Mnt Protein Research Articles

Structure-Based Screening Reveals a Ligand That Stabilizes the [2Fe-2S] Clusters of Human mitoNEET and Reduces Ovarian Cancer Cell Proliferation.

Human NEET proteins play an important role in a variety of diseases, including cancer. Using the recently published X-ray structure of the human mNT-M1 complex, we screened a commercial chemical compound library and identified a new human mitoNEET (mNT) binding ligand (NTS-01). Biochemical investigations revealed that NTS-01 specifically binds to the human mNT protein and stabilizes its [2Fe-2S] clusters under oxidative conditions in vitro. Treatment of ovarian cancer cells with NTS-01 induces ovarian cancer (SKOV-3) mitochondrial fragmentation (fission) and reduces ovarian cancer cell proliferation in a 2D single-layer cell culture, as well as in a 3D-spheroids culture. The NTS-01 molecule represents therefore a new lead compound for further drug design studies attempting to develop efficient treatment against ovarian cancer.

The role of the Myc family member, Mnt, in OX40-promoted T cell survival (P4526)

Abstract Together with T cell receptor recognition of cognate MHC-peptide complexes, signaling through OX40 promotes late-stage proliferation, survival, and enhanced memory formation of T cells. Despite its known role in co-stimulation, the intracellular signaling pathways regulated by OX40 are not well understood. Recently, we demonstrated that stimulation with an OX40 agonist increases Mnt protein levels by promoting Mnt protein stability. Mnt is thought to play a role in apoptosis, cell survival, and cell proliferation by opposing Myc activity through competition for binding to Max and shared target genes. Using siRNA-directed knockdown of Mnt, we demonstrated that Mnt is necessary for T cell survival ex vivo. In these current studies, we have examined the necessity of Mnt in OX40 signaling and function in the primary T cell response following vaccination. Using OX40-cre/ Mnt-floxed mouse model, we found that Mnt is required for the OX40-mediated enhancement of the primary CD4 T cell response following Listeria monocytogenes vaccination. In addition, we found that OX40 signaling decreased glucose uptake in T cells, a predicted consequence of Mnt upregulation and its antagonism of Myc-mediated glycolytic activity. Hence, future studies will examine if the glycolytic pathway is altered in Mnt knock-out T cells stimulated with OX40 agonists.

OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen‐stimulated T cells leading to an increase in cell survival

OX40 engagement on activated T cells leads to increased proliferation, expansion and survival of Ag-specific T cells. Direct ex vivo examination of Ag-stimulated murine T cells show that the Myc antagonists, Mxd4 and Mnt, are transiently upregulated and translocated to the nucleus following OX40 engagement and may be involved in suppressing cell death. Both Mxd4 and Mnt are upregulated following OX40 stimulation through increased protein stability and we identify a critical phosphorylation site in Mxd4 that controls Mxd4 stability. The upregulation of Mxd4 and Mnt contributes to OX40-mediated T-cell survival because siRNA knockdown of Mxd4 and Mnt led to increased cell death. We hypothesize the upregulation of c-Myc following OX40 engagement drives T-cell proliferation and that upregulation of Mxd4 and Mnt suppresses Myc-dependent cell death. Thus, Mxd4 and Mnt upregulation following OX40 engagement most likely increases T-cell survival.

Abstract A65: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT

Abstract Intratumoral hypoxia is well known as a hallmark of most solid tumors. Here, we studied expression level of about 200 microRNAs in a panel of cancer cell lines from different tissues, and identified a microRNA miR210, its expression was prominently up-regulated by hypoxia. Over expression of miR210 in cancer cell lines partially reversed the hypoxic gene expression signature and allowed cells to escape from hypoxia-induced cell-cycle arrest. We then identified MNT, a known MYC antagonist, as a key miR-210 target. miR-210 directly down-regulated MNT transcript and protein via recognition sites in the transcript 3′ UTR. Consistent with a role for MNT as a miR-210 target, RNA interference-mediated knockdown of MNT phenocopied miR-210 over expression cell cycle effect. Furthermore, loss of MYC abolished the ability of miR-210 to override hypoxia-induced cell-cycle arrest. Thus, miR-210 could function as a modulator in cancer cells in response to hypoxic conditions through Myc network. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A65.

