Development and Optimization of Therapeutic Analogues of Anti-TNFα Antibody Infliximab

Previously, we have reported the crystal structures of Fab fragment of Infliximab in complex with TNFα. The structurally identified epitope on TNFα revealed the mechanism of TNFα inhibition by partially overlapping with the TNFα-receptor interface and the possibility to optimize the binding affinity. In this study, we launched a screen of a phage display library to isolate novel anti-TNFα antibodies based on the infliximab epitope. To develop novel anti-TNFα antibodies, structural analysis, the phage display antibody isolation, step by step antibody optimization, CDR residues random mutagenesis, and binding affinity characterization were performed. One of the novel antibodies generated on the backbone of infliximab, Inf3D6, has the superior binding affinity to TNFα, thus, demonstrating the potential for structure guided optimization for improvement of existing antibody-based therapeutics.     

Characterization of Mamalian GS-α Proteins Expressed in Yeast

Abstract

The guanine nucleotide regulatory protein, G_s, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the Mp 42-45,000, short form (S), and M₁= 46-52,000, long form (L), of the a-subunits of rat G_s were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP 1 promoter and transformed into Saccharomvces cerevisiae. In the presence of 100 pM CuSOq, the transformed yeast expressed Gs-a mRNAs and proteins. In reconstitution experiments, rat Gs-a(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol, and guanine nudeotidedependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous G_s-a. G_s-a(S) demonstrated twice the activity of Gs-a(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of Gs-a(S) expressed in yeast with Gs purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as puriiied G,. Addition of bovine brain py subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for Gq-a(S and L) obtained from yeaa. In contrast, transducin py only enhanced agonist-stimulated adenylyl cyclase activity for Gs-a(S and L) following reconstitution. These results demonstrate that the expression of functional mammalian Gs-a subunits in yeast may be useful for their biochemical characterization.     

X-Ray Structures of the LXRα LBD in Its Homodimeric Form and Implications for Heterodimer Signaling

Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRα. These compounds are GW3965, F₃methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRα ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRβ homodimer and LXRα:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.     

Phosphorylation of Glutathione-S-transferase by Protein Kinase C-α Implications for Affinity-Tag Purification

Expression and purification of proteins as fusions with glutathione S-transferase (GST) is a standard and widely employed system. In more than 2,500 published studies, GST has been used to facilitate the purification of recombinant proteins, assess protein-protein interactions, and establish protein function. In this report, we provide evidence that GST can be phosphorylated in vitro by protein kinase C-alpha (PKC-alpha) at Ser-93. Therefore, since GST itself may be a target for a number of catalytic enzymes, failure to remove the GST tag from the recombinant protein may lead to inaccurate conclusions.     

Expression of HIF-1α and Its Target Genes in the Nanorana parkeri Heart:Implications for High Altitude Adaptation

Hypoxia-inducible factor 1 alpha(HIF-1α) and its target genes vascular endothelial growth factor(VEGF) and transferrins(TF) play an important role in native endothermic animals' adaptation to the high altitude environments. For ectothermic animals – especially frogs – it remains undetermined whether HIF-1α and its target genes(VEGF and TF) play an important role in high altitude adaptation, too. In this study, we compared the gene sequences and expression of HIF-1α and its target genes(VEGF and TF) between three Nanorana parkeri populations from different altitudes(3008 m a.s.l., 3440 m a.s.l. and 4312 m a.s.l.). We observed that the c DNA sequences of HIF-1A exhibited high sequence similarity(99.38%) among the three altitudinally separated populations; but with increasing altitude, the expression of HIF-1A and its target genes(VEGF and TF) increased significantly. These results indicate that HIF-1α plays an important role in N. parkeri adaptation to the high altitude, similar to its role in endothermic animals.     

