Lindsay Burch

Codimension and analytic spread

In this paper, I shall establish the sufficiency of certain conditions on an ideal A of a local ring Q , and on a set { g 1 …, g k } of elements of Q generating a proper ideal G , for the ideals A and G to be analytically disjoint. Hence I shall establish an upper bound for the analytic spread of A . The maximal ideal of Q will be denoted throughout by M , and it will be assumed that the field Q / M is infinite.

Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo.

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2·p53 complex using a p53 mutant lacking theBOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA·MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA·MDM2 complex within p53 is SGXLLGESXF, which links the S9–S10 β-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser261 and Leu264, which bridges the S9–S10 β-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe270 within the S10 β-sheet resulted in a mutant p53, which binds more stably to RNA·MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala19 mutation into the p53F270A protein abolished both RNA·MDM2 complex binding and hyper-ubiquitinationin vivo, thus indicating that p53F270A protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9–S10 β-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.

Loss‐of‐Function CYP2C9 Variants Improve Therapeutic Response to Sulfonylureas in Type 2 Diabetes: A Go‐DARTS Study

Sulfonylureas are metabolized mainly by the cytochrome p450 2C9 (CYP2C9) enzyme. Two CYP2C9 variants—*2 (Arg144Cys) and *3 (Ile359Leu)—are associated with reduced enzyme activity and impaired substrate metabolism. We identified 1,073 incident users of sulfonylureas in Tayside, Scotland, and assessed the impact of the combined CYP2C9*2 and CYP2C9*3 genotypes on early and sustained sulfonylurea response. We found that patients with two copies of a loss‐of‐function allele were 3.4 times (P = 0.0009) more likely to achieve a treatment hemoglobin A1c (HbA1c) level <7% than patients with two wild‐type CYP2C9 alleles. This corresponds to a 0.5% (P = 0.003) greater reduction in HbA1c concentration. In addition, *2 and *3 allele carriers were less likely to experience treatment failure with sulfonylurea monotherapy (P = 0.04; per‐allele hazard ratio 0.79; 95% confidence interval 0.63–0.99). In conclusion, CYP2C9 loss‐of‐function alleles are associated with greater response to sulfonylureas and decreased failure of therapy consistent with the pharmacokinetic role of CYP2C9.

The Proline Repeat Domain of p53 Binds Directly to the Transcriptional Coactivator p300 and Allosterically Controls DNA-Dependent Acetylation of p53

ABSTRACT The transcription coactivator p300 cannot acetylate native p53 tetramers, thus revealing intrinsic conformational constraints on p300-catalyzed acetylation. Consensus site DNA is an allosteric effector that promotes acetylation of p53, suggesting that p300 has an undefined conformationally flexible interface within the p53 tetramer. To identify such conformationally responsive p300-binding sites, p300 was subjected to peptide selection from a phage-peptide display library, a technique that can define novel protein-protein interfaces. The enriched p300-binding peptides contained a proline repeat (PXXP/PXPXP) motif, and five proline repeat motifs actually reside within the p53 transactivation domain, suggesting that this region of p53 may harbor the second p300 contact site. p300 binds in vitro to PXXP-containing peptides derived from the proline repeat domain, and PXXP-containing peptides inhibit sequence-specific DNA-dependent acetylation of p53, indicating that p300 docking to both the LXXLL and contiguous PXXP motif in p53 is required for p53 acetylation. Deletion of the proline repeat motif of p53 prevents DNA-dependent acetylation of p53 by occluding p300 from the p53-DNA complex. Sequence-specific DNA places an absolute requirement for the proline repeat domain to drive p53 acetylation in vivo. Chromatin immunoprecipitation was used to show that the proline repeat deletion mutant p53 is bound to the p21 promoter in vivo, but it is not acetylated, indicating that proline-directed acetylation of p53 is a post-DNA binding event. The PXXP repeat expands the basic interface of a p300-targeted transactivation domain, and proline-directed acetylation of p53 at promoters indicates that p300-mediated acetylation can be highly constrained by substrate conformation in vivo.

Paradoxical Lower Serum Triglyceride Levels and Higher Type 2 Diabetes Mellitus Susceptibility in Obese Individuals with the PNPLA3 148M Variant

