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Supplementary Figure 2 from MDM2 Regulates Dihydrofolate Reductase Activity through Monoubiquitination

MDM2 is a ubiquitin ligase that is best known for its essential function in the negative regulation of p53. In addition, MDM2 expression is associated with tumor progression in a number of common cancers, and in some cases, this has been shown to be independent of p53 status. MDM2 has been shown to promote the degradation of a number of other proteins involved in the regulation of normal cell growth and proliferation, including MDM4 and RB1. Here, we describe the identification of a novel substrate for the MDM2 ubiquitin ligase: dihydrofolate reductase (DHFR). MDM2 binds directly to DHFR and catalyses its monoubiquitination and not its polyubiquitination. In addition, MDM2 expression reduces DHFR activity in a p53-independent manner, but has no effect upon the steady-state level of expression of DHFR. We show that changes in MDM2 expression alter folate metabolism in cells as evidenced by MDM2-dependent alteration in the sensitivity of cells to the antifolate drug methotrexate. Furthermore, we show that the ability of MDM2 to inhibit DHFR activity depends upon an intact MDM2 RING finger. Our studies provide for the first time a link between MDM2, an oncogene with a critical ubiquitin ligase activity and a vital one-carbon donor pathway involved in epigenetic regulation, and DNA metabolism, which has wide ranging implications for both cell biology and tumor development. [Cancer Res 2008;68(9):3232–41]

Supplementary Figure 1 from MDM2 Regulates Dihydrofolate Reductase Activity through Monoubiquitination

The Serious Illness Care Programme is a complex intervention developed in the US to improve communication between clinicians and patients with serious illness. In collaboration with Aridane Labs the programme has been developed for use within the UK, with pilot implementation funded by NHS England. Within the programme, clinicians are trained in use of a Serious Illness Conversation Guide (the guide), to structure conversations with patients/families. A feasibility of the UK programme is underway, within one North West cancer centre. Aim To present interim results from the feasibility study Methods Data on the use and experience of the UK programme, from the perspective of clinicians and patients Quantitative Data: Questionnaires: Clinicians – pre/post training: confidence; attitudes to care of the dying; acceptability of serious illness care programme Patients – pre/post serious illness conversation: Quality of clinical communication; Quality of life; Anxiety; depression. Qualitative Data: In depth interviews: Clinicians: experience of the UK programme, education and training and engaging patients in serious illness conversations Patients: experience of engaging in serious illness conversations, including their acceptability of the approach Results Quantitative Data: Results will be presented to assess the feasibility and acceptability of the UK programme: Proportion of patients identified conversations and participation in study measures Illustrate any indicative improvements in communication and patient outcomes. Qualitative Data: Thematic Analysis to explore perceptions and experience of the programme: results will highlight main themes, such as: Conversation Value; Holistic and Compassionate; Opening up conversations with family members; Breaking the ice; Timing of conversation and Prognosis. Conclusion Results from this study suggest these conversations promote shared decision making and individualised care planning, and espouse 9holistic9 and 9person centred9 care. Further research and evaluation is required to understand the effect of the UK programme on patient outcomes.

MDM2 expression, combined with increased p53 expression, is associated with reduced survival in several cancers, but is particularly of interest in renal cell carcinoma (RCC) where evidence suggests the presence of tissue-specific p53/MDM2 pathway defects. We set out to identify MDM2-interacting proteins in renal cells that could act as mediators/targets of MDM2 oncogenic effects in renal cancers. We identified the non-metastatic cells 2, protein; NME2 (NDPK-B, NM23-B/-H2), a nucleoside diphosphate kinase, as an MDM2-interacting protein using both a proteomic-based strategy [affinity chromatography and tandem mass spectrometry [MS/MS] from HEK293 cells] and a yeast two-hybrid screen of a renal carcinoma cell-derived complementary DNA library. The MDM2-NME2 interaction is highly specific, as NME1 (87.5% amino acid identity) does not interact with MDM2 in yeast. Specific NME proteins display well-documented cell motility and metastasis-suppressing activity. We show that NME2 contributes to motility suppression under conditions where MDM2 is expressed at normal physiological/low levels. However, up-regulation of MDM2 in RCC cells abolishes the ability of NME2 to suppress motility. Significantly, when MDM2 expression is down-regulated in these cells using small interfering RNA, the motility-suppressing activity of NME2 is rescued, confirming that MDM2 expression causes the loss of NME2 cell motility regulatory function. Thus MDM2 up-regulation in renal cancer cells can act in a dominant manner to abrogate the function of a potent suppressor of motility and metastasis. Our studies identify a novel protein-protein interaction between MDM2 and NME2, which suggests a mechanism that could explain the link between MDM2 expression and poor patient survival in RCC.

