Delegates from seventeen countries attended. The formal program included forty verbal presentations by invited speakers and sixty miniposter presentations, and was formulated by the Organizing and Scientific Committee: E. Carmeliet Leuven , J. Collin Poitiers , S. Forsen Lund , C. Heizmann Ziirich , D. Lawson Cambridge , P.
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Calcium binding proteins in normal and transformed cells, Perugia, May
Pochet Brussels. This volume contains the papers prepared by the invited speakers. The highlights of the symposium are numerous. Bulky hydrophobic residues of H5 including V86, F90, and I94 are key residues that associate with the hydrophobic pocket and act as anchoring residues Fig. The EF-hand domain and H5 are directly connected by a short flexible linker Fig. Next, we investigated the possible interaction between H5 and ABD2.
Again, no chemical shift changes were observed, indicating no interaction. The 30 lowest energy structures are superimposed using the backbone atoms in the well folded region left panels.
Ribbon representation of the lowest energy structure right panels. The key residues V86, F90, and I94 which associate with the hydrophobic pocket of the EF domain are highlighted. In order to study the structural effects caused by the Ser5 phosphorylation, we have prepared a S5E mutant of the EF-H5 construct.
The S5E mutation has been utilized previously to mimic the phosphorylation at Ser 5 The few residues that experience chemical shift changes were all located in the immediate vicinity of the mutation. Therefore, phosphorylation does not seem to affect the interaction between the EF domain and H5 and the global structure of the EF domain remains unchanged. This result is resonable as this phosphorylation site is located in the flexible N-terminal portion of EF-H5 in both forms Supplementary Fig. Figure 3a shows the ITC isotherm obtained during a calcium-titration experiment and the fitting to determine the thermodynamic parameters for EF-H5.
The isotherm shows that it contains two binding events which are very close in their Kds. However, we were not able to unambiguously derive two sets of ITC parameters from these data. When we analyzed these data as a single binding event, a Kd value of 0. In this case, the curve fitting with a two sites model produced two very different Kd values, 0. These data are summarized in Table 1.
The baseline corrected ITC titrations are shown in the top panel. Derived binding isotherms were fitted to obtain thermodynamic parameters bottom panel. In order to investigate the role of H5 in actin-bundling, we incubated platelet actin and various LPL constructs depicted in Fig. This result is in line with previous reports 9. It was unexpected that the ABD12 construct, which only contains four CH-domains, produced only a very small amount of actin bundles Fig. However, the inclusion of H5 in the construct H5-ABD12 dramatically increased the amount of actin bundles to almost the same level with the full-length LPL, suggesting that H5 is indispensable for actin-bundling of LPL.
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The Kd values derived were compared to that of an H5 synthetic peptide representing the H5 region of LPL residues 83— The affinity between EF and the H5 peptide was found to be relatively weak with a Kd value of 3. Interestingly, the melittin peptide bound one order of magnitude stronger than the H5 peptide.
Many signals disappeared as we added the peptide, indicating an interaction with an intermediate exchange behavior on the NMR time scale. As we expected, most of the signals that disappeared originated from the residues in the bound H5 region Fig. As a control, a titration experiment was also performed with the CaMKIp peptide, which has almost the same Kd as the H5 peptide Table 2.
There were no substantial chemical shift changes detected in this titration, indicating that the CaMKIp peptide was unable to displace H5. Finally, we have examined whether the presence of melittin peptide affects the actin-bundling of LPL Fig. The actin-bundling induced by the melittin peptide alone was negligible data not shown. The assignments for the affected signals are indicated. The flexible side chains of methionine allow the target binding pockets of CaM to accommodate widely different targets This implies that the hydrophobic pocket of the LPL EF-hands is devoid of methionine side-chains and seems to be designed to be rather specific for the H5 sequence.
Importantly, amino-acid sequence alignments show that the H5 region is highly conserved among all three human plastin isoforms data not shown. Indeed this sequence produced a high theoretically predicted score for CaM binding Indeed, CaM binding to CH-domains from many different actin-binding proteins has been reported before 49 , 50 , 51 , The decreased localization level of LPL in the contact zone by removing the H5 region may then be attributed to the reduced actin-bundling activity of LPL see below.
