Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47)

Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin _V3/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin _V3 is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing.     

Metazoan motor models: kinesin superfamily in C. elegans

In eukaryotic cells members of the kinesin family mediate intracellular transport by carrying cellular cargo on microtubule tracks. The nematode Caenorhabditis elegans genome encodes 21 members of the kinesin family, which show significant homology to their mammalian orthologs. Based on motor domain sequence homology and placement of the motor domain in the protein, the C. elegans kinesins have been placed in eight distinct groups; members of which participate in embryonic development, protein transport, synaptic membrane vesicles movement and in the axonal growth. Among 21 kinesins, at least 11 play a central role in spindle movement and chromosomal segregation. Understanding the function of C. elegans kinesins and related proteins may help navigate through the intricacies of intracellular traffic in a simple animal.     

Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

Lipids in blood-brain barrier models in vitro I: Thin-layer chromatography and high-performance liquid chromatography for the analysis of lipid classes and long-chain polyunsaturated fatty acids

The objectives of this study were to optimize a sensitive high-performance liquid chromatography (HPLC) method for fatty acid (FA) analysis for the quantification of polyunsaturated FAs (PUFAs) in cell lipid extracts and to analyze the lipid and FA patterns of three cell lines used in blood-brain barrier (BBB) models: RBE4, ECV304, and C6. Thin-layer chromatographic analysis revealed differences in the phosphatidylcholine-phosphatidylethanolamine (PC:PE) ratios and the triglyceride (TG) content. The PC:PE ratio was <1 for RBE4 cells but >1 for ECV304 and C6 cells. ECV304 cells displayed up to 9% TG depending on culture time, whereas the other cell lines contained about 1% TG. The percentages of docosahexaenoic acid were 9.4 +/- 1.7% of the unsaturated FAs in RBE4 cells (n = 5; 4 d in culture; 9.9% after 10 d), 8.1 +/- 2.0% in ECV304 cells (n = 11; 10 to 14 d), and 6.7 +/- 0.6% in C6 cells (n = 6; 10 to 14 d) and were close to the published values for rat brain microvascular endothelium. The percentage of arachidonic acid (C20:4) was about half that in vivo. ECV304 cells contained the highest fraction of C20:4, 17.8 +/- 2.2%; RBE4 cells contained 11.6 +/- 2.4%; and C6 cells 15.8 +/- 1.9%. It is concluded that a sensitive HPLC method for FAs is now optimized for the analysis of long-chain PUFAs. The results provide a useful framework for studies on the effects of lipid modulation and give reference information for the development of further BBB models.     

Importance of the propeptide in the biosynthetic maturation of rat cathepsin C

Cathepsin C is a cysteine dipeptidyl-aminopeptidase. Active cathepsin C is found in lysosomes as a 200-kDa multimeric enzyme. Subunits constituting this assembly all arise from the proteolytic cleavage of a single precursor giving rise to three peptides: the propeptide, the alpha- and the beta-chains. Some features of the propeptide such as its length, its high level of glycosylation and its retention in the active lysosomal form of the enzyme suggest an important contribution of the proregion in the transport, maturation and expression of cathepsin C. In order to assess some aspects of this contribution, we transiently expressed mutant molecules of rat cathepsin C either lacking three of the four glycosylation sites, partially deleted in the proregion, or mutated at tryptophan 39 also located in the proregion, and studied their biosynthesis. Our results show that at least one of the three glycosylation sites in the propeptide must be glycosylated in order to obtain targeting and maturation of cathepsin C. We also show that a deletion of 14 amino acids and mutation W39S in the propeptide totally abolishes the biosynthetic processing of the enzyme. These results demonstrate that in addition to its role as a chaperone or in maintaining the latency of the enzymatic activity, the propeptide is required for proper transport and expression of newly synthesized cathepsin C.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Molecular motions and conformational changes of HPPK

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) belongs to a class of catalytic enzymes involved in phosphoryl transfer and is a new target for the development of novel antimicrobial agents. In the present study, the fundamental consideration is to view the overall structure of HPPK as a network of interacting residues and to extract the most cooperative collective motions that define its global dynamics. A coarse-grained model, harmonically constrained according to HPPK's crystal structure is used. Four crystal structures of HPPK (one apo and three holo forms with different nucleotide and pterin analogs) are studied with the goal of providing insights about the function-dynamic correlation and ligand induced conformational changes. The dynamic differences are examined between HPPK's apo- and holo-forms, because they are involved in the catalytic reaction steps. Our results indicate that the palm-like structure of HPPK is nearly rigid, whereas the two flexible loops: L2 (residues 43-53) and L3 (residues 82-92) exhibit the most concerted motions for ligand recognition and presumably, catalysis. These two flexible loops are involved in the recognition of HPPKs nucleotide and pterin ligands, whereas the rigid palm region is associated with binding of these cognate ligands. Six domains of collective motions are identified, comprised of structurally close but not necessarily sequential residues. Two of these domains correspond to the flexible loops (L2 and L3), whereas the remaining domains correspond to the rigid part of the molecule.