Proteolytic cleavage of p53 mutants in response to mismatched DNA

Interaction of p53 with mismatched DNA induces proteolytic cleavage with release of a 35-kDa protein fragment from the p53–DNA complexes. The 35-kDa cleavage product is activated for specific biochemical function(s) and may play a role in the cellular response to DNA damage (Molinari et al (1996) Oncogene13: 2077–2086; Okorokov et al (1997) EMBO J16: 6008–6017). In the present study we have asked if mutants of p53 retain the ability to undergo similar proteolytic cleavage, and compared sequence-specific ‘DNA contact’ with ‘structural’ mutants commonly found in human cancer. In addition, a series of phosphorylation site mutants were generated to investigate the possible effects of phosphorylation/dephosphorylation on the proteolytic cleavage of p53. All mutants tested bound to a mismatched DNA target in vitro. Moreover, studies in vitro and in vivo indicate that p53 mutants with intact conformational structure (as determined by immunoreactivity with PAb246 and PAb1620) retain the ability to undergo proteolytic cleavage similar, if not identical, to the wild-type p53 protein. Our results suggest that the capacity for p53 to bind mismatched DNA is independent of structural conformation of the central core domain. Proteolytic cleavage, however, is crucially dependent upon a wild-type conformation of the protein. © 1999 Cancer Research Campaig

DISPARE: DIScriminative PAttern REfinement for Position Weight Matrices

<p>Abstract</p> <p>Background</p> <p>The accurate determination of transcription factor binding affinities is an important problem in biology and key to understanding the gene regulation process. Position weight matrices are commonly used to represent the binding properties of transcription factor binding sites but suffer from low information content and a large number of false matches in the genome. We describe a novel algorithm for the refinement of position weight matrices representing transcription factor binding sites based on experimental data, including ChIP-chip analyses. We present an iterative weight matrix optimization method that is more accurate in distinguishing true transcription factor binding sites from a negative control set. The initial position weight matrix comes from JASPAR, TRANSFAC or other sources. The main new features are the discriminative nature of the method and matrix width and length optimization.</p> <p>Results</p> <p>The algorithm was applied to the increasing collection of known transcription factor binding sites obtained from ChIP-chip experiments. The results show that our algorithm significantly improves the sensitivity and specificity of matrix models for identifying transcription factor binding sites.</p> <p>Conclusion</p> <p>When the transcription factor is known, it is more appropriate to use a discriminative approach such as the one presented here to derive its transcription factor-DNA binding properties starting with a matrix, as opposed to performing <it>de novo </it>motif discovery. Generating more accurate position weight matrices will ultimately contribute to a better understanding of eukaryotic transcriptional regulation, and could potentially offer a better alternative to <it>ab initio </it>motif discovery.</p

Challenges in Whole Exome Sequencing: An Example from Hereditary Deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ∼6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease

Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

BRCT domains: Easy as one, two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity

Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

The p53 gene has been implicated in many cancers due to its frequent mutations as well as mutations in other genes whose proteins directly affect p53’s functions. In addition, high expression of p53 [wild-type (WT) or mutant] has been found in the cytoplasm of many tumor cells, and studies have associated these observations with more aggressive tumors and poor prognosis. Cytoplasmic mis-localization of p53 subsequently reduced its transcriptional activity and this loss-of-function (LOF) was used to explain the lack of response to chemotherapeutic agents. However, this hypothesis seemed inadequate in explaining the apparent selection for tumor cells with high levels of p53 protein, a phenomenon that suggests a gain-of-function (GOF) of these mis-localized p53 proteins. In this study, we explored whether the direct involvement of p53 in the apoptotic response is via regulation of the caspase pathway in the cytoplasm. We demonstrate that p53, when present at high levels in the cytoplasm, has an inhibitory effect on caspase-9. Concurrently, knockdown of endogenous p53 caused an increase in the activity of caspase-9. p53 was found to interact with the p35 fragment of caspase-9, and this interaction inhibits the caspase-9 activity. In a p53-null background, the high-level expression of both exogenous WT and mutant p53 increased the resistance of these cells to cisplatin, and the data showed a correlation between high p53 expression and caspase-9 inhibition. These results suggest the inhibition of caspase-9 as a potential mechanism in evading apoptosis in tumors with high-level p53 expression that is cytoplasmically localized.Jacqueline L.Y. Chee, Suzan Saidin, David P. Lane, Sai Mun Leong, Jacqueline E. Noll, Paul M. Neilsen, Yi Ting Phua, Hani Gabra and Tit Meng Li