Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain

Suppression of Tumorigenicity 2 (ST2), a member of the interleukin-1 receptor (IL-1R) family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC) analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2^ECD) models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2^ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2^ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2^ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2^ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2^ECD (containing D1-D3 domains) in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2^ECD contributes predominantly to the interaction between ST2^ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2^ECD and modulation sites conformed to ST2^ECD conformations. Our study provides structural models and analyses of ST2^ECD that could be useful for inhibitor discovery.     

Alginate as a support ligand for enhanced colloidal liquid aphron immobilization of proteins and drug delivery

Research within the field of colloidal liquid aphrons (CLAs) for enzyme immobilization has often used ionic surfactants for the retention of enzymes. Although these charged interactions allow for enhanced immobilization, they can often lead to denaturation of enzyme activity, and even release of the protein. Sodium alginate has been used in drug delivery applications due to its low toxicity and charged interactions that allow for encapsulation. Hence, alginate systems can be used as an alternative to ionic surfactants in CLA immobilization. This paper presents, for the first time, the use of sodium alginate as potential ligand for enhanced CLA immobilization. The use of five model proteins; lysozyme, bovine serum albumin, ovalbumin, insulin, and α-chymotrypsin, of various pIs and hydrophobicities, showed the relevance of electrostatic interactions in promoting binding with sodium alginate when the pH < pI, with 100% immobilization attributed to alginate incorporated CLAs over general nonionic formulations. Furthermore, above their pI, >80% protein recovery was observed, with activity and conformation comparable to their native counterparts. Finally, the use of proteolysis showed that as the degree of ionic bonding increased between the protein and sodium alginate, the degree of protease resistance decreased due to conformational changes experienced during binding.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Co-extraction during reactive extraction of phenylalanine using Aliquat 336: interfacial mass transfer

Reactive liquid-liquid extraction can be used to separate hydrophilic fermentation products that would not otherwise partition into nonpolar solvents. However, during extraction of the target solute other compounds present in the extraction medium will also react with the ion exchange reagent and are thus co-extracted. In this study the effect of co-extraction on the interfacial flux of the target solute phenylalanine has been investigated for reactive extraction using Aliquat 336. The effect of co-extracting compounds has been included in a new interfacial flux balance, and experimental results reveal that the interfacial concentrations are equal to the final equilibrium conditions of the system. Using this information a simple mass transfer model has been developed from which film mass transfer coefficients may be determined. Co-extraction of other compounds present in the feed was found to reduce the interfacial flux of the target solute by reducing the driving force. Co-extraction did not affect the value of the film mass transfer coefficient, and therefore, co-extraction does not effect the transport properties of the solute to the interface. Extraction from a multicomponent fermentation broth resulted in a reduced flux, which arises from a reduction in the driving force caused by high levels of co-extraction. Furthermore, the flux was also reduced as the result of a mass transfer resistance caused by soluble surface-active compounds present in the fermentation broth adsorbing to the interface. The biomass associated with the fermentation broth was also found to reduce the solute flux, and it is believed that this is due to blockage of the interfacial area.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.