Regeneration of Articular Cartilage Using Membranes of Polyester Scaffolds in a Rabbit Model.

One promising method for cartilage regeneration involves combining known methods, such as the microfracture technique with biomaterials, e.g., scaffolds (membranes). The most important feature of such implants is their appropriate rate of biodegradation, without the production of toxic metabolites. This study presents work on two different membranes made of polyester (L-lactide-co-ε-caprolactone-PLCA) named "PVP and "Z". The difference between them was the use of different pore precursors-polyvinylpyrrolidone in the "PVP" scaffold and gelatin in the "Z" scaffold. These were implemented in the articular cartilage defects of rabbit knee joints (defects were created for the purpose of the study). After 8, 16, and 24 weeks of observation, and the subsequent termination of the animals, histopathology and gel permeation chromatography (GPC) examinations were performed. Statistical analysis proved that the membranes support the regeneration process. GPC testing proved that the biodegradation process is progressing exponentially, causing the membranes to degrade at the appropriate time. The surgical technique we used meets all the requirements without causing the membrane to migrate after implantation. The "PVP" membrane is better due to the fact that after 24 weeks of observation there was a statistical trend for higher histological ratings. It is also better because it is easier to implant due to its lower fragility then membrane "Z". We conclude that the selected membranes seem to support the regeneration of articular cartilage in the rabbit model.

Solution X-Ray Scattering for Membrane Proteins.

While X-ray crystallography remains the most popular and productive technique for protein structure determination, it very often produces incomplete models, either due to truncations introduced by the scientists or locally weak experimental data. This problem is even more common for transmembrane proteins, owing to the difficulties inherent in their crystallization. By the virtue of operating in solution, SAXS bypasses the problems with crystallization and allows for easier work with full-length constructs and, thus, can potentially be used to fill the missing (and often crucial) details. Here, we describe a complete procedure to build a complete model of a transmembrane protein based on a truncated crystallographic model and experimental SEC-SAXS data using refractometry and UV absorption for internal validation of the measurements.

New Perspectives in Joint Cartilage Treatment with Special Emphasis on New Types of Hydrogels: a Review.

Joints are a necessary anatomic and functional element of the organ of locomotion. Hyaline cartilage is a very important element of a joint in physiological terms. Joint cartilage is subjected to injuries associated with non-physiological loading and excessive abnormal mobility caused by ligament instability. These can lead to damage to the surface of the cartilage and the development of defects. Until now there has been no "golden standard" for treating injuries to joint cartilage. The goal of this treatment is to sustain knee function at a level that is tolerable and acceptable to the patient. Three major minimally invasive techniques for treating damage of the surface of the joints are currently available, namely 1) the microfracture technique, which stimulates bone marrow. Regenerative processes promote the formation of fibrohyaline cartilage. 2) transplantation of osteocartilaginous allo- and autogeneic cylinders. 3) in vitro chondrocyte culture and transplantation of these chondrocytes to sites with cartilage defects. This review describes both the historical and modern techniques of joint cartilage treatment as well as new perspectives related to the use of biomaterials in the healing of cartilage defects.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.

A unified coarse-grained model of biological macromolecules based on mean-field multipole-multipole interactions.

A unified coarse-grained model of three major classes of biological molecules--proteins, nucleic acids, and polysaccharides--has been developed. It is based on the observations that the repeated units of biopolymers (peptide groups, nucleic acid bases, sugar rings) are highly polar and their charge distributions can be represented crudely as point multipoles. The model is an extension of the united residue (UNRES) coarse-grained model of proteins developed previously in our laboratory. The respective force fields are defined as the potentials of mean force of biomacromolecules immersed in water, where all degrees of freedom not considered in the model have been averaged out. Reducing the representation to one center per polar interaction site leads to the representation of average site-site interactions as mean-field dipole-dipole interactions. Further expansion of the potentials of mean force of biopolymer chains into Kubo's cluster-cumulant series leads to the appearance of mean-field dipole-dipole interactions, averaged in the context of local interactions within a biopolymer unit. These mean-field interactions account for the formation of regular structures encountered in biomacromolecules, e.g., α-helices and ß-sheets in proteins, double helices in nucleic acids, and helicoidally packed structures in polysaccharides, which enables us to use a greatly reduced number of interacting sites without sacrificing the ability to reproduce the correct architecture. This reduction results in an extension of the simulation timescale by more than four orders of magnitude compared to the all-atom representation. Examples of the performance of the model are presented.

Improvement of the treatment of loop structures in the UNRES force field by inclusion of coupling between backbone- and side-chain-local conformational states.

The UNited RESidue (UNRES) coarse-grained model of polypeptide chains, developed in our laboratory, enables us to carry out millisecond-scale molecular-dynamics simulations of large proteins effectively. It performs well in ab initio predictions of protein structure, as demonstrated in the last Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP10). However, the resolution of the simulated structure is too coarse, especially in loop regions, which results from insufficient specificity of the model of local interactions. To improve the representation of local interactions, in this work we introduced new side-chain-backbone correlation potentials, derived from a statistical analysis of loop regions of 4585 proteins. To obtain sufficient statistics, we reduced the set of amino-acid-residue types to five groups, derived in our earlier work on structurally optimized reduced alphabets, based on a statistical analysis of the properties of amino-acid structures. The new correlation potentials are expressed as one-dimensional Fourier series in the virtual-bond-dihedral angles involving side-chain centroids. The weight of these new terms was determined by a trial-and-error method, in which Multiplexed Replica Exchange Molecular Dynamics (MREMD) simulations were run on selected test proteins. The best average root-mean-square deviations (RMSDs) of the calculated structures from the experimental structures below the folding-transition temperatures were obtained with the weight of the new side-chain-backbone correlation potentials equal to 0.57. The resulting conformational ensembles were analyzed in detail by using the Weighted Histogram Analysis Method (WHAM) and Ward's minimum-variance clustering. This analysis showed that the RMSDs from the experimental structures dropped by 0.5 Å on average, compared to simulations without the new terms, and the deviation of individual residues in the loop region of the computed structures from their counterparts in the experimental structures (after optimum superposition of the calculated and experimental structure) decreased by up to 8 Å. Consequently, the new terms improve the representation of local structure.

Sequential duplications of an ancient member of the DnaJ-family expanded the functional chaperone network in the eukaryotic cytosol.

Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, and biochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinary functional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones to form a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all three kingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteins diverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversification and thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimental analyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, which emerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modeling revealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However, despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in the translocation of proteins from the cytosol into mitochondria. Apj1's specialized role is related to degradation of sumolyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specialized functions, allowing expansion of the chaperone functional network, while maintaining the overall structural organization of their parental gene.