A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.     

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.     

A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.     

Proposed follow up programme after curative resection for lower third oesophageal cancer

Cyclins are indispensable elements of the cell cycle and derangement of their function can lead to cancer formation. Recent studies have also revealed more mechanisms through which cyclins can express their oncogenic potential. This review focuses on the aberrant expression of G1/S cyclins and especially cyclin D and cyclin E; the pathways through which they lead to tumour formation and their involvement in different types of cancer. These elements indicate the mechanisms that could act as targets for cancer therapy.