The role of Mad, Mnt and c-Myc proteins in OX40 stimulated T cells (33.26)

Abstract We sought to determine the molecular mechanisms by which OX40 mediates T cell function. Gene array analysis of OX40-activated Ag-specific T cells suggested a role for the transcription factor c-Myc and antagonist Mad family proteins. D011.10 T cells were adoptively transferred into BALB/c recipients and immunized with soluble OVA and an agonist anti-OX40 antibody, which enhances Ag-specific T cell proliferation and survival. Western blot analysis of Ag-specific T cells purified ex vivo revealed that Mad4, Mnt and c-Myc proteins were up-regulated after OX40 engagement. Mad4 and Mnt protein levels peaked at days 3-4 and decreased as the cells contracted in size. C-Myc protein levels remained unchanged over time after OX40 stimulation. We hypothesize that the up-regulation of c-Myc in activated T cells drives proliferation and consequently activation-induced cell death, which is counteracted by Mad4 and Mnt proteins after anti-OX40 engagement, allowing T cells to survive the "blast crisis" phase. Indeed, siRNA knockdown of Mad4 and Mnt proteins led to decreased survival of OX40-activated T cells. We also found that OX40 stabilized both Mad4 and Mnt proteins against degradation and we demonstrated the presence of a serine residue in Mad4 that was essential in mediating accelerated protein degradation. These data provide evidence that Mad4 and Mnt play a role to enhance memory T cell survival after OX40 engagement.

Changes in Differential Gene and Protein Expression Patterns during Development of Cord Blood (CB) Versus Adult Peripheral Blood (APB) Monocyte (Mo) into Mature Dendritic Cells (mDC): Insight into Immaturity of CB DC Immunity.

It has been recognized that dysfunction of CB immune system is in part due to the immaturity of CB cellular immunity (Cairo, Blood,1997). The molecular mechanisms associated with the immaturity of CB cellular immunity including DC subset remain to be defined. The maturation status of DC greatly influences its antigen presentation capacity. Recently, we have utilized oligonucleotide microarray to demonstrate differential gene expression profiles of CB vs APB Mo (Jiang/Cairo, JI, 2004). In the current study, differential expressed genes and proteins were examined in Mo-derived CB vs. APB DC during DC developmental stages: Mo, immature DC (iDC) and mDC, by utilizing oligonucleotide microarray and proteomics. Briefly, Mo isolated from CB or APB and cultured for 8 days with GM-CSF/IL-4 (iDC) and further stimulated with LPS (mDC). Oligonucleotide microarray was carried out using U133A gene chip (Affymetrix). The representative differentially expressed genes resulted from microarray analysis were selected and analyzed by quantitative RT-PCR (Roche). The proteomic technique was conducted by liquid chromatography (LC) and mass spectrometry (MS) (Lim, Mol Cell Proteomics, 2006). The differentially expressed proteins were compared in CB vs. APB for iDC and mDC. We identified different gene expression patterns that were significantly lower in CB vs. APB in different stages during DC differentiation: Mo, iDC and mDC. These differentially expressed genes included RELA (5F), JUNB (6F), IRF-1 (3F) in Mo; CREB5 (3F), MAP7 (5F), IL1R2 (6F) in iDC; and HLA-DQA1 (4F), CD80 (3F), IRF-5 (3F) in mDC. The proteomic studies demonstrated Tyrosine Kinase Fer (12.5F), Actin regulator 3 (2.5F), Rap guanine nucleotide exchange factor 1 (2.4F) and Myeloid cell nuclear differentiation antigen (1.5F) were expressed higher in APB vs.CB iDC, while MAX binding protein MNT (5.5F), IRS2 (2.2F) and Zinc-Finger Proteins (514, 212, 462) (3–14F) were expressed higher in CB vs. APB iDC. Further, the proteomic results also indicated other Zinc-Finger Proteins (292, 221, 474) (2–5F), Fibrillin 1 precursor (2.5F) and interleukin-4 (7.7F) were expressed higher in APB vs. CB mDC. In contrast, cyclin I (3F), Rb-like protein 2 (4.35 F) and PKC theta (2F) were significantly lower in APB vs. CB DC. Moreover, the comparison of CB vs. APB DC antigen presenting activity by ELISPOT was performed and the influenza-peptide loaded CB-mDC demonstrated weaker ability to induce T cells to produce IFNg compared with APB-mDC. In summary, these differentially expressed genes in Mo (RELA, JUN) may play key roles in initiating Mo differentiation toward DC. The increased expression of genes in APB vs. CB iDC, like CREB5, IL1R2, may be involved in mediating maturation process of iDC to mDC. Lastly, the elevated expression of genes in APB vs. CB mDC, such as HLA-DQA1, CD80, IRF5 among others, may be likely to control mDC functionality as demonstrated by weaker antigen presenting activity of CB vs. APB mDC. We postulate that decreased expression of specific genes in CB vs. APB DC during DC developmental stages may in part be responsible for the lack of maturity of CB, and ultimately may partially be responsible for differential CB vs. APB innate and adaptive immunity.

Of Myc and Mnt

Deregulation of Myc expression is a common feature in cancer and leads to tumor formation in experimental model systems. There are several potential barriers that Myc must overcome in order to promote tumorigenesis, including its propensity to sensitize many cell types to apoptotic cell death. Myc activities appear also to be constrained and fine-tuned by a set of proteins that include the Mxd (formerly named Mad) family and the related protein Mnt. Like Myc-family proteins, Mxd and Mnt proteins use Max as a cofactor for DNA binding. But Mnt-Max and Mxd-Max complexes are transcriptional repressors and can antagonize the transcriptional activation function of Myc-Max. Studies examining the relationship between Myc, Mxd and Mnt proteins suggest that whereas Mnt plays a general role as a Myc antagonist, Mxd proteins have more specialized roles as Myc antagonist that is probably related to their more restricted expression patterns. The interplay between these proteins is postulated to fine-tune Myc activity for cell-cycle entry and exit, proliferation rate and apoptosis.

The Transcriptional Repressor dMnt Is a Regulator of Growth in Drosophila melanogaster

The Myc-Max-Mad/Mnt network of transcription factors has been implicated in oncogenesis and the regulation of proliferation in vertebrate cells. The identification of Myc and Max homologs in Drosophila melanogaster has demonstrated a critical role for dMyc in cell growth control. In this report, we identify and characterize the third member of this network, dMnt, the sole fly homolog of the mammalian Mnt and Mad family of transcriptional repressors. dMnt possesses two regions characteristic of Mad and Mnt proteins: a basic helix-loop-helix-zipper domain, through which it dimerizes with dMax to form a sequence-specific DNA binding complex, and a Sin-interacting domain, which mediates interaction with the dSin3 corepressor. Using the upstream activation sequence/GAL4 system, we show that expression of dMnt results in an inhibition of cellular growth and proliferation. Furthermore, we have generated a dMnt null allele, which results in flies with larger cells, increased weight, and decreased life span compared to wild-type flies. Our results demonstrate that dMnt is a transcriptional repressor that regulates D. melanogaster body size.

Genomic Binding and Transcriptional Regulation by the Drosophila Myc and Mnt Transcription Factors

