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.

Ubiquitin-associated (UBA) domains are found in a large number of proteins with diverse functions involved in ubiquitination, DNA repair, and signaling pathways. Recent studies have shown that several UBA domain proteins interact with ubiquitin (Ub), specifically p62, the phosphotyrosine-independent ligand of the SH2 domain of p56 lck ; HHR23A, a human nucleotide excision repair protein; and DDI1, another damage-inducible protein. NMR chemical shift mapping reveals that Ub binds specifically but weakly to a conserved hydrophobic epitope on HHR23A UBA(1) and UBA(2) and that the UBA domains bind on the hydrophobic patch on the surface of the five-stranded ␤-sheet of Ub. Models of the UBA(1)-Ub and UBA(2)-Ub complexes obtained from de novo docking reveal different orientations of the UBA domains on the Ub surface compared with those obtained by homology modeling with the related CUE domains, which also bind Ub. Our results suggest that UBA domains may interact with Ub as well as other proteins in more than one way while utilizing the same binding surface.The lifespan of proteins inside and outside a cell is tightly regulated by the ubiquitin-proteasome system, and numerous studies show that protein degradation is tightly interlocked with cell cycle progression and is therefore an integral part of transduction pathways and other cellular processes (1-4). For protein degradation by the Ub/proteasome 1 system, the target proteins need to be tagged with a poly-Ub chain. These covalent complexes are then recognized and degraded by the 26 S proteasome (1, 2). The principle mechanism of this covalent modification has been identified: an enzyme cascade known as E1-E2-E3 is responsible for activation and transfer of Ub onto the target protein in a linkage-specific manner (1, 5).The 26 S proteasome is formed by a 20 S cylindrical proteolytically active subunit and two 19 S regulatory subunits (1, 2, 6, 7). The 19 S particles represent the lid of the proteasome and regulate the access to the proteolysis (8). Although the polyubiquitinated substrate seems to be recognized by the S5a subunit in the 19 S particle (9 -11), additional contacts between poly-Ub chains and parts of the 19 S regulatory subunit have been identified (12). Deletion studies indicate that other polyubiquitin-binding sites must exist (13).Monoubiquitination is not sufficient for targeting proteins to the proteasome, however; assembly of a poly-Ub chain of at least four Ub moieties is required to create a degradation signal (11,14,15). Although Ub contains seven lysine residues, they are not used with the same frequency in poly-Ub chain assembly. Although key steps of Ub activation and transfer to a substrate as well as the structure of the 20 S subunit of the proteasome are known, the question of how proteins are targeted to the proteasome remains unanswered. It is not known whether there is an additional mechanism to regulate the time point of degradation. One possibility is that monoubiquitination leads to a "point of no return," which proceeds to substra...

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

Low cost and simplicity of cultivating bacteria make the E. coli expression system a preferable choice for production of therapeutic proteins both on a lab scale and in industry. In addition straightforward recombinant DNA technology offers engineering tools to produce protein molecules with modified features. The lack of posttranslational modification mechanisms in bacterial cells such as glycosylation, proteolytic protein maturation or limited capacity for formation of disulfide bridges may, to a certain extent, be overcome with protein engineering. Protein engineering is also often employed to improve protein stability or to modulate its biological action. More sophisticated modifications may be achieved by genetic fusions of two proteins. This article presents a variety of examples of genetic engineering of therapeutic proteins. It emphasizes the importance of designing a construct without any unnecessary amino acid residues.

Rad23 proteins are involved both in the ubiquitin-proteasome pathway and in nucleotide excision repair (NER), but the relationship between these two pathways is not yet understood. The two human homologs of Rad23, hHR23A and B, are functionally redundant in NER and interact with xeroderma pigmentosum complementation group C (XPC) protein. The XPC-hHR23 complex is responsible for the specific recognition of damaged DNA, which is an early step in NER. The interaction of the XPC binding domain (XPCB) of hHR23A/B with XPC protein has been shown to be important for its optimal function in NER. We have determined the solution structure of XPCB of hHR23A. The domain consists of five amphipathic helices and reveals hydrophobic patches on the otherwise highly hydrophilic domain surface. The patches are predicted to be involved in interaction with XPC. The XPCB domain has limited sequence homology with any proteins outside of the Rad23 family except for sacsin, a protein involved in spastic ataxia of CharlevoixSaguenay, which contains a domain with 35% sequence identity.Keywords: Rad23; xeroderma pigmentosum; NER; DNA repair; NMR structure; chaperone; sacsin Nucleotide excision repair (NER) plays a crucial role in the prevention of mutagenesis and consequent carcinogenesis by the elimination of a wide variety of DNA lesions (Friedberg et al. 1995;Sarasin 1999;Berneburg and Lehmann 2001). NER deficiency in humans is associated with the rare disorder xeroderma pigmentosum (XP). Patients with XP exhibit increased photosensitivity, suffer from skin abnormalities, and have a >1000-fold increased frequency of sunlight-induced skin cancers (Kraemer et al. 1987;Friedberg et al. 1995). About seven different variants of the XP syndrome have been discovered (XP complementation group A to G). The XP group C (XP-C) is particularly interesting because its defect is limited to the global genome repair pathway, which eliminates lesions from the entire genome, and in contrast to other XP groups, it is not involved in the transcription-coupled repair (Friedberg et al. 1995;Sugasawa et al. 1998Sugasawa et al. , 2001). All variants of the XP syndrome appear to be caused by defects in proteins that function in NER. The protein factor related to the XP-C-variant of XP has been identified as a complex of the XPC protein and its interaction partner hHR23 (human homolog of the yeast Rad23 protein; Masutani et al. 1994). Thus, understanding how hHR23 interacts with XPC protein is important for elucidating the molecular basis of XP. Recently, some successful trials were performed to reconstitute genetically corrected skin in vitro (Arnaudeau-Begard et al. 2003). A retroviral expression of the wild-type XPC protein in the keratinocytes from patients with XP-C led to restoration of the normal DNA repair and cell survival properties after UV irradiation. This result demonstrates that knowledge about the molecular mechanism of XP syndrome may ultimately be useful in gene therapy.XPC/hHR23 was shown to bind specifically and preferentially to a n...