Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed by structural and biochemical studies on WCI mutants

The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated in the maintenance of the binding loop conformation of Kunitz (STI) inhibitors, its possible involvement in the prevention of proteolysis is still unexplored. In this paper, we have investigated the specific role of the spacer Asn in the prevention of proteolysis through structural and biochemical studies on the mutants where Asn14 of winged bean chymotrypsin inhibitor (WCI) has been replaced by Gly, Ala, Thr, Leu, and Gln. A residue having no side chain or beta-branching at the 14th position creates deformation and insufficient protrusion of the binding loop, and as a result N14G and N14T lose the ability to recognize proteases. Although the reactive site loop conformation of N14A and N14Q are almost identical to WCI, biochemical results present N14A as a substrate indicating that the methyl group of Ala14 is not suitable to capture the cleaved parts together for religation. The poor inhibitory power of N14L points toward the chemical incompatibility of Leu at the 14th position, although its size is the same as Asn; on the other hand, slight loss of inhibitory potency of N14Q is attributed to the inappropriate placement of the Gln14 polar head, caused by the strained accommodation of its bigger side chain. These observations collectively allow us to conclude that the side chain of spacer Asn fits snugly into the concave space of the reactive site loop cavity and its ND2 atom forms hydrogen bonds with the P2 and P1' carbonyl O at either side of the scissile bond holding the cleaved products together for religation. Through database analysis, we have identified such spacer asparagines in five other families of serine protease inhibitors with a similar disposition of their ND2 atoms, which supports our proposition.     

Spectroscopic evidence for Ca2+ involvement in the assembly of the Mn4Ca cluster in the photosynthetic water-oxidizing complex

Biogenesis and repair of the inorganic core (Mn4CaO(x)Cl(y)), in the water-oxidizing complex of photosystem II (WOC-PSII), occurs through the light-induced (re)assembly of its free elementary ions and the apo-WOC-PSII protein, a reaction known as photoactivation. Herein, we use electron paramagnetic resonance (EPR) spectroscopy to characterize changes in the ligand coordination environment of the first photoactivation intermediate, the photo-oxidized Mn3+ bound to apo-WOC-PSII. On the basis of the observed changes in electron Zeeman (g(eff)), 55Mn hyperfine (A(Z)) interaction, and the EPR transition probabilities, the photogenerated Mn3+ is shown to exist in two pH-dependent forms, differing in terms of strength and symmetry of their ligand fields. The transition from an EPR-invisible low-pH form to an EPR-active high-pH form occurs by deprotonation of an ionizable ligand bound to Mn3+, implicated to be a water molecule: [Mn3+ (OH2)] <--> [Mn3+ (OH-)]. In the absence of Ca2+, the EPR-active Mn3+ exhibits a strong pH dependence (pH approximately 6.5-9) of its ligand-field symmetry (rhombicity Delta delta = 10%, derived from g(eff)) and A(Z) (DeltaA(Z) = 22%), attributable to a protein conformational change. Binding of Ca2+ to its effector site eliminates this pH dependence and locks both g(eff) and A(Z) at values observed in the absence of Ca2+ at alkaline pH. Thus, Ca2+ directly controls the coordination environment and binds close to the high-affinity Mn3+, probably sharing a bridging ligand. This Ca2+ effect and the pH-induced changes are consistent with the ionization of the bridging water molecule, predicting that [Mn3+-(mu-O(-2))-Ca2+] or [Mn3+-(mu-OH(-))2-Ca2+] is the first light intermediate in the presence of Ca2+. The formation of this intermediate templates the apo-WOC-PSII for the subsequent rapid cooperative binding and photo-oxidation of three additional Mn2+ ions, forming the active water oxidase.     

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

Formation of dityrosine (DT) cross‐linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein‐polyphenol cross‐linkages. To prevent the formation of such cross‐linkages we have used β‐cyclodextrin (β‐CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β‐cyclodextrin (β‐CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β‐CD and are no longer available for protein cross‐linking.     