Mycobacterium tuberculosis produces two homologous chaperonin (Cpn)60 proteins, Cpn60.1 and Cpn60.2 (Hsp65). Both proteins stimulate human and murine monocyte cytokine synthesis but, unlike Cpn60 proteins from other microbial species, fail to stimulate the breakdown of cultured murine bone. Here, we have examined the mechanism of action of these proteins on bone remodelling and osteoclastogenesis, induced in vitro in murine calvarial explants and the murine monocyte cell line RAW264.7. Additionally, we have determined their effect on bone remodelling in vivo in an animal model of arthritis. Recombinant Cpn60.1 but not Cpn60.2 inhibited bone breakdown both in vitro, in murine calvaria and in vivo, in experimental arthritis. Analysis of the mechanism of action of Cpn60.1 suggests that this protein works by directly blocking the synthesis of the key osteoclast transcription factor, nuclear factor of activated T cells c1. The detection of circulating immunoreactive intact Cpn60.1 in a small number of patients with tuberculosis but not in healthy controls further suggests that the skeleton may be affected in patients with tuberculosis. Taken together, these findings reveal that M. tuberculosis Cpn60.1 is a potent and novel inhibitor of osteoclastogenesis both in vitro and in vivo and a potential cure for bone-resorptive diseases like osteoporosis.

We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems.

Heat-shock protein (hsp)60/chaperonin 60 is a potent immunogen which has recently been claimed to have cell-signalling actions upon myeloid and vascular endothelial cells. The literature is controversial with different chaperonin 60 proteins producing different patterns of cellular activation and the ever-present criticism that activity is the result of bacterial contaminants. To clarify the situation we have cloned, expressed and purified to homogeneity the chaperonin 60 proteins from Chlamydia pneumoniae, Helicobacter pylori and the human mitochondrion. These highly purified proteins were compared for their ability to stimulate human peripheral blood mononuclear cell (PBMC) cytokine synthesis and vascular endothelial cell adhesion protein expression. In spite of their significant sequence homology, the H. pylori protein was the most potent PBMC activator with the human protein the least potent. PBMC activation by C. pneumoniae and human, but not H. pylori, chaperonin 60 was blocked by antibody neutralization of Toll-like receptor-4. The C. pneumoniae chaperonin 60 was the most potent endothelial cell activator, with the human protein being significantly less active than bacterial chaperonin 60 proteins. These results have implications for the role of chaperonin 60 proteins as pathological factors in autoimmune and cardiovascular disease, and raise the possibility that each of these proteins may result in different pathological effects in such diseases.

The Chaperonin 60 (Cpn60) proteins have, in addition to their well-known functions of protein folding and protection, a range of intercellular signalling activities. As part of a study to investigate the biological activity of the Cpn60 proteins, particularly from pathogenic organisms, we have cloned and expressed three Cpn60 proteins from Homo sapiens, Helicobacter pylori and Chlamydia pneumoniae. The Cpn60 proteins were purified to apparent homogeneity using a combination of nickel column affinity chromatography and Reactive Red dye affinity columns. Insoluble protein was solubilised using 8 M urea and then re-folded on the nickel column by stepwise removal of the urea. The immunostimulant LPS was removed by addition of the antibiotic polymyxin B as part of the purification process.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. To improve prognostication, a better understanding of critical genes associated with disease progression is required. The objective of this review was to focus attention on 2 such genes, p53 and murine double minute 2 (MDM2), and to provide a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation. First, increased p53 expression is tightly linked with increased MDM2 expression; and, second, patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. Further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions.