Topological Frustration in βα-Repeat Proteins: Sequence Diversity Modulates the Conserved Folding Mechanisms of α/β/α Sandwich Proteins

The thermodynamic hypothesis of Anfinsen postulates that structures and stabilities of globular proteins are determined by their amino acid sequences. Chain topology, however, is known to influence the folding reaction, in that motifs with a preponderance of local interactions typically fold more rapidly than those with a larger fraction of nonlocal interactions. Together, the topology and sequence can modulate the energy landscape and influence the rate at which the protein folds to the native conformation. To explore the relationship of sequence and topology in the folding of βα-repeat proteins, which are dominated by local interactions, we performed a combined experimental and simulation analysis on two members of the flavodoxin-like, α/β/α sandwich fold. Spo0F and the N-terminal receiver domain of NtrC (NT-NtrC) have similar topologies but low sequence identity, enabling a test of the effects of sequence on folding. Experimental results demonstrated that both response-regulator proteins fold via parallel channels through highly structured submillisecond intermediates before accessing their cis prolyl peptide bond-containing native conformations. Global analysis of the experimental results preferentially places these intermediates off the productive folding pathway. Sequence-sensitive Gō-model simulations conclude that frustration in the folding in Spo0F, corresponding to the appearance of the off-pathway intermediate, reflects competition for intra-subdomain van der Waals contacts between its N- and C-terminal subdomains. The extent of transient, premature structure appears to correlate with the number of isoleucine, leucine, and valine (ILV) side chains that form a large sequence-local cluster involving the central β-sheet and helices α2, α3, and α4. The failure to detect the off-pathway species in the simulations of NT-NtrC may reflect the reduced number of ILV side chains in its corresponding hydrophobic cluster. The location of the hydrophobic clusters in the structure may also be related to the differing functional properties of these response regulators. Comparison with the results of previous experimental and simulation analyses on the homologous CheY argues that prematurely folded unproductive intermediates are a common property of the βα-repeat motif.     

Plasma Levels of Inter-α Inhibitor Proteins in Children with Acute Dengue Virus Infection

Background

Inter-α inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the concentration of IaIp during DENV infections and evaluated its potential as a biomarker.

Methods and Findings

Concentrations of IaIp were measured in patients with acute DENV infections using a quantitative, competitive enzyme linked immunoassay. Concentrations of IaIp measured in pediatric patients suffering from severe DENV infections were significantly lower than in healthy controls.

Conclusions

This is the first report to demonstrate changes in concentration of IaIp during viral infections. The data also highlight the potential of IaIp as a biological marker for severity of DENV infections.     

Factors Influencing Diversity of Farmers’ Varieties of Sweet Potato in Uganda: Implications for Conservation

Factors Influencing Diversity of Farmers’ Varieties of Sweet Potato in Uganda: Implications for Conservation. There is increasing concern that agricultural intensification is causing loss of crop biodiversity due to displacement of traditional farmers’ varieties by a small number of improved cultivars. Using ethnobotanical surveys, we assessed the implication of adoption of new sweet potato (Ipomoea batatas) cultivars on the maintenance of farmers’ varieties in Uganda. Other factors influencing varietal diversity were also assessed. A total of 102 farmer households distributed in the top three sweet potato production agro-ecological zones were interviewed. With the exception of released cultivars, very few varieties appeared in more than one region. The majority of the respondents indicated that they continue to plant some of the existing varieties when they adopt new cultivars. Loss of planting materials due to drought was a major constraint to maintaining varietal diversity for this vegetatively propagated crop. Limited land and lack of access to best management practices were also key constraints to maintenance of farmers’ varieties. The primary criteria for adopting new cultivars were higher yield, taste, and duration to maturity. Yield stability, tolerance to native biotic and abiotic stresses, and good taste were important for maintenance of currently grown varieties. Overall, criteria for variety selection varied with household characteristics including farmer age and gender, uses of the crop, micro-climatic conditions in the farmers’ fields, and level of access to agricultural extension. The observed heterogeneity in selection criteria, influence of social ties, and the role of environment in varietal maintenance have important implications for establishing breeding priorities and preservation of crop diversity.     

The λ Red Proteins Promote Efficient Recombination between Diverged Sequences: Implications for Bacteriophage Genome Mosaicism

Genome mosaicism in temperate bacterial viruses (bacteriophages) is so great that it obscures their phylogeny at the genome level. However, the precise molecular processes underlying this mosaicism are unknown. Illegitimate recombination has been proposed, but homeologous recombination could also be at play. To test this, we have measured the efficiency of homeologous recombination between diverged oxa gene pairs inserted into λ. High yields of recombinants between 22% diverged genes have been obtained when the virus Red Gam pathway was active, and 100 fold less when the host Escherichia coli RecABCD pathway was active. The recombination editing proteins, MutS and UvrD, showed only marginal effects on λ recombination. Thus, escape from host editing contributes to the high proficiency of virus recombination. Moreover, our bioinformatics study suggests that homeologous recombination between similar lambdoid viruses has created part of their mosaicism. We therefore propose that the remarkable propensity of the λ-encoded Red and Gam proteins to recombine diverged DNA is effectively contributing to mosaicism, and more generally, that a correlation may exist between virus genome mosaicism and the presence of Red/Gam-like systems.     

Nα-Methylation of arginine: Implications for cell-penetrating peptides

The field of cell-penetrating peptides is dominated by the use of oligomers of arginine residues. Octanol–water partitioning in the presence of an anionic lipid is a validated proxy for cell-penetrative efficacy. Here, we add one, two, or three N-methyl groups to Ac-Arg-NH₂ and examine the effects on octanol–water partitioning. In the absence of an anionic lipid, none of these arginine derivatives can be detected in the octanol layer. In the presence of sodium dodecanoate, however, increasing N-methylation correlates with increasing partitioning into octanol, which is predictive of higher cell-penetrative ability. We then evaluated fully N^α-methylated oligoarginine peptides and observed an increase in their cellular penetration compared with canonical oligoarginine peptides in some contexts. These findings indicate that a simple modification, N^α-methylation, can enhance the performance of cell-penetrating peptides.     

Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.     

Does sex of children matter? Implications for fertility in Pakistan

Preference for children of either sex is considered a constraint on fertility decline as it induces many couples to keep adding on surviving children in the hope of having a desired sex composition of children. However, preferences for children of a particular sex may differ in relation to demographic and socioeconomic characteristics of women, traditional values and cultural practices, such as propagating a family name, providing economic advantages, and obtaining a medium of social and economic security in times of illness, unemployment and old age. Utilizing the Pakistan Integrated Household Survey (2001-02), this paper aims at investigating the existence of sex preference and examines sex preference differentials by different attributes of women in Pakistan. The results reveal that there is a desire to have another child in the presence of all children of one sex, either sons or daughters. The desire to have a son with only or mostly daughters, however, is stronger than the desire to have a daughter with only or mostly sons. This behaviour will retard fertility decline unless there is a shift in the desire to have children of both sexes in Pakistan.     

Synthesis and anti-BVDV activity of novel δ-sultones in vitro: Implications for HCV therapies

In this study we report the synthesis and activity against bovine viral diarrhea virus (BVDV) of a novel series of bicycle δ-sultones containing γ-lactones. BVDV is responsible for major losses in cattle. Some of the synthesized δ-sultones showed pronounced anti-BVDV activity with EC₅₀ values of 0.12–1.0 μM and no significant cytotoxicity. Among them, the ortho bromosubstituted derivative 4f (EC₅₀ = 0.12 μM) showed better antiviral activity than other derivatives and was 10 fold more that of than positive control ribavirin (EC₅₀ = 1.3 μM). BVDV is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. The above results provided a novel candidate for the development of anti-HCV agents.     

Broad Distribution of TPI-GAPDH Fusion Proteins among Eukaryotes: Evidence for Glycolytic Reactions in the Mitochondrion?