Glucose transporter isoform-3 mutations cause early pregnancy loss and fetal growth restriction

Glucose transporter isoform-3 (GLUT3) is the trophoblastic facilitative glucose transporter. To investigate the role of this isoform in embryonic development, we created a novel GLUT3-null mouse and observed arrested early embryonic development and loss at neurulation stage when both alleles were mutated. This loss occurred despite the presence of other related isoforms, particularly GLUT1. In contrast, when a single allele was mutated, despite increased embryonic cell apoptosis, adaptive changes in the subcellular localization of GLUT3 and GLUT1 in the preimplantation embryo led to postimplantation survival. This survival was compromised by decreased GLUT3-mediated transplacental glucose transport, causing late-gestation fetal growth restriction. This yielded young male and female adults demonstrating catch-up growth, with normal basal glucose, insulin, insulin-like growth factor-I and IGF-binding protein-3 concentrations, fat and lean mass, and glucose and insulin tolerance. We conclude that GLUT3 mutations cause a gene dose-dependent early pregnancy loss or late-gestation fetal growth restriction despite the presence of embryonic and placental GLUT1 and a compensatory increase in system A amino acid placental transport. This critical life-sustaining functional role for GLUT3 in embryonic development provides the basis for investigating the existence of human GLUT3 mutations with similar consequences during early pregnancy.     

Community Based Case-Control Study of Rotavirus Gastroenteritis among Young Children during 2008-2010 Reveals Vast Genetic Diversity and Increased Prevalence of G9 Strains in Kolkata

Background

Group A Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children (<5 years) worldwide. Although rotavirus vaccines have been successfully administered in many countries, in India the introduction of rotavirus vaccine in national immunization program was approved in 2014. Since high disease burden and large number of genetic variants have been reported from low income countries including India, monitoring of rotavirus was initiated prior to implementation of the vaccine in the region.

Methods

A total number of 3,582 stool samples were collected from an urban slum community in Kolkata, among which 1,568 samples were obtained from children of ≤5 years of age, with moderate to severe diarrhoea and 2,014 samples were collected from age-sex matched healthy neighbourhood controls. Rotavirus positive samples were typed by multiplex semi-nested PCR and nucleotide sequencing. Circulating strains were phylogenetically analyzed.

Results

Among 1,568 children with diarrhoea, 395 (25.2%), and among 2,014 asymptomatic children, 42 (2%) were rotavirus positive. G1P[8] was identified as the most common strain (32%) followed by G9P[8] (16.9%), G2P[4] (13.5%) and G9P[4] (10.75%). G12 strains with combinations of P[4], P[6] and P[8] comprised 11.9% of total positive strains. The rest (<10%) were rare and uncommon strains like G1P[4], G1P[6], G2P[8] and animal-like strains G4P[6], G6P[14] and G11P[25]. The 42 rotavirus positive samples from asymptomatic children revealed common genotypes like G1, G2 and G9.

Conclusion

This community based case-control study showed increased predominance of genotype G9 in Kolkata. It also confirmed co-circulation of a large number of genetic variants in the community. Asymptomatic rotavirus positive children though low in number can also be a source of dispersal of infection in the community. This study provides background information to the policy makers for implementation of rotavirus vaccines in this region.     

p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.     

Protein synthesis in rabbit reticulocytes XXI. Purification and properties of a protein factor (Co-EIF-1) which stimulates Met-tRNAf binding to EIF-1.

The peptide chain initiation factor, Co-EIF-1 has been purified to homogeneity. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous preparation gives a single protein band corresponding to a molecular weight of approximately 20,000. In the crude preparation, Co-EIF-1 exists in two molecular forms: Co-EIF-1H (Mr = 200,000) and Co-EIF-1L (Mr = 20,000). Both forms are equally active in all the reactions studied. Upon heating, the heavy form (Co-EIF-1H) is completely converted into the light form (Co-EIF-1L). Radioactively labeled [14C]Co-EIF-1 was prepared by reductive alkylation using [14C]formaldehyde and borohydride. [14C]Methyl-Co-EIF-1 binds specifically to EIF-1; EIF-1.[14C]Co-EIF-1 complex was analyzed by gel (Sephadex G-100) filtration. EIF-1.Co-EIF-1 complex is distinctly more stable towards heat than EIF-1 alone and the quarternary complex, Met-tRNAf.EIF-1.Co-EIF-1.GTP is more resistant to aurintricarboxylic acid than the ternary complex, Met-tRNAf.EIF-1.GTP. Both the quarternary complex, Met-tRNAf.EIF-1.Co-EIF-1.GTP, and the ternary complex, Met-tRNAf.EIF-1.GTP, are equally sensitive to Mg2+ in the presence of EIF-2 (TDF). In the presence of Co-EIF-1, the initial rate of Met-tRNAf binding to 40 S ribosomes was significantly increased.