The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.
Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three-and ®ve-stranded b-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel b-sheet and two disul®de bonds. MIA is also the ®rst extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with ®bronectin and that the peptide ligands identi®ed for MIA exhibit a matching sequence to type III human ®bronectin repeats, especially to FN14, which is close to an integrin a 4 b 1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of ®bronectin competes with integrin binding, thus detaching cells from the extracellular matrix.
The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC).The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.
The selective removal of high-abundance proteins is considered to be an important prerequisite for a sensitive proteome analysis in plasma. In this study, we examined the "multiaffinity removal system", an immunoaffinity depletion column targeted against six plasma proteins. As determined by sandwich ELISA, the depletion rate for each target protein is >99% over 200 cycles of regeneration. Our data give evidence that two column antibodies are slowly inactivated during the repeated use of the column; however, the individual depletion rate meets the specification of the manufacturer. To estimate a potential loss of analytes after the immunodepletion, we performed spiking/recovery experiments with a selection of tumor markers at concentrations in the lower to medium ng/mL range. The average recovery of 9 out of 11 markers is 78%. A significant proportion of two other markers binds to the column. Based on the average marker recovery and a depletion of ;85% of the total protein we estimate a five-fold enrichment of a potential biomarker by the use of this depletion column. We conclude that the selective depletion of plasma proteins by immunoaffinity chromatography is a valid strategy for the enrichment of potential biomarkers sought by proteomics methodologies.
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