, we demonstrated that miR-127-3p was significantly down-regulated after spinal cord transection (SCT). Then, bioinformatics analyses and experimental detection showed that miR-127-3p exhibited specific effects on the regulation of neurite outgrowth and the induction of neuronal apoptosis by regulating the expression of the mitochondrial membrane protein mitoNEET. Moreover, knockdown of MitoNEET leaded to neuronal loss and apoptosis in primary cultured spinal neurons. This study therefore revealed that miR-127-3p, which targets mitoNEET, plays a vital role in regulating neurite outgrowth and neuronal apoptosis after SCT. Thus, modificatioin of the mitoNEET expression, such as mitoNEET activition may provide a new strategy for the treatment of SCI in preclinical trials.