Chaperone-mediated inhibition of tubulin self-assembly

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.     

Recognition of functional genetic polymorphism using ESE motif definition: a conservative evolutionary approach to CYP2D6/CYP2C19 gene variants

Genetica - Although predicting the effects of variants near intron-exon boundaries is relatively straightforward, predicting the functional Exon Splicing Enhancers (ESEs) and the possible effects...     

Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex

Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.     

Design and synthesis of caffeoyl-anilides as portmanteau inhibitors of HIV-1 integrase and CCR5

Designing multi-functional ligands is a recent strategy by which multiple targets can be inhibited by a single entity. A series of caffeoyl-anilide compounds based on structures of various integrase and CCR-5 inhibitors have been designed and synthesized as anti-HIV agents in the present study. Most of the compounds exhibited potent anti-HIV activity at micromolar concentration in CEM-GFP CD4+ T cells infected with HIV-1NL4.3 virus. Compound 14 showed a lower EC(50) and better TI as compared to AZT. Mechanism based studies suggest that these compounds inhibit either one or in some cases, both the targets. The experimental data and the docking results showed that these compounds are potential inhibitors for both HIV-1 IN and CCR5.     

Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer

DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered to be the key molecular mechanisms mediating FEN1's key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock-in of a point mutation rather than knock-out of a whole gene.     

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C?G? repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.     

Multilayered microspheres for the controlled release of growth factors in tissue engineering

Tissue regeneration may be stimulated by growth factors but to be effective, this delivery must be sustained and requires delivery vehicles that overcome the short half-life of these molecules in vivo. One promising approach is to couple growth factors to the biomaterial surface so that they are readily bioavailable. Here the layer-by-layer process was used to construct a multilayered polyelectrolyte delivery system on the surface of poly(lactic-co-glycolic) acid constructs. The system was first optimized on a planar surface before translation to a 3D microsphere system. The layers incorporated heparin to facilitate the loading of basic fibroblast growth factor and increase growth factor stability. Cross-linked capping layers also reduced any burst release. The model growth factor was released in a sustained manner and stimulated significantly higher cell proliferation in vitro on release compared with the addition of the growth factor heparin complex free in solution, demonstrating the promise of this approach.