Deregulated expression of members of the myc oncogene family has been linked to the genesis of a wide range of cancers, whereas their normal expression is associated with growth, proliferation, differentiation, and apoptosis. Myc proteins are transcription factors that function within a network of transcriptional activators (Myc) and repressors (Mxd/Mad and Mnt), all of which heterodimerize with the bHLHZ protein Mad and bind E-box sequences in DNA. These transcription factors recruit coactivator or corepressor complexes that in turn modify histones. Myc, Mxd/Max, and Mnt proteins have been thought to act on a specific subset of genes. However, expression array studies and, most recently, genomic binding studies suggest that these proteins exhibit widespread binding across the genome. Here we demonstrate by immunostaining of Drosophila polytene chromosome that Drosophila Myc (dMyc) is associated with multiple euchromatic chromosomal regions. Furthermore, many dMyc-binding regions overlap with regions containing active RNA polymerase II, although dMyc can also be found in regions lacking active polymerase. We also demonstrate that the pattern of dMyc expression in nuclei overlaps with histone markers of active chromatin but not pericentric heterochromatin. dMyc binding is not detected on the X chromosome rDNA cluster (bobbed locus). This is consistent with recent evidence that in Drosophila cells dMyc regulates rRNA transcription indirectly, in contrast to mammalian cells where direct binding of c-Myc to rDNA has been observed. We further show that the dMyc antagonist dMnt inhibits rRNA transcription in the wing disc. Our results support the view that the Myc/Max/Mad network influences transcription on a global scale.

Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells.

Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation.

Simulation and analysis of in vitro DNA evolution.

We study theoretically the in vitro evolution of a DNA sequence by binding to a transcription factor. Using a simple model of protein-DNA binding and available binding constants for the Mnt protein, we perform large-scale, realistic simulations of evolution starting from a single DNA sequence. Varying the evolution parameters reveals three different regimes characterized by distinct evolutionary behaviors, and for each regime we find analytical estimates which agree well with simulation results. We also study how the details of the DNA-protein interaction affect the evolution.

Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.

The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes.

Analysis of the Max-binding protein MNT in human medulloblastomas.

Medulloblastomas (MBs) are the most frequent malignant brain tumors in children. The molecular pathogenesis of these tumors is still poorly understood. Microsatellite and restriction-fragment-length polymorphism studies have revealed allelic loss of genetic material on the short arm of chromosome 17 in the region 17p13 in approximately 50% of MBs, suggesting the presence of a tumor-suppressor gene in this region. A candidate for this putative tumor-suppressor is the MNT gene, located at 17p13.3 and encoding a Max-interacting nuclear protein with transcriptional-repressor activity. In this study, we analyzed MNT mRNA and protein expression in 44 MB samples, including 32 primary tumors, 3 recurrent tumors and 9 MB cell lines. Allelic loss at 17p13.3 was found in 49% of informative cases. RT-PCR showed MNT mRNA expression in all cases analyzed. Endogenous Mnt protein with an apparent molecular weight of 72 to 74 kDa was detected in lysates from MB cell lines. The presence and functional integrity of Mnt in MBs were tested in electrophoretic mobility-shift assays. These experiments demonstrated that Mnt interacts with Max, and that this heterodimer binds DNA specifically, suggesting a functional bHLHZip domain of MB-derived Mnt. In support, single-strand conformation-polymorphism (SSCP) analyses revealed no mutation in the bHLHZip region. Deletion of the Mnt Sin3 interaction domain was shown to convert Mnt from an inhibitor of myc/ras-co-transformation into a molecule capable of cooperating with Ras in transformation. This region therefore was screened for mutation by SSCP: again, no alterations were found. These findings indicate that the MNT gene located at 17p13.3 is not likely to be involved in the molecular pathogenesis of MBs.

Quantitative specificity of the Mnt repressor

The Mnt protein of Salmonella phage P22 binds site-specifically to its operator. To better understand this binding we used dideoxy DNA sequencing in a quantitative manner to determine the relative binding constants, and hence the relative free energies, of wild-type Mnt protein to a substantial number of variants of its operator. These measurements were supported by experiments which used the SELEX procedure to generate a set of operators from an initially randomized population. In the Discussion we show that the present model of Mnt protein/operator binding, due to Sauer and co-workers, along with the assumption of an independent contribution of each position in the operator to the total binding, provides a reasonably accurate description of the system. We also discuss the use of information content as a measure of DNA-protein binding specificity with the Mnt protein/operator system serving as an example and show again that the assumption of independence supports the current view of this case of site-specific binding.

Genetic analysis of the Rhizobium meliloti nifH promoter, using the P22 challenge phage system.

In several genera of bacteria, the sigma54-RNA polymerase holoenzyme (E sigma54) is a minor form of RNA polymerase that is responsible for transcribing genes whose products are involved in diverse metabolic processes. E sigma54 binds to the promoters of these genes to form a closed promoter complex. An activator protein is required for the transition of this closed promoter complex to an open complex that is transcriptionally competent. In this study, the P22-based challenge phage system was used to investigate interactions between E sigma54 and the Rhizobium meliloti nifH promoter. Challenge phages were constructed in which the R. meliloti nifH promoter replaced the binding site for the Mnt protein, a repressor of the phage P22 ant gene. When a Salmonella typhimurium strain that overexpressed sigma54 was infected with these challenge phages, E sigma54 bound to the nifH promoter and repressed transcription of the ant gene as seen by the increased frequency of lysogeny. Following mutagenesis of challenge phages that carried the R. meliloti nifH promoter, mutant phages that could form plaques on an S. typhimurium strain that overexpressed sigma54 were isolated. These phages had mutations within the nifH promoter that decreased the affinity of the promoter for E sigma54. The mutations were clustered in seven highly conserved residues within the -12 and -24 regions of the nifH promoter.

Mnt: a novel Max-interacting protein and Myc antagonist.

We have identified a novel Max-binding protein, Mnt, which belongs to neither the Myc nor the Mad families (Hurlin et al. 1997). Mnt interacts with Max in vivo and functions as a transcriptional repressor of reporter genes containing promoter-proximal CACGTG sites. Mnt:Max complexes also efficiently suppress Myc-dependent activation from the same promoter. Transcription repression by Mnt maps to a 13 amino acid N-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. This region of Mnt mediates interaction with mSin3 corepressor proteins and its deletion converts Mnt from a repressor to an activator and from a suppressor of Myc-dependent transformation to a cooperating oncogene. This latter result suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. Expression of mnt RNA is observed in many tissues and in both proliferating and differentiating cells. Likewise, Mnt protein is expressed in many proliferating cell types in culture where both Myc:Max and Mnt:Max complexes are detected. An exception is P19 embryonal carcinoma cells, where Mnt is expressed and in a complex with Max, but Myc proteins are not detected. Mnt is likely to be a key regulator of Myc activities in vivo and, in addition, may possess Myc-independent functions.

Specificity of the Mnt protein determined by binding to randomized operators.

The relative binding affinities of Mnt protein from bacteriophage P22 are determined for each possible base pair at position 17 of the operator. These are determined from the partitioning of randomized operators into bound and unbound fractions; quantitation is provided by restriction enzyme analysis. Mnt protein is found to have an unusual specificity at this position: a C.G base pair (the wild-type operator) has the highest affinity, a G.C base pair has the lowest affinity, and both orientations of A.T base pairs are intermediate and nearly equivalent. A specific binding constant and specific binding free energy are defined and shown to be directly related to the information content of the operator sequences bound to the protein, taking into account the quantitative differences in binding affinities.

Primary structure of the immI immunity region of bacteriophage P22

The DNA sequence of the immI immunity region of bacteriophage P22 has been determined. This region includes the ant gene, which encodes the P22 antirepressor protein, and the mnt and arc genes, which encode proteins that negatively regulate antirepressor synthesis. We have purified antirepressor protein and selected tryptic peptides of antirepressor, and have determined the amino terminal sequences and amino acid composition of these molecules. These data, in combination with the DNA sequence, locate the ant gene and define the complete amino acid sequence of antirepressor (300 residues). The mnt and arc genes have been located by sequencing the mnt-am343 and arc-amH1605 mutations. The Mnt and Arc proteins are predicted to be small, basic polypeptides that are homologous in amino acid sequence. The Mnt protein also shows significant sequence homology with the lambda Cro protein. The arc and ant genes are transcribed rightward from the Pant promoter, while mnt is transcribed leftward from a promotor that may overlap Pant. The Mnt protein apparently acts by binding to an operator site located immediately adjacent to the startpoint of Pant transcription.