Cell biological roles of αB-crystallin

αB-crystallin, also called HspB5, is a molecular chaperone able to interact with unfolding proteins. By interacting, it inhibits further unfolding, thereby preventing protein aggregation and allowing ATP-dependent chaperones to refold the proteins. αB-crystallin belongs to the family of small heat-shock proteins (sHsps), which in humans consists of 10 different members. The protein forms large oligomeric complexes, containing up to 40 or more subunits, which in vivo consist of heterooligomeric complexes formed by a mixture of αB-crystallin and other sHsps. αB-crystallin is highly expressed in the lens and to a lesser extent in several other tissues, among which heart, skeletal muscle and brain. αB-crystallin plays a role in several cellular processes, such as signal transduction, protein degradation, stabilization of cytoskeletal structures and apoptosis. Mutations in the αB-crystallin gene can have detrimental effects, leading to pathologies such as cataract and cardiomyopathy. This review describes the biological roles of αB-crystallin, with a special focus on its function in the eye lens, heart muscle and brain. In addition its therapeutic potential is discussed.     

Characterization and sequence prediction of structural variations in α-helix

Background

Centralised resources such as GenBank and UniProt are perfect examples of the major international efforts that have been made to integrate and share biological information. However, additional data that adds value to these resources needs a simple and rapid route to public access. The Distributed Annotation System (DAS) provides an adequate environment to integrate genomic and proteomic information from multiple sources, making this information accessible to the community. DAS offers a way to distribute and access information but it does not provide domain experts with the mechanisms to participate in the curation process of the available biological entities and their annotations.

Results

We designed and developed a Collaborative Annotation System for proteins called DAS Writeback. DAS writeback is a protocol extension of DAS to provide the functionalities of adding, editing and deleting annotations. We implemented this new specification as extensions of both a DAS server and a DAS client. The architecture was designed with the involvement of the DAS community and it was improved after performing usability experiments emulating a real annotation task.

Conclusions

We demonstrate that DAS Writeback is effective, usable and will provide the appropriate environment for the creation and evolution of community protein annotation.     

Effects of macromolecular crowding on the structural stability of human α-lactalbumin

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. α-Lactalbumin, as a model system for protein-folding studies, has been used extensively because it can form stable molten globule states under a range of conditions. Here we report that the crowding agents Ficoll 70, dextran 70, and polyethylene glycol (PEG) 2000 have different effects on the structural stability of human α-lactalbumin (HLA) represented by the transition to a molten globule state: dextran 70 dramatically enhances the thermal stability of Ca(2+)-depleted HLA (apo-HLA) and Ficoll 70 enhances the thermal stability of apo-HLA to some extent, while PEG 2000 significantly decreases the thermal stability of apo-HLA. Ficoll 70 and dextran 70 have no obvious effects on trypsin degradation of apo-HLA but PEG 2000 accelerates apo-HLA degradation by trypsin and destabilizes the native conformation of apo-HLA. Furthermore, no interaction is observed between apo-HLA and Ficoll 70 or dextran 70, but a weak, non-specific interaction between the apo form of the protein and PEG 2000 is detected, and such a weak, non-specific interaction could overcome the excluded-volume effect of PEG 2000. Our data are consistent with the results of protein stability studies in cells and suggest that stabilizing excluded-volume effects of crowding agents can be ameliorated by non-specific interactions between proteins and crowders.     

Locus assignment of human α-globin structural mutants by selective enzymatic amplification of α1 and α2-globin cDNAs

Summary We have used the powerful methodology of DNA enzymatic amplification in order to assign human -globin structural mutants to one of the two highly homologous -globin genes. Selectively amplified 1 and 2-globin cDNAs were dot-blotted and further hybridized to synthetic oligonucleotides encompassing either the normal or the mutated sequences. The generated signals corresponded specifically to one of the two -globin genes. Using this approach the -globin structural mutants J-Buda and G-Pest were found to be encoded by the 2 and the 1-globin genes, respectively. Furthermore, the exact nucleotide changes were determined. We propose this technique to serve as a simple and definitive method for assigning -globin structural mutants.     

