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41.
The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers. 相似文献
42.
Oliva A Borriello A Zeppetelli S Di Feo A Cortellazzi P Ventriglia V Criscuolo M Zappia V Della Ragione F 《Molecular and cellular biochemistry》2003,247(1-2):55-60
The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27Kip1 and p16INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity. 相似文献
43.
Mello GC Oliva ML Sumikawa JT Machado OL Marangoni S Novello JC Macedo ML 《Journal of Protein Chemistry》2001,20(8):625-632
A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors. 相似文献
44.
DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein α. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in α (αcr) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on λ immunity in E.coli for in vivo detection of protein–protein interactions, we found that: (i) α protein interacts with Cnr, whereas αcr proteins do not; (ii) both α–α and αcr–αcr interactions occur and the interaction domain is located within the C-terminal of α; (iii) Cnr–Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both α–α and αcr–αcr dimerization. Our data suggest that Cnr and α interact in at least two ways, which may have different functional roles in P4 replication control. 相似文献
45.
Many of the protein precursors traversing the secretory pathway undergo cleavage at multibasic sites to generate their bioactive forms. The proprotein convertases (PCs), a family of subtilisin-like proteases, are the major endoproteases that serve this function. Genes encoding seven distinct members of this family have so far been characterized in vertebrates: furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6 and PC7/PC8/LPC. Multiple PC genes have also been cloned from a number of invertebrates, including Drosophila melanogaster and Caenorhabditis elegans. These findings suggest that gene duplication and diversification of the PCs have occurred throughout metazoan evolution. To investigate the structural and functional changes which have occurred during vertebrate development, we have analyzed the expression of PC genes in the protochordate amphioxus. We have previously shown that amphioxus express homologous PC2 and PC1/PC3 genes [Proc. Natl. Acad. Sci. USA 92 (1995) 3591]. Here we report the characterization of amphioxus cDNAs encoding proteases with a high degree of similarity to mammalian PC6. Three cDNAs encoding three PC6 isoforms differing only in their carboxy-terminal sequences were found, derived by alternative splicing. Two isoforms appear to be soluble enzymes, whereas the third contains a transmembrane hydrophobic segment and thus is likely to be membrane-bound. All three variants contain many repeats of a cysteine-rich motif that is found in several other PC family members. Thus, amphioxus, like the vertebrates, expresses two types of PCs, e.g., PC2 and PC1/PC3 which function in the regulated secretory pathway in neuroendocrine cells, and the more widely expressed PC6 which functions mainly in the constitutive pathway. 相似文献
46.
Marc A. Martí-Renom José M. Mas Patrick Aloy Enrique Querol Francesc X. Avilés Baldomero Oliva 《Journal of molecular modeling》1998,4(11):347-354
The conformations of protein loops from a non-redundant set of 347 proteins with less than 25% sequence homology have been studied in order to clarify the topological variation of protein loops. Loops have been classified in five types (α-α, α-β, β-α, β-links and β-hairpins) depending on the secondary structures that they embrace. Four variables have been used to describe the loop geometry (3 angles and the end-to-end distance between the secondary structures embracing the loop). Loops with well defined geometry are identified by means of the internal dependency between the geometrical variables by application of information-entropy theory. From this it has been deduced that loops formed by less than 10 residues show an intrinsic dependency on the geometric variables that defines the motif shape. In this interval the most stable loops are found for short connections owing to the entropic energy analysed. 相似文献
47.
Luciana Di Ciero Maria L. V. Oliva Ricardo Torquato Peter Köhler Jürgen K. P. Weder José Camillo Novello Claudio A. M. Sampaio Benedito Oliveira Sergio Marangoni 《Journal of Protein Chemistry》1998,17(8):827-834
Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M
r
of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K
i
values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64). 相似文献
48.
Joan Segura Manuel Alejandro Marín-López Pamela F. Jones Baldo Oliva Narcis Fernandez-Fuentes 《PloS one》2015,10(3)
The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers. 相似文献
49.
Diana Lanza Alessandra F. Perna Adriana Oliva Raymond Vanholder Anneleen Pletinck Salvatore Guastafierro Annarita Di Nunzio Carmela Vigorito Giovambattista Capasso Vera Jankowski Joachim Jankowski Diego Ingrosso 《PloS one》2015,10(1)
Human mesenchymal stem cells (hMSCs), the precursors of osteoblasts during osteogenesis, play a role in the balance of bone formation and resorption, but their functioning in uremia has not been well defined. To study the effects of the uremic milieu on osteogenic properties, we applied an in vitro assay culturing hMSCs in osteogenic medium supplemented with serum from healthy donors and from uremic patients on hemodialysis. Compared to control, serum from uremic patients induces, in hMSC cultures, a modification of several key regulators of bone remodeling, in particular a reduction of the ratio Receptor Activator of Nuclear factor Kappa B Receptor (RANKL) over osteoprotegerin, indicating an adaptive response of the system to favor osteogenesis over osteoclastosis. However, the levels of osteopontin, osteocalcin, and collagen type I, are increased in cell medium, while BMP-2, and alizarin red staining were decreased, pointing to a reduction of bone formation favoring resorption. Selected uremic toxins, such as p-cresylsulfate, p-cresylglucuronide, parathyroid hormone, indoxyl sulfate, asymmetric dimethylarginine, homocysteine, were able to mimic some of the effects of whole serum from uremic patients. Serum from cinacalcet-treated patients antagonizes these effects. Hydrogen sulfide (H2S) donors as well as hemodialysis treatment are able to induce beneficial effects. In conclusion, bone modifications in uremia are influenced by the capability of the uremic milieu to alter hMSC osteogenic differentiation. Cinacalcet, H2S donors and a hemodialysis session can ameliorate the hampered calcium deposition. 相似文献
50.
Thierry Jardé Lisa Kass Margaret Staples Helen Lescesen Peter Carne Karen Oliva Paul J. McMurrick Helen E. Abud 《PloS one》2015,10(9)
Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts. 相似文献