Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.Multiple endocrine neoplasia type 1 is a rare, autosomal dominant tumor syndrome, typically defined by the presence of tumors in at least two of the following three tissues: the parathyroid, enteropancreatic endocrine tissue, and the anterior pituitary (reviewed in reference 46). The human MEN1 gene was identified by positional cloning in 1997 (13) and shown to encode a 610-amino-acid product (menin) with predominantly nuclear localization (24). Loss of heterozygosity in the region containing the MEN1 locus has been observed in tumor tissue obtained from individuals with multiple endocrine neoplasia type 1 (39) and from mice with an engineered deletion in one of their Men1 alleles (16), predictive of a tumor suppressor function. This is supported by the detection of MEN1 frameshift or nonsense mutations in approximately 70% of human multiple endocrine neoplasia type 1 tumors (46). Menin overexpression has also been shown to diminish the tumorigenic phenotype of Ras-transformed NIH 3T3 cells (36), consistent with its putative tumor suppressor function. Knockout of both Men1 alleles in mice has been shown to result in embryonic lethality (16), suggesting that menin is also important for early development. However, the absence of significant homology to other proteins has complicated efforts to elucidate the function(s) of menin and/or the mechanisms of its tumor suppressor activity.A number of menin-interacting proteins have been identified in an effort to obtain clues about menin function, including the AP-1 transcription factor JunD (2), the putative tumor metastasis suppressor/nucleoside diphosphate kinase nm23 (54), the...
Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1-like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF-kB, PEM, SMAD3, RPA2, FANCD2, NM23beta, nonmuscle myosin heavy chain II-A, GFAP, and/or vimentin. These partners have not clarified menin's pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun-kinase pathway. The Men1+/- mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.
SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.
Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.
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