Protein folding mediated by solvation: water expelling and formation of the hydrophobic core occurs after the structure collapse

The interplay between structure-search of the native structure and desolvation in protein folding has been explored using a minimalist model. These results support a folding mechanism where most of the structural formation of the protein is achieved before water is expelled from the hydrophobic core. This view integrates water expulsion effects into the funnel energy landscape theory of protein folding. Comparisons to experimental results are shown for the SH3 protein. After the folding transition, a near-native intermediate with partially solvated hydrophobic core is found. This transition is followed by a final step that cooperatively squeezes out water molecules from the partially hydrated protein core.Comment: Proceedings of the National Academy of Science, 2002, Vol.99. 685-69

Temperature dependence of ESR intensity for the nanoscale molecular magnet V15

The electron spin resonance (ESR) of nanoscale molecular magnet ${\rm V}_{15}$ is studied. Since the Hamiltonian of ${\rm V}_{15}$ has a large Hilbert space and numerical calculations of the ESR signal evaluating the Kubo formula with exact diagonalization method is difficult, we implement the formula with the help of the random vector technique and the Chebyshev polynominal expansion, which we name the double Chebyshev expansion method. We calculate the temperature dependence of the ESR intensity of ${\rm V}_{15}$ and compare it with the data obtained in experiment. As another complementary approach, we also implement the Kubo formula with the subspace iteration method taking only important low-lying states into account. We study the ESR absorption curve below $100{\rm K}$ by means of both methods. We find that side peaks appear due to the Dzyaloshinsky-Moriya interaction and these peaks grows as temperature decreases.Comment: 9 pages, 4 figures. To appear in J. Phys. Soc. Jpn. Supp

Secondary-Structure Design of Proteins by a Backbone Torsion Energy

We propose a new backbone-torsion-energy term in the force field for protein systems. This torsion-energy term is represented by a double Fourier series in two variables, the backbone dihedral angles phi and psi. It gives a natural representation of the torsion energy in the Ramachandran space in the sense that any two-dimensional energy surface periodic in both phi and psi can be expanded by the double Fourier series. We can then easily control secondary-structure-forming tendencies by modifying the torsion-energy surface. For instance, we can increase/decrease the alpha-helix-forming-tendencies by lowering/raising the torsion-energy surface in the alpha-helix region and likewise increase/decrease the beta-sheet-forming tendencies by lowering/raising the surface in the beta-sheet region in the Ramachandran space. We applied our approach to AMBER parm94 and AMBER parm96 force fields and demonstrated that our modifications of the torsion-energy terms resulted in the expected changes of secondary-structure-forming-tendencies by performing folding simulations of alpha-helical and beta-hairpin peptides.Comment: 13 pages, (Revtex4), 5 figure

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

Targeting BRAF in thyroid cancer

Activating mutations in the gene encoding BRAF are the most commonly identified oncogenic abnormalities in papillary thyroid cancer. In vitro and in vivo models have demonstrated that overexpression of activated BRAF induces malignant transformation and aggressive tumour behaviour. BRAF and other RAF kinases are frequently activated by other thyroid oncogenes and are important mediators of their biological effects including dedifferentiation and proliferation. Because current therapeutic options for patients with thyroid cancers that are aggressive and/or do not respond to standard therapies are limited, BRAF and its downstream effectors represent attractive therapeutic targets. In this review, data supporting a role for BRAF activation in thyroid cancer development and establishing the potential therapeutic efficacy of BRAF-targeted agents in patients with thyroid cancer will be reviewed

Copy Number Alteration and Uniparental Disomy Analysis Categorizes Japanese Papillary Thyroid Carcinomas into Distinct Groups

The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAFV600E, 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAFV600E and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA+, UPD+, and no chromosomal aberrations. BRAFV600E mutational status did not correlate with any parameters of chromosomal defects

Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas

<p>Abstract</p> <p>Background</p> <p>p75^NTRhas been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75^NTRin esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75^NTR+cells.</p> <p>Methods</p> <p>p75^NTRexpression in ESCC was assessed by immunohistochemistry. p75^NTR+and p75^NTR-cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75^NTR+and p75^NTR-cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, ⁶⁴copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75^NTR+cells.</p> <p>Results</p> <p>In ESCC specimens, p75^NTRwas found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75^NTR+cells was 1.6%–3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75^NTR+cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75^NTR+cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75^NTR+cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, β1-integrin and β4-integrin, were lower in p75^NTR+cells. In addition, p75^NTR+cells generated both p75^NTR+and p75^NTR-cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75^NTR+cells were found to be more resistant to DDP and exhibited lower ⁶⁴copper accumulation than p75^NTR-cells.</p> <p>Conclusion</p> <p>Our results demonstrated that p75^NTR+cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75^NTR+cells may probably be attributable to decreased expression of CTR1.</p

Cross-Regulation between Oncogenic BRAFV600E Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma

BACKGROUND:The BRAF(V600E) mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAF(V600E) and MST1,which activates Foxo3,has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAF(V600E) positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAF(V600E)markedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAF(V600E)is strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAF(V600E)was able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereas addition of BRAF(V600E) inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAF(V600E)in the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAF(V600E)transgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. CONCLUSIONS/SIGNIFICANCE:The results of this study revealed that the oncogenic effect of BRAF(V600E) is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAF(V600E) tumors

Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations

<p>Abstract</p> <p>Background</p> <p>Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.</p> <p>Methods</p> <p>Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.</p> <p>Results</p> <p>Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF^V600Emutation. In BRAF^V600Emutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27^Kip1. Specific inhibition of BRAF by RNAi in cells with BRAF^V600Emutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF^V600Emutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.</p> <p>Conclusion</p> <p>Our results in thyroid cancer cells, namely those harbouring BRAF^V600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF^V600Emutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.</p

In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

Background: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. Methodology/Principal Findings: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133pos cells only in ARO and KAT-4 (6469% and 57612%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/ CD133pos cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133neg. Furthermore, ARO/CD133pos showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133neg was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133pos in comparison to ARO/CD133neg cells. The stem cell markers c- KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133pos when compared with ARO/CD133neg cells. Conclusions/Significance: We describe CD133pos cells in ATC cell lines. ARO/CD133pos cells exhibit stem cell-like features - such as high proliferation, self-renewal ability, expression of OCT-4 - and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches