| 1. |
- Roblick, U J, et al.
(author)
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Sequential proteome alterations during genesis and progression of colon cancer.
- 2004
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In: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 61:10
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Journal article (peer-reviewed)abstract
- Changes in the proteome of colon mucosal cells accompany the transition from normal mucosa via adenoma and invasive cancer to metastatic disease. Samples from 15 patients with sporadic sigmoid cancers were analyzed. Proteins were separated by two-dimensional gel electrophoresis. Relative differences in expression levels between normal tissue, adenoma, carcinoma and metastasis were evaluated in both intra- and inter-patient comparisons. Up- and down-regulated proteins (> twofold) during development to cancer or metastasis were excised and submitted to peptide mass fingerprinting and MS/MS sequence analysis, facilitated by the use of a compact disc workstation. In total, 112 protein spots were found to be differentially regulated, of which 72 were determined as to protein identity, 46 being up-regulated toward the progression of cancer, and 26 down-regulated. Several of the identifications correlate with proteins of the cell cycle, cytoskeleton or metabolic pathways. The pattern changes now identified have the potential for design of marker panels for assistance in diagnostics and therapeutic strategies in colorectal cancer.
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| 2. |
- Benach, J, et al.
(author)
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Structure of bacterial 3 beta/17 beta-hydroxysteroid dehydrogenase at 1.2 angstrom resolution : A model for multiple steroid recognition
- 2002
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:50, s. 14659-14668
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Journal article (peer-reviewed)abstract
- The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Conramonas testosteroni. It catalyzes the reversible reduction/ dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 Angstrom resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.
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| 3. |
- Bergman, T, et al.
(author)
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C-terminal sequence analysis
- 2003
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In: Current protocols in protein science. - : Wiley. - 1934-3663 .- 1934-3655. ; Chapter 11, s. 11.8-
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Journal article (peer-reviewed)
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| 4. |
- Bjellerup, P, et al.
(author)
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Limited neuropeptide Y precursor processing in unfavourable metastatic neuroblastoma tumours
- 2000
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In: British Journal of Cancer. - 0007-0920 .- 1532-1827. ; 83:2, s. 171-176
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Journal article (peer-reviewed)abstract
- Neuropeptide Y (NPY) is found at high concentrations in neural crest-derived tumours and has been implicated as a regulatory peptide in tumour growth and differentiation. Neuroblastomas, ganglioneuromas and phaeochromocytomas with significant concentrations of NPY-like immunoreactivity were investigated for different molecular forms of NPY and for significance of proNPY processing. Gel-permeation chromatography identified intact NPY (1-36) in all tumours, whereas proNPY (69 amino acids) was detected only in control adrenal tissue and malignant neuroblastomas. Purification of NPY-like immunoreactivity in tumour extracts and structural characterization revealed that both NPY (1-36) and the truncated form NPY (3-36) was present. The degree of processing of proNPY to NPY in tumour tissue was lower in advanced neuroblastomas with regional or metastatic spread (stage 3 and 4) (n = 6), (41%, 12-100%, median, range), compared to the less aggressive stage 1, 2 and 4S tumours (n = 12), (93%, 69-100%), (P = 0.012). ProNPY processing of less than 50% was correlated with poor clinical outcome (P = 0.004). MYCN oncogene amplification was also correlated to a low degree of proNPY processing (P = 0.025). In summary, a low degree of proNPY processing was correlated to clinical advanced stage and poor outcome in neuroblastomas. ProNPY/NPY processing generated molecular forms of NPY with known differences in NPY-receptor selectivity, implicating a potential for in vivo modulation of NPY-like effects in tumour tissue. (C) 2000 Cancer Research Campaign.
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| 5. |
- Filling, C, et al.
(author)
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Critical residues for structure and catalysis in short-chain dehydrogenases/reductases
- 2002
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:28, s. 25677-25684
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Journal article (peer-reviewed)abstract
- Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Angstrom resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.
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| 6. |
- Filling, C, et al.
(author)
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Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold
- 2001
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 289:3, s. 712-717
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Journal article (peer-reviewed)abstract
- Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3 beta /17 beta -hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-Angstrom resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11 beta -hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.
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| 7. |
- Hirschberg, Daniel, et al.
(author)
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N-terminal acetylation in a third protein family of vertebrate alcohol dehydrogenase/retinal reductase found through a 'proteomics' approach in enzyme characterization.
- 2001
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In: Cellular and Molecular Life Sciences (CMLS). - 1420-682X .- 1420-9071. ; 58:9
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Journal article (peer-reviewed)abstract
- A recent finding of a novel class of retinol-active alcohol dehydrogenase (ADH) in frog prompted analysis of this activity in other vertebrate forms. Surprisingly, yet another and still more unrelated ADH was identified in chicken tissues. It was found to be a member of the aldo-keto reductase (AKR) enzyme family, not previously known as an ADH in vertebrates. Its terminal blocking group and the N-terminal segment, not assigned by protein and cDNA structure analysis, were determined by electrospray tandem mass spectrometry after protein isolation by two-dimensional gel electrophoresis. The N terminus is Acetyl-Ala- and the N-terminal segment contains two consecutive Asn residues. The results establish the new ADH enzyme of the AKR family and show the usefulness of combined gel separation and mass spectrometry in enzyme-characterization.
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| 8. |
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| 9. |
- Jonsson, AP, et al.
(author)
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A novel Ser O-glucuronidation in acidic proline-rich proteins identified by tandem mass spectrometry.
- 2000
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In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 475:2, s. 131-4
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Journal article (peer-reviewed)abstract
- Human acidic proline-rich salivary protein PRP-1 and its C-terminally truncated form PRP-3 were analyzed by electrospray tandem mass spectrometry. Post-translational modifications were detected and characterized. A pyroglutamic acid residue was demonstrated at the N-terminus, Ser-8 and Ser-22 were shown to be phosphorylated and an O-linked glucuronic acid conjugation was identified. The latter modification was located to Ser-17 and found to be present in approximately 40% of the polypeptides.
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| 10. |
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| 11. |
- Sima, AA, et al.
(author)
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Proinsulin C-peptide--a consensus statement
- 2001
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In: International journal of experimental diabetes research. - : Hindawi Limited. - 1560-4284. ; 2:2, s. 145-51
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Journal article (peer-reviewed)abstract
- In recent years the physiological role of the proinsulin C-peptide has received increasing attention, focusing on the potential therapeutic value of C-peptide replacement in preventing and ameliorating type 1 diabetic complications. In order to consolidate these new data and to identify the immediate directions of C-peptide research and its clinical usefulness, an International Symposium was held in Detroit, Michigan, on October 20–21, 2000, under the auspices of the Wayne State University/Morris Hood Jr. Comprehensive Diabetes Center. In this communication, we review the cellular, physiological and clinical effects of C-peptide replacement in animal models and in patients with type 1 diabetes. Finally, recommendations are presented as to the most urgent studies that should be pursued to further establish the biological action of C-peptide and its therapeutic value.
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| 12. |
- Wu, X., et al.
(author)
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Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli : RNA stability and secondary structure but not tRNA abundance
- 2004
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 313:1, s. 89-96
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Journal article (peer-reviewed)abstract
- Expression patterns in Escherichia coli of two small archaeal proteins with a natural content of about 30% rare codons were analyzed. The proteins, a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx), were produced with expression plasmids encoding wild-type genes, codon-optimized synthetic, and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative, and modified strains carrying copies for rare codon tRNAs or deletions in the RNAseE gene. Both Ssh10 and mtGrx expression levels were constitutively high in BL21(DE3) and its derivatives, with the exception of the LysS phenotype, which prevented high level expression of the Ssh10 wild-type gene. Surprisingly, a codon-optimized mtGrx gene construct displayed undetectable levels of protein production. The translational block observed with the synthetic mtGrx gene could be circumvented by using a synthetic mtGrx-glutathione S-transferase (GST) fusion construct or by in vitro translation. Taken together, the results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli.
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