Glycolysis is a central metabolic pathway in eukaryotic and prokaryotic cells. In eukaryotes, the textbook view is that glycolysis occurs in the cytosol. However, fusion proteins comprised of two glycolytic enzymes, triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were found in members of the stramenopiles (diatoms and oomycetes) and shown to possess amino-terminal mitochondrial targeting signals. Here we show that mitochondrial TPI-GAPDH fusion protein genes are widely spread across the known diversity of stramenopiles, including non-photosynthetic species (Bicosoeca sp. and Blastocystis hominis). We also show that TPI-GAPDH fusion genes exist in three cercozoan taxa (Paulinella chromatophora, Thaumatomastix sp. and Mataza hastifera) and an apusozoan protist, Thecamonas trahens. Interestingly, subcellular localization predictions for other glycolytic enzymes in stramenopiles and a cercozoan show that a significant fraction of the glycolytic enzymes in these species have mitochondrial-targeted isoforms. These results suggest that part of the glycolytic pathway occurs inside mitochondria in these organisms, broadening our knowledge of the diversity of mitochondrial metabolism of protists.     

Muscling in on PGC-1α for improved quality of life in ALS

Impaired activity of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)-1α has been implicated in the pathophysiology of several neurodegenerative disorders. In this issue, Da Cruz et al. (2012) show improved muscle function, but not survival, with increased PGC-1α activity in muscle in a mouse model of amyotrophic lateral sclerosis.     

The multifaceted activity of VEGF in angiogenesis – Implications for therapy responses

Vascular endothelial growth factor (VEGF) is a key growth factor driving angiogenesis (i.e. the formation of new blood vessels) in health and disease. Pharmacological blockade of VEGF signaling to inhibit tumor angiogenesis is clinically approved but the survival benefit is limited as patients invariably acquire resistance. This is partially mediated by the intrinsic flexibility of tumor cells to adapt to VEGF-blockade. However, it has become clear that tumor stromal cells also contribute to the resistance. Originally, VEGF was thought to specifically target endothelial cells (ECs) but it is now clear that many stromal cells also respond to VEGF signaling, making anti-VEGF therapy more complex than initially anticipated. A more comprehensive understanding of the complex responses of stromal cells to VEGF-blockade might inform the design of improved anti-angiogenic agents.     

Alternate Positions for the Sulphydryl Group in β-Lactoglobulin: the Significance for Sulphydryl Location in Other Proteins

EARLIER studies of the location of the single cysteine residue and the two disulphide bridges in bovine β-lactoglobulins A and B¹, for each of which the monomer is a single chain of 162 residues and 18,000 molecular weight^2,3, led to the conclusion that the sulphydryl group is at position 69 and that the disulphides bridge positions 123 to 160 and 57 to 70. These results were based on diagonal peptide studies⁴ and on the composition of peptides in which the sulphydryl group had been labelled with ¹⁴C-iodoacetamide, the disulphide bridges being left intact. Use was made of the partial amino-acid sequence given by Frank and Braunitzer⁵ and the reasonable assumption was made that the sulphydryl occurred in only one position. Subsequently, Shaw⁶ has shown that the sequence of Frank and Braunitzer⁵ showing Cys residues adjacent at positions 69 and 70 is incorrect and that they are separated by a glutamine, the sequence for positions 67 to 71 for the Bvariant being Ala.Cys.Gln.Cys.Leu. Autoradiography of the dansyl amino-acid derivatives formed during the sequence determination of this pentapeptide indicated that both residues 68 and 70 seemed to have been labelled and so we have given further consideration to the sulphydryl location. It has been found that although it does occur at 68, with 57 and 70 disulphide bridged, there is also an equal amount of protein present with the sulphydryl at 70, with 57 and 68 disulphide bridged. We discuss this additional finding here and the significance for the determination of the location of sulphydryl groups in other proteins.