Synthesis and biological evaluation of α-hydroxyalkylphosphonates as new antimicrobial agents

A set of α-quaternary 3-chloro-1-hydroxyalkylphosphonates, analogues of fosfomycin and fosfonochlorin, some of which are new compounds, was synthesized. The compounds were screened for bioactivity against several clinical and standard microbial isolates. Some were found to have moderate activity. The activity was higher with phenyl protection of the phosphoryl ester groups and α-phenyl substitution. Compound 11 was as effective or more potent than fosfomycin or chloramphenicol against several Gram-negative bacteria as well as against some Gram-positive ones.     

Mice with podocyte-specific overexpression of wild type α-actinin-4 are healthy controls for K256E-α-actinin-4 mutant transgenic mice

Mutations in the gene ACTN4 encoding the actin bundling protein—α-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant α-actinin-4 (K256E-ACTN4 ^pod+). However, whether α-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype α-actinin-4 (wt-ACTN4 ^pod+). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype α-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged α-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 ^pod+ mice are similar to K256E-ACTN4 ^pod+ mice. In contrast to K256E-ACTN4 ^pod+ mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 ^pod+ mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of α-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.     

Structure of α-conotoxin BuIA: influences of disulfide connectivity on structural dynamics

Background  

α-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in α-conotoxins is variable, leading to the classification of sub-families. BuIA is the only α-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin.     

Thermal denaturation and structural changes of α-helical proteins in keratins

To gain insight into the thermal stability of intermediate filaments and matrix in the biological composite structure of α-keratins, the thermal denaturation performance of human hair fibers was investigated by Modulated Differential Scanning Calorimetry (MDSC) in the dry and the wet state. Denaturation enthalpy ΔH(D) in water was found to be independent of heating rate (11.5J/g) and to be approximately double as high as in the dry state (5.2J/g). The lower enthalpy (dry) and its dependency on heating rate are attributed to effects of pyrolysis. The stepwise change of reversing heat capacity ΔC(p) marks the denaturation process as a classic two-stage transition. The increase of ΔC(p) with heating rate reflects a continuous shift of the nature of the denaturation of the α-helical material, first, into random coil and then towards random β-materials for lower heating rates. Denaturation temperatures follow Arrhenius relationships with heating rate, yielding activation energies of 416kJ/mol (dry) and 263kJ/mol (wet), respectively. A decrease of activation energy (wet) for high heating rates supports the hypothesis of systematic changes of the pathway of denaturation.     

Synthesis,structural characterization,and biological properties of pentyl- and isopentyl-α-D-glucosides

The study was devoted to the synthesis of pentyl glucosides (PenG_n) and isopentyl glucosides (Iso-PenG_n) by transglycosylation using recombinant cyclodextrin glycosyltransferase from Bacillus circulans A11, β-cyclodextrin as a glucosyl donor and 1-pentanol and isopentanol as acceptors. TLC and MS analysis indicated at least 3 products which were in accordance with PenG_n and IsoPenG_n having glucose, maltose and maltotriose attached to the alkyl groups of both alcohols. Two products of each glucoside were purified by preparative TLC and their structures were identified by NMR technique to be pentyl-α-D-glucopyranoside (PenG₁), pentyl-α-D-maltopyranoside (PenG₂), isopentyl-α-D-glucopyranoside (IsoPenG1) and isopentyl- α-D-maltopyranoside (IsoPenG₂). The effect of water-in-hexadecane emulsion on emulsion-forming properties showed that PenG₂ had the highest emulsifying activity. Adding PenG₂ to the insoluble Corynebacterium glutamicum amylomaltase from Escherichia coli transformants (A406R), helped to perform it to more soluble conformation. Moreover, it was found that PenG_1,2 exhibited a higher antibacterial activity against E. coli ATCC 25922 than that of IsoPenG_1,2. Hence, the biological properties of the synthesized products may be useful for their applications as emulsifying, solubilizing and antibacterial agents.     