Background Obesity is highly associated with elevated serum triglycerides, hepatic steatosis and type 2 diabetes (T2D). The I148M (rs738409) genetic variant of patatin-like phospholipase domain-containing 3 gene (PNPLA3) is known to modulate hepatic triglyceride accumulation, leading to steatosis. No association between PNPLA3 I148M genotype and T2D in Europeans has been reported. Aim of this study is to examine the relationship between PNPLA3 I148M genotypes and serum triglycerides, insulin resistance and T2D susceptibility by testing a gene-environment interaction model with severe obesity. Methods and Findings PNPLA3 I148M was genotyped in a large obese cohort, the SOS study (n = 3,473) and in the Go-DARTS (n = 15,448), a T2D case-control study. Metabolic parameters were examined across the PNPLA3 I148M genotypes in participants of the SOS study at baseline and at 2- and 10-year follow up after bariatric surgery or conventional therapy. The associations with metabolic parameters were validated in the Go-DARTS study. Serum triglycerides were found to be lower in the PNPLA3 148M carriers from the SOS study at baseline and from the Go-DARTS T2D cohort. An increased risk for T2D conferred by the 148M allele was found in the SOS study (O.R. 1.09, 95% C.I. 1.01-1.39, P = 0.040) and in severely obese individuals in the Go-DARTS study (O.R. 1.37, 95% C.I. 1.13-1.66, P = 0.001). The 148M allele was no longer associated with insulin resistance or T2D after bariatric surgery in the SOS study and no association with the 148M allele was observed in the less obese (BMI<35) individuals in the Go-DARTS study (P for interaction  = 0.002). This provides evidence for the obesity interaction with I48M allele and T2D risk in a large-scale cross-sectional and a prospective interventional study. Conclusions Severely obese individuals carrying the PNPLA3 148M allele have lower serum triglyceride levels, are more insulin resistant and more susceptible to T2D. This study supports the hypothesis that obesity-driven hepatic lipid accumulation may contribute to T2D susceptibility.

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain.

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX‐I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX‐I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA‐binding domain of p53 bind to CHK2 and stimulate phosphorylation of full‐length p53 at Thr 18 and Ser 20, thus identifying CHK2‐docking sites. CHK2 can be fully activated in trans by the two p53 DNA‐binding‐domain peptides, and can phosphorylate BOX‐I transactivation‐domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA‐binding‐domain peptides. The CHK2‐docking site in the BOX‐V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX‐V peptide prevents CHK2 activation. A database search identified a p53 BOX‐I‐homology motif in p21WAF1 and although CHK2 is inactive towards this protein, the p53 DNA‐binding‐domain peptides induce phosphorylation of p21WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.

Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation

Induction of the transactivation function of p53 after cellular irradiation was studied under conditions in which upstream signaling events modulating p53 activation were uncoupled from those regulating stabilization. This investigation prompted the discovery of a novel radiation-responsive kinase pathway targeting Ser20 that results in the masking of the DO-1 epitope in undamaged cells. Unmasking of the DO-1 epitope via dephosphorylation occurs in response to low doses of non-ionizing radiation. Our data show that phosphorylation at Ser20 reduces binding of the mdm2 protein, suggesting that a function of the Ser20-kinase pathway may be to produce a stable pool of inactive p53 in undamaged cells which can be readily activated after cellular injury. Phospho-specific monoclonal antibodies were used to determine whether the Ser20 signaling pathway is coupled to the Ser15 and Ser392 radiation-responsive kinase pathways. These results demonstrated that: (1) dephosphorylation at Ser20 is co-ordinated with an increased steady-state phosphorylation at Ser392 after irradiation, without p53 protein stabilization, and (2) stabilization of p53 protein can occur without Ser15 phosphorylation at higher doses of radiation. These data show that the Ser20 and Ser392 phosphorylation sites are both targeted by an integrated network of signaling pathways which is acutely sensitive to radiation injury.

A note on the homology of ideals ǵenerated by three elements in local rings

In this note, I show that, if Q is a local ring and s an integer, 1 s Q , then there is an ideal A s of Q , generated by three or fewer elements of Q , such that and that, if Q is not regular, then there is an ideal B of Q , generated by three or fewer elements and of infinite homological dimension over Q .

Peroxisome Proliferator-Activated Receptor-δ Genotype Influences Metabolic Phenotype and May Influence Lipid Response to Statin Therapy in Humans: A Genetics of Diabetes Audit and Research Tayside Study

CONTEXT Previous studies have identified a single-nucleotide polymorphism in the gene encoding peroxisome proliferator-activated receptor-delta (PPARD), rs2016520, that is associated with changes in metabolic disease in some but not all studies, which suggests that PPARD agonists may have therapeutic benefits for the treatment of metabolic disorders, including dyslipidemia, type 2 diabetes, and obesity. OBJECTIVE The objective of the study was to determine whether rs2016520 or other single-nucleotide polymorphism in the PPARD locus influenced the risk of developing various characteristics of metabolic disease. DESIGN Haplotype tagging analysis across PPARD was performed in 11,074 individuals from the Welcome Trust U.K. Type 2 Diabetes Case Control Collection. RESULTS In subjects with and without type 2 diabetes, rs2016520 was associated with body mass index, high-density lipoprotein cholesterol, leptin, and TNFalpha and was dependent on gender. CONCLUSION The current results suggest differential effects of PPARdelta in males and females.