MDM2 is a ubiquitin ligase that is best known for its essential function in the negative regulation of p53. In addition, MDM2 expression is associated with tumor progression in a number of common cancers, and in some cases, this has been shown to be independent of p53 status. MDM2 has been shown to promote the degradation of a number of other proteins involved in the regulation of normal cell growth and proliferation, including MDM4 and RB1. Here, we describe the identification of a novel substrate for the MDM2 ubiquitin ligase: dihydrofolate reductase (DHFR). MDM2 binds directly to DHFR and catalyses its monoubiquitination and not its polyubiquitination. In addition, MDM2 expression reduces DHFR activity in a p53-independent manner, but has no effect upon the steady-state level of expression of DHFR. We show that changes in MDM2 expression alter folate metabolism in cells as evidenced by MDM2-dependent alteration in the sensitivity of cells to the antifolate drug methotrexate. Furthermore, we show that the ability of MDM2 to inhibit DHFR activity depends upon an intact MDM2 RING finger. Our studies provide for the first time a link between MDM2, an oncogene with a critical ubiquitin ligase activity and a vital one-carbon donor pathway involved in epigenetic regulation, and DNA metabolism, which has wide ranging implications for both cell biology and tumor development. [Cancer Res 2008;68(9):3232–41]

It is established that the molecular chaperone, chaperonin 60, from various bacteria and from Homo sapiens has cell-cell signalling activity and is able to induce proinflammatory cytokine synthesis. We previously reported that chaperonin 60 proteins from Gram-negative bacteria, but not mycobacteria, have the capacity to resorb cultured murine calvarial bone. We now report that lipopolysaccharide-low human recombinant chaperonin 60 (Hsp60) is a relatively weak cytokine-inducing agonist but is a potent stimulator of murine calvarial bone resorption. The osteolytic activity of Hsp60 was significantly inhibited by indomethacin, interleukin-1 receptor antagonist, and osteoprotegerin, but 5-lipoxygenase inhibitors were less effective. Analysis of Hsp60 truncation mutants revealed that N-terminal mutants (Delta1-137, Delta1-358, and Delta1-465) retained bone resorbing activity. In contrast, a C-terminal truncation mutant (Delta1-26 + Delta466-573) was inactive. This suggests that the active domain in this protein is found within residues 466-573. It is now established that Hsp60 is present in the blood of the majority of the population with the normal range encompassing levels able to activate bone cells. The possibility exists that this protein could play a role in bone remodelling.

The cell biology of the chaperonins (Cpns) has been intensively studied over the past 25 years. These ubiquitous and essential molecules assist proteins to fold into their native state and function to protect proteins from denaturation after stress. The structure of the most widely studied Cpn60, Escherichia coli GroEL, has been solved and its mechanism of protein folding action largely established. But in the last decade, evidence has accumulated to suggest that the Cpn60s have functions in addition to intracellular protein folding, particularly the ability to act as intercellular signals with a wide variety of biological effects. Cpn60 has the ability to stimulate cells to produce proinflammatory cytokines and other proteins involved in immunity and inflammation and may, therefore, provide a link between innate and adaptive immunity. Cpn60s are also thought to be pathogenic factors in a wide range of diseases and have recently been reported to be present in the circulation of normal subjects and those with heart disease. An interesting facet of these proteins is the finding that in spite of significant sequence conservation, individual Cpn60 proteins can express very different biological activities. This review discusses the work to date, which has revealed the cell-cell signaling actions of Cpn60 proteins.

The cell biology of the chaperonins (Cpns) has been intensively studied over the past 25 years. These ubiquitous and essential molecules assist proteins to fold into their native state and function to protect proteins from denaturation after stress. The structure of the most widely studied Cpn60, Escherichia coli GroEL, has been solved and its mechanism of protein folding action largely established. But in the last decade, evidence has accumulated to suggest that the Cpn60s have functions in addition to intracellular protein folding, particularly the ability to act as intercellular signals with a wide variety of biological effects. Cpn60 has the ability to stimulate cells to produce proinflammatory cytokines and other proteins involved in immunity and inflammation and may, therefore, provide a link between innate and adaptive immunity. Cpn60s are also thought to be pathogenic factors in a wide range of diseases and have recently been reported to be present in the circulation of normal subjects and those with heart disease. An interesting facet of these proteins is the finding that in spite of significant sequence conservation, individual Cpn60 proteins can express very different biological activities. This review discusses the work to date, which has revealed the cell-cell signaling actions of Cpn60 proteins.