The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

• MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• genetická transkripce MeSH
• imidazoly farmakologie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• melanom genetika   metabolismus   patologie   MeSH
• MFC-7 buňky MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• piperaziny farmakologie   MeSH
• pluripotentní kmenové buňky metabolismus   MeSH
• proteiny buněčného cyklu biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny biosyntéza   genetika   metabolismus   MeSH
• roskovitin farmakologie   MeSH
• sulfonamidy farmakologie   MeSH
• synergismus léků MeSH
• transfekce MeSH
• triaziny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atuveciclib MeSH Prohlížeč
• CDK9 protein, human MeSH Prohlížeč
• Cdk9 protein, mouse MeSH Prohlížeč
• cyklin-dependentní kinasa 9 MeSH
• imidazoly MeSH
• inhibitory proteinkinas MeSH
• MDM2 protein, human MeSH Prohlížeč
• Mdm2 protein, mouse MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• Mdm4 protein, mouse MeSH Prohlížeč
• nutlin 3 MeSH Prohlížeč
• piperaziny MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• roskovitin MeSH
• sulfonamidy MeSH
• triaziny MeSH

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.

• Klíčová slova
• acute myeloid leukaemia, murine double minute 2 (MDM2), murine double minute 4 (MDM4), myelodysplastic syndrome, p53,
• MeSH
• akutní myeloidní leukemie * genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• myelodysplastické syndromy * genetika   MeSH
• myši MeSH
• nádorový supresorový protein p53 genetika   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MDM4 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH

BACKGROUND: HDM2 and HDMX proteins are key negative regulators of the tumor suppressor p53. Under normal conditions, p53 protein expression is maintained at a low level, whereas under stress conditions, this negative regulation is alleviated to increase the p53 level. HDM2 and HDMX are overexpressed in many cancer types, mainly in tumors with wild type p53, such as sarcomas. In addition to an inactivating mutation in the TP53 gene, HDM2 and HDMX overexpression represents another kind of p53 inactivation pathway. AIM: In this review, we first briefly describe the roles of HDM2 and HDMX proteins and then the increased occurrence of their overexpression and the possible causes of this overexpression in different human cancer types as well as therapeutic approaches targeting HDM2 and HDMX for the treatment of human cancer. CONCLUSION: HDM2 and HDMX are important therapeutic targets. The interruption of their negative effect on p53 pathway by compounds such as nutlins, leads to the reactivation of the p53 pathway. However, a deeper understanding of HDM2-HDMX-p53 structure and function will enable the identification of new therapeutic strategies that could help to provide more specific and more efficient therapies for cancer patients. Several small molecules and peptides are the subject of clinical testing in phase I, II and even III trials. Key words: HDM2 - HDMX - p53 signalling pathway - oncogenes - MDM2 - MDMX This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 7. 2018.

• Klíčová slova
• HDM2 - HDMX - p53 signalling pathway - oncogenes - MDM2 - MDMX This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 7. 2018, products,
• MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory farmakoterapie   metabolismus   MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.

• Klíčová slova
• E3 ubiquitin ligase substrate specificity, HDM2, HDMX, allosteric interactions, p53,
• MeSH
• buněčné linie MeSH
• imidazoly farmakologie   MeSH
• jaderné proteiny chemie   nedostatek   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• piperaziny farmakologie   MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 antagonisté a inhibitory   nedostatek   metabolismus   MeSH
• protoonkogenní proteiny chemie   nedostatek   metabolismus   MeSH
• ubikvitin chemie   metabolismus   MeSH
• ubikvitinace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imidazoly MeSH
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• nutlin 3 MeSH Prohlížeč
• piperaziny MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• ubikvitin MeSH

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

• Klíčová slova
• E3 ubiquitin ligase, MAGE, melanoma-associated antigen, MAGEA1, MDM4, MHD, MAGE homology domain, NSE, non-SMC element, NSE1-NSE3-NSE4 complex, NSE4/EID family, PCGF6, RING-finger proteins, RNF166, SMC, structure maintenance of chromosomes, TRAF6, TRIM family, TRIM, tripartite motif, TRIM31, TRIM41, TRIM8, WH, winged helix, Y2H, yeast 2-hybrid., melanoma-associated antigen family, protein evolution, ubiquitination,
• MeSH
• biologické modely MeSH
• chromatografie kapalinová MeSH
• domény RING-prstů MeSH
• HEK293 buňky MeSH
• imunoprecipitace MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• multiproteinové komplexy metabolismus   MeSH
• nádorové proteiny chemie   metabolismus   MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• peptidy chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny metabolismus   MeSH
• TRIM protein MeSH
• ubikvitinligasy chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAGE-A1 protein (278-286), human MeSH Prohlížeč
• multiproteinové komplexy MeSH
• nádorové proteiny MeSH
• NSE4 protein, human MeSH Prohlížeč
• NSMCE1 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• peptidy MeSH
• transportní proteiny MeSH
• TRIM protein MeSH
• TRIM31 protein, human MeSH Prohlížeč
• ubikvitinligasy MeSH