Structure–activity relationships of αs-casein peptides with multifunctional biological activities

Multifunctional bioactive peptides have a wider role in modulating physiological functions and possess multiple biological activities. Peptides from bovine milk with sequences QKALNEINQF [p10] and TKKTKLTEEEKNRL [p14] from α-_S2 casein f (79–88) and α-_S2 casein f (148–161) were identified to be having multifunctional biological activities and were synthesized. These synthesized peptides show various biological activities like angiotensin-converting enzyme inhibition, prolyl endopeptidase inhibition, antioxidant, and antimicrobial activities. The mode of antimicrobial mechanism was studied and p10 shows depolarization of cell membrane, whereas p14 was found to display DNA-binding activity. Structural studies envisaged backbone flexibility, for differences in their mode of action. Peptide structure function studies were correlated to understand their multifunctional biological activity.     

A biological cosmos of parallel universes: Does protein structural plasticity facilitate evolution?

While Darwin pictured organismal evolution as "descent with modification" more than 150 years ago, a detailed reconstruction of the basic evolutionary transitions at the molecular level is only emerging now. In particular, the evolution of today's protein structures and their concurrent functions has remained largely mysterious, as the destruction of these structures by mutation seems far easier than their construction. While the accumulation of genomic and structural data has indicated that proteins are related via common ancestors, naturally occurring protein structures are often considered to be evolutionarily robust, thus leaving open the question of how protein structures can be remodelled while selective pressure forces them to function. New information on the proteome, however, increasingly explains the nature of local and global conformational diversity in protein evolution, which allows the acquisition of novel functions via molecular transition forms containing ancestral and novel structures in dynamic equilibrium. Such structural plasticity may permit the evolution of new protein folds and help account for both the origins of new biological functions and the nature of molecular defects.     

Synthesis and structural study of a fully protected α-C-galactosyl model dipeptide

The synthesis of three N-alkyl-6,7-dideoxy-1,2:3,4-di-O-isopropylidene-7-[(alkyl-carbonyl)amino]-L-glycero--D-galacto-octopyranuronamides6a–c, analogous model dipeptides containing two amide groups connected to the -carbon bearing the fully protected galactose as a side chain, has been realized with the aim of determining the conformational influence of the galactosyl moiety on the peptide backbone. Molecular modeling of 6a, X-ray crystallography of 6c, and IR and NMR experiments on 6a–c in organic solvents show that the carbohydrate ring assumes a twist boat conformation. In non-polar organic solvents, the NH of the left amide group interacts with one ketal oxygen of the galactosyl group.     

The thermal structural transition of α-crystallin inhibits the heat induced self-aggregation

α-crystallin, the major constituent of human lens, is a member of the heat-shock proteins family and it is known to have a quaternary structural transition at . The presence of calcium ions and/or temperature changes induce supramolecular self-aggregation, a process of relevance in the cataractogenesis. Here we investigate the potential effect of the bovine α-crystallin's structural transition on the self-aggregation process. Along all the temperatures investigated, aggregation proceeds by forming intermediate molecular assemblies that successively aggregate in clusters. The final morphology of the aggregates, above and below Tc, is similar, but the aggregation kinetics are completely different. The size of the intermediate molecular assemblies, and their repulsive energy barrier show a marked increase while crossing . Our results highlight the key role of heat modified form of α-crystallin in protecting from aggregation and preserving the transparency of the lens under hyperthermic conditions.     

Synthesis and structural study of a fully protected α-C-galactosyl model dipeptide

Summary The synthesis of three N-alkyl-6,7-dideoxy-1,2:3,4-di-O-isopropylidene-7-[(alkyl-carbonyl)amino]-L-glycero-α-D-galacto-octopyranuronamides6a-c, analogous model dipeptides containing two amide groups connected to the α-carbon bearing the fully protected galactose as a side chain, has been realized with the aim of determining the conformational influence of the galactosyl moiety on the peptide backbone. Molecular modeing of6a, X-ray crystallography of6c and IR and NMR experiments on6a-c in organic solvents show that the carbohydrate ring assumes a twist boat conformation. In non-polar organic solvents, the NH of the left amide group interacts with one ketal oxygen of the galactosyl group.     

Synthesis and biological evaluation of caffeic acid derivatives as potent inhibitors of α-MSH-stimulated melanogenesis

We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin medication including a cosmetic skin-whitening agent. The SAR studies revealed that cyclohexyl ester and secondary amide derivatives of caffeic acid showed significant inhibitory activities.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.