Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Matrix imbalance by inducing expression of metalloproteinase and oxidative stress in cochlea of hyperhomocysteinemic mice

Molecular and Cellular Biochemistry - Glycation is a process closely related to the aging and pathogenesis of diabetic complications. Reactive α-dicarbonyl compounds (e.g., methylglyoxal) are...     

Activation of GABA‐A receptor ameliorates homocysteine‐induced MMP‐9 activation by ERK pathway

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase‐9 (MMP‐9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ‐aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA‐A receptor in Hcy‐induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP‐9 activity via the extracellular signal‐regulated kinase (ERK) signaling pathway and by inhibition of GABA‐A receptors, thus behaving as an inhibitory neurotransmitter. Hcy‐induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′‐dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate‐oxidase‐4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP‐9, measured by gelatin zymography. The GABA‐A receptor agonist, muscimol ameliorated the Hcy‐mediated MMP‐9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase‐4 (TIMP‐4). Treatment of the endothelial cell with muscimol restored the levels of TIMP‐4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy‐induced MMP‐9 via ERK signaling pathway. These results suggest that Hcy competes with GABA‐A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. J. Cell. Physiol. 220: 257–266, 2009. © 2009 Wiley‐Liss, Inc.     

Interaction of Vitamin D Receptor with HLA DRB1*0301 in Type 1 Diabetes Patients from North India

Background

Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where interaction and integration of immune response genes along with environmental factors play a role in autoimmune destruction of the insulin producing Pancreatic Beta cells.

Methodology/Principal Findings

We have studied four single nucleotide polymorphisms (FokI site in Exon 2, BsmI and ApaI sites in Intron 8 and TaqI site in exon 9) in the vitamin D receptor (VDR) gene using PCR-RFLP and HLA-DRB1 alleles using PCR and hybridization with sequence specific oligonucleotide probes and studied their interaction using LD based statistics for non-linked loci followed by sequence analysis of the vitamin D response element (VDRE) present in the promoter region of HLA-DRB1*0301. Haplotypes, constructed using SHEsis program for four single nucleotide polymorphisms in the VDR gene, were studied for their interaction with HLA-DRB1 alleles in 233 T1D patients and 191 healthy controls from North India. A significant increase of haplotypes FBAt and fBAT (p<0.02, OR = 1.44 and p<0.002, OR = 3.23 respectively) was observed in the patients. Both the haplotypes FBAt and fBAT were significantly increased in male patients with age at onset less than 18 years; however, fBAT was significantly increased in female patients irrespective of their age at onset. LD based statistics showed significant interaction between the high producer F and T alleles with HLA-DRB1*0301. F and T alleles of VDR have been shown to contribute to VDR mRNA independently. The promoter sequence analysis of HLA-DRB1*0301 showed presence of VDRE involved in higher expression of HLA-DRB1*030, which was confirmed by flow cytometry and real time PCR analysis.

Conclusions/Significance

These data suggest that the interaction between VDR and HLA alleles is mediated by VDRE present in the promoter region of HLA-DRB1*0301 allele, which may be detrimental for the manifestation of T1D in the absence of 1,25-(OH)₂D₃ in early childhood due to poor expression of DRB1*0301 in the thymus resulting in autoimmunity.     

Deciphering evolution of immune recognition in antibodies

Background

Antibody, the primary effector molecule of the immune system, evolves after initial encounter with the antigen from a precursor form to a mature one to effectively deal with the antigen. Antibodies of a lineage diverge through antigen-directed isolated pathways of maturation to exhibit distinct recognition potential. In the context of evolution in immune recognition, diversity of antigen cannot be ignored. While there are reports on antibody lineage, structural perspective with respect to diverse recognition potential in a lineage has never been studied. Hence, it is crucial to evaluate how maturation leads to topological tailoring within a lineage enabling them to interact with significantly distinct antigens.

Results

A data-driven approach was undertaken for the study. Global experimental mouse and human antibody-antigen complex structures from PDB were compiled into a coherent database of germline-linked antibodies bound with distinct antigens. Structural analysis of all lineages showed variations in CDRs of both H and L chains. Observations of conformational adaptation made from analysis of static structures were further evaluated by characterizing dynamics of interaction in two lineages, mouse V_H1–84 and human V_H5–51. Sequence and structure analysis of the lineages explained that somatic mutations altered the geometries of individual antibodies with common structural constraints in some CDRs. Additionally, conformational landscape obtained from molecular dynamics simulations revealed that incoming pathogen led to further conformational divergence in the paratope (as observed across datasets) even while maintaining similar overall backbone topology. MM-GB/SA analysis showed binding energies to be in physiological range. Results of the study are coherent with experimental observations.

Conclusions

The findings of this study highlight basic structural principles shaping the molecular evolution of a lineage for significantly diverse antigens. Antibodies of a lineage follow different developmental pathways while preserving the imprint of the germline. From the study, it can be generalized that structural diversification of the paratope is an outcome of natural selection of a conformation from an available ensemble, which is further optimized for antigen interaction. The study establishes that starting from a common lineage, antibodies can mature to recognize a wide range of antigens. This hypothesis can be further tested and validated experimentally.

     

Influence of ancillary binding and nonspecific adsorption on bioresponsive hydrogel microlenses

We report investigations of specific and nonspecific adsorption effects on bioresponsive hydrogel microlenses to better understand their utility and potential advantages for biosensing. Bioresponsive microgels were prepared from stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-co-AAc) microgels after functionalization with both biotin and ABP (as a photoaffinity label) via carbodiimide chemistry. Bioresponsive hydrogel microlenses were then constructed from the microgels via Coulombic assembly of the anionic microgels on a positively charged, silane-modified, glass substrate. Specific and nonspecific protein binding on the hydrogel microlenses was studied by monitoring the optical properties using brightfield and fluorescence optical microscopies. The bioresponsivity, as determined by changes in the microlensing power, is strongly coupled to the formation of cross-links via ligand-protein and/or antigen-antibody binding. However, the microlensing phenomenon and the intrinsic bioresponsivity of the hydrogels are completely insensitive to simple adsorption via nonspecific protein binding from reconstituted human serum. These results suggest that the hydrogel microlens construct may be a good candidate for a wide range of applications in which the bioresponsive material would be required to operate in complex biological media.