This observation confirmed that the steric hindrance caused by the reduced distance is not the major reason for the reduced actin bundling.
Exome-wide association study of plasma lipids in >300,000 individuals.
Previous cryo-EM studies of LPL constructs, when they are decorated on the F-actin filament, have provided significant low resolution structural information 37 , 60 , 61 , 62 , Indeed, the crystal structure of the actin-binding core ABDs of Arabidopsis thaliana fimbrin AtFim1 and Schizosaccharomyces pombe fimbrin sac6 have revealed that the relative domain orientation between the CH1 and CH2 cannot be defined with a single orientation, which suggests some flexibility between the domains Indeed, the relative domain orientation in the crystal structure was not consistent with that determined by the cyro-EM studies 60 , 61 , 63 and was not supported by previous mutational studies These observations suggest that a significant rearrangement of relative domain orientation of the four CH domains is required prior to actin-binding.
According to our results, H5 does not interact directly with the ABDs in the absence of any the actin filaments Supplementary Fig. However, from the cryo-EM structure 60 , the H5 region may interact with ABD1 between CH1 and CH2 upon binding to the actin-filament where H5 acts as a wedge to stabilize domain orientation to form a favorable actin-binding interface.
The possible sterical hindrance caused by the reduced distance between EF and ABDs may play a role in the regulation as discussed above. This may lead to the development of potential drugs that could contribute to suppressing the metastatic activity of cancer cells. Considering that H5 is directly linked to the EF domain in the actual protein, it would require much higher affinity to outcompete.
Among the peptides we have examined, only melittin showed a much higher affinity than the H5 peptide Table 2. Among all three human plastin isoforms, LPL is the only isoform that is currently known to be phosporylated in vivo Janji and coworkers demonstrated that compared to the non-phosphorylated LPL protein, the S5E mutant had increased actin bundling activity Our results show that the phosphorylation of Ser5 does not induce a conformational change in the EF domain.
Therefore, we propose that the phosphorylation of Ser5 directly enhances this effect. Development of metastasis provides the most serious challenge for cancer treatment and is responsible for most cancer related deaths. A number of recent studies have shown that ectopically expressed LPL is largely responsible for this process Therefore, LPL presents itself as a promising target for drug development to prevent the metastatic activity of the cancer cells. It is also well known that inhibition of fascin, another actin-bundling protein, is a viable approach to block tumor metastasis 66 , The melittin peptide, which is isolated from bee venom, has been reported to have anticancer activity including the inhibition of metastasis by reducing cell motility and it has been proposed as an agent for anticancer therapy 68 , However, melittin is also a strong hemolytic peptide that is toxic to all normal cells.
Therefore, it is not a suitable peptide for direct clinical applications.
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Nevertheless, drugs that can block the binding pocket of the EF domain of LPL and that are safe for clinical application could be discovered via high-throughput screening. Therefore, we believe that our findings may be a significant first step towards the future development of drugs that target the metastatic events that occur in many types of cancer cells.
A synthetic gene with optimized codons for the expression of the full-length human L-plastin protein LPL in E. All the recombinant plasmids were transformed into competent E. Uniformly 15 N- or 15 N, 13 C-labelled proteins were prepared in M9 minimal medium supplemented with 0. The supernatant was applied onto the column equilibrated with the extraction buffer. The column was washed with the extraction buffer supplemented with 0.
The eluted fractions were passed through the cOmplete column to remove the TEV protease. TEV protease was expressed and purified from the pRK plasmid as previously described CYANA version 2.
Phosphorylation of tyrosine residues of calmodulin in Rous sarcoma virus-transformed cells.
The 30 lowest-energy structures from a total of were used for analysis. The calcium free buffer was prepared via 1 week incubation with Chelex chelating agent. Each protein sample was exchanged into the calcium free buffer, and then passed through a Calcium Sponge S column Life Technologies. The BIAevaluation software 2. Two different concentrations in each experiment were injected twice to obtain the fitting errors SEM.
How to cite this article : Ishida, H.