72 research outputs found
De novo backbone and sequence design of an idealized α/β-barrel protein: evidence of stable tertiary structure
We have designed, synthesized, and characterized a 216 amino acid residue
sequence encoding a putative idealized α/β-barrel protein. The
design was elaborated in two steps. First, the idealized backbone was
defined with geometric parameters representing our target fold: a central
eight parallel-stranded β-sheet surrounded by eight parallel α-helices,
connected together with short structural turns on both sides of the barrel.
An automated sequence selection algorithm, based on the dead-end elimination
theorem, was used to find the optimal amino acid sequence fitting
the target structure. A synthetic gene coding for the designed sequence
was constructed and the recombinant artificial protein was expressed in
bacteria, purified and characterized. Far-UV CD spectra with prominent
bands at 222 nm and 208 nm revealed the presence of α-helix secondary
structures (50%) in fairly good agreement with the model. A pronounced
absorption band in the near-UV CD region, arising from immobilized aromatic
side-chains, showed that the artificial protein is folded in solution.
Chemical unfolding monitored by tryptophan fluorescence revealed a
conformational stability (ΔGH_2O) of 35 kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T_m of 65 °C. Moreover, the artificial protein did not exhibit any affinity
for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic
acid (ANS), providing additional evidence that the artificial barrel is not
in the molten globule state, contrary to previously designed artificial a/
b-barrels. Finally, ^1H NMR spectra of the folded and unfolded proteins
provided evidence for specific interactions in the folded protein. Taken
together, the results indicate that the de novo designed α/β-barrel protein
adopts a stable three-dimensional structure in solution. These encouraging
results show that de novo design of an idealized protein structure of
more than 200 amino acid residues is now possible, from construction of
a particular backbone conformation to determination of an amino acid
sequence with an automated sequence selection algorithm
ORAI3 (ORAI calcium release-activated calcium modulator 3)
Review on ORAI3 (ORAI calcium release-activated calcium modulator 3), with data on DNA, on the protein encoded, and where the gene is implicated
Highly Efficient Elimination of Colorectal Tumor-Initiating Cells by an EpCAM/CD3-Bispecific Antibody Engaging Human T Cells
With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs) are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof
Mechanisms of hypoxic up-regulation of versican gene expression in macrophages
Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression
A New Method for Isolation of Interstitial Fluid from Human Solid Tumors Applied to Proteomic Analysis of Ovarian Carcinoma Tissue
Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample's origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na+, two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods
The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression
International audienc
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