ChIP-Sequencing of TET Proteins.

TET proteins are methylcytosine dioxygenases that interact directly with chromatin to shape the DNA methylation landscape. To increase the understanding of TET protein function in a specific cellular context, it is important to be able to map the interactions between TET proteins and DNA. This ChIP-seq protocol details our procedure to analyze TET2 bound DNA in disuccinimidyl glutarate (DSG) and formaldehyde-crosslinked chromatin but can also be adapted to study other TET enzymes.

Mas-Related G Protein-Coupled Receptors Offer Potential New Targets for Pain Therapy.

The founding member of the Mas-related G-protein-coupled receptor (Mrgpr) family was discovered in 1986. Since then, many more members of this receptor family have been identified in multiple species, and their physiologic functions have been investigated widely. Because they are expressed exclusively in small-diameter primary sensory neurons, the roles of Mrgpr proteins in pain and itch have been best studied. This review will focus specifically on the current knowledge of their roles in pathological pain and the potential development of new pharmacotherapies targeted at some Mrgprs for the treatment of chronic pain. We will also discuss the limitations and future scope of this receptor family in pain treatment.

Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells.

Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.

Companion-diagnostic testing limited to KRAS codons 12 and 13 misses 17% of potentially relevant RAS mutations in colorectal cancer.

BACKGROUND: KRAS codons 12 and 13 mutations are commonly used to identify colorectal carcinoma (CRC) patients who are unlikely to benefit from anti-EGFR therapy. However, humans have four different homologous RAS proteins and no routine screening is performed for the other mutation sites. Non-screened mutations may still be present in a significant subset of patients without KRAS codon 12 and 13 mutations. METHODS: We developed a LightCycler screening assay that encompasses codons 12, 13 and 61 of all RAS genes. Screen-positive specimens were characterized by Sanger sequencing. 130 CRC specimens were screened for all RAS genes. The results for KRAS codons 12 and 13 were compared with an FDA approved method (Qiagen). RESULTS: Twenty-nine of 130 specimens (22.3%) were positive for KRAS codons 12 and 13, with 100% congruence with the Qiagen method. Six additional specimens were identified to have mutations. One mutation in HRAS codon 61, two in KRAS codon 61, and three in NRAS codon 61. CONCLUSION: Limiting RAS testing to only KRAS codons 12 and 13 in companion diagnostic testing of CRC results in nearly 1/5 of patients with RAS mutations not being excluded from costly EGFR antagonist treatment, despite likely futility. Inclusion of all RAS genes in companion diagnostic screening is warranted.

Genomics and biological activity of neutrophil gelatinase-associated lipocalin in several clinical settings.

Recent literature has shown that neutrophil gelatinase-associated lipocalin (NGAL) is one of the most interesting and promising biomarkers in case of acute kidney injury. However, several studies indicated that this protein may be applied beyond the boundaries of renal pathophysiology and may be used in other pathophysiological settings since it is also expressed in neutrophils, and respiratory, bowel and prostate epithelia. In this review, we report NGAL genomics and biology and its possible use in several clinical settings. In particular, we review the genomic organization of the NGAL gene, the lipocalin family structure, the interaction between NGAL and ligands, and the induction and expression of NGAL in different conditions.

Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

c-Ski in health and disease.

c-Ski is an evolutionary conserved protein that is involved in diverse cellular processes such as proliferation, differentiation, transformation, and tumor progression. A large range of cellular partners of c-Ski, including transcription factors, chromatin-remodeling molecules, tumor suppressors, and nuclear hormone receptors, has been identified. Moreover, numerous mechanisms have been described by which c-Ski regulates essential signaling pathways, e.g., the TGFß pathway. In this review, we summarize the diverse roles attributed to c-Ski during normal development and in cancer progression and discuss future strategies to unravel further the complex nature of c-Ski actions in a context-dependent manner.

Recent insights into cerebral cavernous malformations: the molecular genetics of CCM.

Cerebral cavernous malformations (CCM) are vascular lesions which can occur as a sporadic (80% of the cases) or familial autosomal dominant form (20%). Three CCM genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Almost 80% of CCM patients affected with a genetic form of the disease harbor a heterozygous germline mutation in one of these three genes. Recent work has shown that a two-hit mechanism is involved in CCM pathogenesis which is caused by a complete loss of any of the three CCM proteins within endothelial cells lining the cavernous capillary cavities. These data were an important step towards the elucidation of the mechanisms of this condition.

Genetic background of MEN1: from genetic homogeneity to functional diversity.

Multiple Endocrine Neoplasia Type 1 corresponds to a monogenic predisposition syndrome inherited as a dominant trait that affects a variety of endocrine tissues, in particular parathyroids, endocrine pancreas and anterior pituitary. It is caused by mutations in the MEN1 tumor suppressor gene that inactivate menin, the MEN1 encoded protein. Menin is involved in cell cycle control and apoptosis through its participation in functional dynamics of chromatin and regulation of transcription. In addition, genetic investigations have implicated menin in the maintenance of genomic integrity. However, the role of menin does not--by far--end here. It plays (too) many roles in the control of cell life and normality, far beyond endocrine oncogenesis, making it unlikely that the function of menin can be deciphered only by genetic investigation. In this context, writing a chapter on the genetic background of MEN1 appears at the same time as a challenge and a paradox. A challenge as everything has been either already written on the topic or included in the present book. A paradox since genetics is simultaneously at the background and at the forefront of MEN1. Our attempts are thus more investigating new--as well as already open issues than delivering a catalog of MEN1 gene mutations.

Cross-talk between the ErbB/HER family and the type I insulin-like growth factor receptor signaling pathway in breast cancer.

Understanding the molecular mechanisms involved in tumorigenesis and their influence on clinical outcome is providing specific molecular markers for targeted therapy. Activation of tyrosine kinase receptors from the human epidermal growth factor receptor family (EGFR, HER2, HER3, HER4) and the insulin-like growth factor receptor I (IGF-IR) plays a key role in the initiation and progression of breast cancer. HER2 overexpression is a validated therapeutic target, as shown by the clinical efficacy of trastuzumab and lapatinib. However, only 25-30% of patients with HER2-overexpressing tumors respond to single-agent trastuzumab or lapatinib, and resistance develops even in responding patients. Therefore, to optimize therapeutic efficacy, it is urgent to elucidate the complex network of signaling pathways that develop in breast cancer cells. Signaling interactions have been reported between ErbB/HER family members and IGF-IR. As increased IGF-IR signaling has been implicated in trastuzumab resistance, agents targeting HER2, and IGF-IR could be potential therapeutic tools in breast cancers that develop resistance to HER2-directed therapy.

Effect of ACP1*C on early life viability.

Associations with past malarial morbidity, season of conception, and common diseases such as obesity, type 2 diabetes, and allergy argue against neutrality of the ACP1 genetic polymorphism. Comparison of ACP1 distribution in mothers and their newborns and analysis of the joint wife-husband ACP1 phenotype distribution in couples with repeated spontaneous abortion suggest a negative effect of the ACP1*C allele on early life viability. Analysis of the polymorphism of the ACP1 gene suggests that, unlike the ACP1*A and ACP1*B alleles, the ACP1*C allele is independent of sequences in the 5' flanking region, resulting in an inverted F/S isoform ratio.

BMI-1 is highly expressed in M0-subtype acute myeloid leukemia.

Recent studies have suggested that one of the polycomb group genes, BMI-1, has an important role in the maintenance of normal and leukemic stem cells by repressing the INK4a/ARF locus. Here, we quantitatively examined BMI-1 expression level in samples from patients with acute myeloid leukemia (AML) and other hematologic malignancies. Moderate to high BMI-1 expression was detected in AML patients, and the BMI-1 expression levels in AML samples were significantly higher than in normal bone marrow controls (P = .0011). Specimens of French-American-British classification subtype M0 showed higher relative expression of the BMI-1 transcript (median, 390.2 3 10(-3)) than the other subtypes (median, 139.0 3 10(-3)) (P < .0001). Leukemia other than AML showed low to moderate expression. INK4a-ARF transcript expression tended to be inverse proportion to that of BMI-1. In an M0 patient with a high BMI-1 transcript level, the INK4a-ARF transcript level fell promptly and maintained a low value after the patient achieved complete remission. These results indicated that a subgroup of M0 patients has a high expression level of polycomb group gene BMI-1, which may contribute to leukemogenesis.

A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling.

More is becoming known about so-called noncanonical Wnt pathways that signal independently of beta-catenin. Here we review recent developments in both the functions and mechanisms of noncanonical Wnt signaling. We also discuss some unresolved and vexing questions. How many noncanonical Wnt pathways are there? How extensive are the parallels between Drosophila planar polarization and vertebrate convergence and extension? Last, we will outline some challenges and difficulties we foresee for this exciting but still very young field.

Consistency checks for characterizing protein forms.

Proteomics enforces the reverse chronological order on the gene to protein dogma and imposes amino acid sequences as a starting point of an investigation relative to function. By this approach, proteomics data can confirm the presence of multiple forms of a protein. Notwithstanding variations attributed specific individual features of organisms and tissues, from two to over ten protein forms can be identified in a given sample. The present work describes some guidelines for tracking the origin of alternative protein forms and attempts to tag the details of sequence data in the literature. Working via these guidelines we have uncovered a third alternative form of the Pim subfamily of oncogenes. The term form is here combined with the qualification alternative to describe any product of a given gene including closely related paralogs. This paper also emphasizes the need for consistency checks in annotation processes, such as gene clustering, to avoid losing important details describing protein alternative forms. By identifying alternative protein forms, we illustrate the fact that rationalizing of protein function via the identification of protein-protein interactions should in reality be that of identifying (alternative) form-form interactions.

Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle.

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.

Protein kinase B/Akt is essential for the insulin- but not progesterone-stimulated resumption of meiosis in Xenopus oocytes.

In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.

The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.