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

• MeSH
• ATM protein metabolismus   MeSH
• fosforylace MeSH
• jaderné proteiny chemie   fyziologie   MeSH
• lidé MeSH
• messenger RNA chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• obrácené repetice MeSH
• poškození DNA MeSH
• posttranskripční úpravy RNA MeSH
• posttranslační úpravy proteinů MeSH
• proteiny buněčného cyklu MeSH
• proteosyntéza MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• protoonkogenní proteiny chemie   fyziologie   MeSH
• regulace genové exprese MeSH
• sbalování RNA MeSH
• sekvence nukleotidů MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč

The exact role of the central acidic domain of Mdm2 in p53 degradation remains unclear. We therefore performed a systematic and comprehensive analysis of the acidic domain using a series of short deletions and found that only a minor part of the domain was indispensable for Mdm2-mediated p53 ubiquitylation. Moreover, we identified a short stretch of acidic amino acids required for p53 degradation but not ubiquitylation, indicating that, in addition to p53 ubiquitylation, the acidic domain might be involved in a critical post-ubiquitylation step in p53 degradation. Rather than representing a single functional domain, different parts of the acidic region perform separate functions in p53 degradation, suggesting that it might be possible to therapeutically target them independently.

• MeSH
• biologické modely MeSH
• delece genu MeSH
• HEK293 buňky MeSH
• imunoprecipitace MeSH
• jaderné proteiny chemie   fyziologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA * MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 chemie   genetika   fyziologie   MeSH
• protoonkogenní proteiny chemie   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů MeSH
• ubikvitin chemie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč
• ubikvitin MeSH

Tumor suppressor p53 is mutated in about 50% of cancers. Most malignant melanomas carry wild-type p53, but p53 activity is often inhibited due to overexpression of its negative regulators Mdm2 or MdmX. We performed high throughput screening of 2448 compounds on A375 cells carrying p53 activity luciferase reporter construct to reveal compounds that promote p53 activity in melanoma. Albendazole and fenbendazole, two approved and commonly used benzimidazole anthelmintics, stimulated p53 activity and were selected for further studies. The protein levels of p53 and p21 increased upon the treatment with albendazole and fenbendazole, indicating activation of the p53-p21 pathway, while the levels of Mdm2 and MdmX decreased in melanoma and breast cancer cells overexpressing these proteins. We also observed a reduction of cell viability and changes of cellular morphology corresponding to mitotic catastrophe, i.e., G2/M cell cycle arrest of large multinucleated cells with disrupted microtubules. In summary, we established a new tool for testing the impact of small molecule compounds on the activity of p53 and used it to identify the action of benzimidazoles in melanoma cells. The drugs promoted the stability and transcriptional activity of wild-type p53 via downregulation of its negative regulators Mdm2 and MdmX in cells overexpressing these proteins. The results indicate the potential for repurposing the benzimidazole anthelmintics for the treatment of cancers overexpressing p53 negative regulators.

• Klíčová slova
• Mdm2, MdmX, benzimidazoles, drug repurposing, melanoma, p53,
• MeSH
• albendazol farmakologie   MeSH
• benzimidazoly farmakologie   MeSH
• down regulace MeSH
• fenbendazol farmakologie   MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• melanom farmakoterapie   metabolismus   MeSH
• MFC-7 buňky MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• přehodnocení terapeutických indikací léčivého přípravku MeSH
• proliferace buněk účinky léků   MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• rychlé screeningové testy MeSH
• screeningové testy protinádorových léčiv MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• albendazol MeSH
• benzimidazoly MeSH
• fenbendazol MeSH
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč

Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance.

• Klíčová slova
• MDM2/MDMX, SNAI2/SLUG, TWIST, epithelial-mesenchymal transition, prostate/breast cancer,
• MeSH
• epitelo-mezenchymální tranzice fyziologie   MeSH
• fenotyp MeSH
• heterografty MeSH
• jaderné proteiny biosyntéza   MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty genetika   metabolismus   patologie   MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 biosyntéza   MeSH
• protoonkogenní proteiny biosyntéza   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH

Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo, suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.

• Klíčová slova
• E3, Mdm2, Mdm4, MdmX, RING domain ubiquitin protein ligase, dimerization, mutagenesis, p53,
• Publikační typ
• časopisecké články MeSH