Protein-chemical identification of the major cleavage sites of the Ca2+ proteinase on murine vimentin, the mesenchymal intermediate filament protein

Neutral thiol proteinases (calpains), activated by calcium are involved in the intracellular turnover of intermediate filaments but the precise position of the cleavage points has remained unknown. Here we identify by direct sequence analysis the major cleavage sites found when murine vimentin is digested by limited proteolysis in vitro with calpain purified from porcine kidney. Contrary to some previous suggestions, no absolute sequence specifity could be detected although 10 specific sites have been identified. This result is in line with the cDNA derived amino-acid sequence of a calpain, which pointed to a similarity of the catalytic site with the active sites in papain, cathepsin and actinidin. However, all major cleavage sites are located within regions of the vimentin molecule, which in current models of intermediate filament structure are thought to be non-helical: the amino-terminal headpiece, the carboxy-terminal tailpiece and the spacer separating the two major coiled-coil domains. The sequence information about the cleavage sites was extended to provide the amino-terminal 119 residues of murine vimentin.     

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.     

Microbiological and Biochemical Characterization of Cassava Retting, a Traditional Lactic Acid Fermentation for Foo-Foo (Cassava Flour) Production

The overall kinetics of retting, a spontaneous fermentation of cassava roots performed in central Africa, was investigated in terms of microbial-population evolution and biochemical and physicochemical parameters. During the traditional process, endogenous cyanogens were almost totally degraded, plant cell walls were lysed by the simultaneous action of pectin methylesterase and pectate lyase, and organic acids (C(inf2) to C(inf4)) were produced. Most microorganisms identified were found to be facultative anaerobes which used the sugars (sucrose, glucose, and fructose) present in the roots as carbon sources. After 24 h of retting, the fermentation reached an equilibrium that was reproducible in all the spontaneous fermentations studied. Lactic acid bacteria were largely predominant (over 99% of the total flora after 48 h) and governed the fermentation. The epiphytic flora was first replaced by Lactococcus lactis, then by Leuconostoc mesenteroides, and finally, at the end of the process, by Lactobacillus plantarum. These organisms produced ethanol and high concentrations of lactate, which strongly acidified the retting juice. In addition, the rapid decrease in partial oxygen pressure rendered the process anaerobic. Strict anaerobes, such as Clostridium spp., developed and produced the volatile fatty acids (mainly butyrate) responsible, together with lactate, for the typical flavor of retted cassava. Yeasts (mostly Candida spp.) did not seem to play a significant role in the process, but their increasing numbers in the last stage of the process might influence the flavor and the preservation of the end products.     

DNA-induced increase in the alpha-helical content of C/EBP and GCN4

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.     

Beta-Actin Is Involved in Modulating Erythropoiesis during Development by Fine-Tuning Gata2 Expression Levels

The functions of actin family members during development are poorly understood. To investigate the role of beta-actin in mammalian development, a beta-actin knockout mouse model was used. Homozygous beta-actin knockout mice are lethal at embryonic day (E)10.5. At E10.25 beta-actin knockout embryos are growth retarded and display a pale yolk sac and embryo proper that is suggestive of altered erythropoiesis. Here we report that lack of beta-actin resulted in a block of primitive and definitive hematopoietic development. Reduced levels of Gata2, were associated to this phenotype. Consistently, ChIP analysis revealed multiple binding sites for beta-actin in the Gata2 promoter. Gata2 mRNA levels were almost completely rescued by expression of an erythroid lineage restricted ROSA26-promotor based GATA2 transgene. As a result, erythroid differentiation was restored and the knockout embryos showed significant improvement in yolk sac and embryo vascularization. These results provide new molecular insights for a novel function of beta-actin in erythropoiesis by modulating the expression levels of Gata2 in vivo.     

The LIM domains of WLIM1 define a new class of actin bundling modules

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.     

The actin cytoskeleton in normal and pathological cell motility

Cell motility is crucial for tissue formation and for development of organisms. Later on cell migration remains essential throughout the lifetime of the organism for wound healing and immune responses. The actin cytoskeleton is the cellular engine that drives cell motility downstream of a complex signal transduction cascade. The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. The multitude of proteins and regulatory mechanisms partaking in this system makes it vulnerable to mutations and alterations in expression levels that ultimately may cause diseases. The most familiar one is cancer that in later stages is characterized by active aberrant cell migration. Indeed tumor invasion and metastasis are increasingly being associated with deregulation of the actin system.     

A method for rapidly screening functionality of actin mutants and tagged actins

Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three β-actin mutants that have been associated with diseases. Published: October 25, 2004.     

TetraThymosinbeta is required for actin dynamics in Caenorhabditis elegans and acts via functionally different actin-binding repeats

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.     

Transcriptome analysis of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>during symbiosis

Background  

Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis.