Molecular dynamics analysis of apolipoprotein-D-lipid hydroperoxide interactions: mechanism for selective oxidation of Met-93

Background

Recent studies suggest reduction of radical-propagating fatty acid hydroperoxides to inert hydroxides by interaction with apolipoprotein-D (apoD) Met93 may represent an antioxidant function for apoD. The nature and structural consequences of this selective interaction are unknown.

Methodology/Principal Findings

Herein we used molecular dynamics (MD) analysis to address these issues. Long-timescale simulations of apoD suggest lipid molecules are bound flexibly, with the molecules free to explore multiple conformations in a binding site at the entrance to the classical lipocalin ligand-binding pocket. Models of 5s- 12s- and 15s-hydroperoxyeicosatetraenoic acids were created and the lipids found to wrap around Met93 thus providing a plausible mechanism by which eicosatetraenoic acids bearing hydroperoxides on different carbon atoms can interact with Met93 to yield Met93 sulfoxide (Met93SO). Simulations of glycosylated apoD indicated that a second solvent exposed Met at position 49 was shielded by a triantennerary N-glycan attached to Asn45 thereby precluding lipid interactions. MD simulations of apoD showed B-factors of the loop containing Met93SO were higher in the oxidized protein, indicating increased flexibility that is predicted to destabilize the protein and promote self-association.

Conclusions/Significance

These studies provide novel insights into the mechanisms that may contribute to the antioxidant function of apoD and the structural consequences that result if Met93SO is not redox-cycled back to its native state.     

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.     

Identification of TLR inducing Th1-responsive Leishmania donovani amastigote-specific antigens

Leishmania is known to elicit Th2 response that causes leishmaniasis progression; on the other hand, Th1 cytokines restricts amastigote growth and disease progression. In this study, we report the potential of two leishmanial antigens (65 and 98?kDa, in combination) which enhance strong macrophage effector functions, viz., production of respiratory burst enzymes, nitric oxide, and Th1 cytokines. The identification of antigens were done by resolving the crude soluble antigens on SDS-PAGE and eluted by reverse staining method. Further, RAW264.7 macrophages were challenged with eluted antigens, and the innate immune response was observed by detecting respiratory burst enzymes, nitric oxide (NOx), TNF-α, IFN-γ, IL-12, toll-like receptors (TLRs) gene expression, and TLR-signaling proteins. These antigens increased the production of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, NOx, TNF-α, IFN-γ, IL-12, TLR2, and p38 mitogen-activated protein kinase. These antigens also induced human peripheral blood mononuclear cells proliferation and Th1 cytokine production. This study concludes that these antigens induce innate immune response as well as have prophylactic efficacy.     

Arsenic (As) Inhibits Radicle Emergence and Elongation in <Emphasis Type="Italic">Phaseolus aureus</Emphasis> by Altering Starch-Metabolizing Enzymes Vis-à-Vis Disruption of Oxidative Metabolism

The present study investigated the effect of Arsenic (As; 5, 10, 50 μM) on protein and sugar metabolism vis-à-vis oxidative damage during early germination process and radicle emergence (at 12, 24 and 48 h stage) in Phaseolus aureus. As-exposure (50 μM) significantly enhanced protein content (by 40–60%), whereas carbohydrate content declined (by 31–44%) over that in the control. It was associated with a decline in the activities of proteases (47–53%), and increase in the activities of α- and β-amylases, starch phosphorylases, and acid invertases by 3.0, 2.6, 4.8, and 1.7 times after 48 h exposure to 50 μM As. The alteration in protein and carbohydrate metabolic machinery was also accompanied by As-induced reactive oxygen species (ROS)-mediated oxidative damage. As treatment enhanced malondialdehyde and hydrogen peroxide content by 46–252% and 23–216%, and hydroxyl and superoxide ion generation by 15–104% and 17–278%, respectively. As-induced lipid peroxidation and membrane disruption was confirmed by enhanced electrolyte leakage (by 49%) and reduced cell viability (by 43%). Furthermore, in response to 50 μM As, the activities of superoxide dismutases, catalases, ascorbate peroxidases, guaiacol peroxidases, and glutathione reductases increased by 77%, 70%, 116%, 43% and 120%, respectively, in radicles at 48 h stage over that in the control. The study concludes that As inhibits radicle emergence and elongation in germinating P. aureus seeds by altering biochemical processes related to sugar metabolism and inducing an ROS-mediated oxidative damage.     

Multiple exposures to unloading decrease bone's responsivity but compound skeletal losses in C57BL/6 mice

A single exposure to mechanical unloading can result in significant bone loss, but the consequences of multiple exposures are largely unknown. Within a 18-wk period, adult C57BL/6 male mice were exposed to 2 wk of hindlimb unloading (HLU) followed by 4 wk of reambulation (RA) once (1x-HLU), twice (2x-HLU), or three times (3x-HLU), or served as ambulatory age-matched controls. In vivo μCT longitudinally tracked changes in trabecular and cortical compartments of the femur. Normally ambulating control mice experienced significant age-related loss in trabecular bone volume fraction throughout the course of the experiment. This loss was compounded by HLU with 2x- and 3x-HLU mice experiencing a 27% and 24% greater reduction in trabecular bone and a 60% and 63% inhibition of age-related trabecular thickening. The recovery of cortical bone was also incomplete during each 4-wk RA period and, at completion of the experiment, cortical area in 3x-HLU mice was 5% smaller than in control and 1x-HLU. When eliminating age as a confounding variable, comparison between individual HLU/RA cycles showed that the magnitude of the response diminished during subsequent exposures. The extent of trabecular thinning in mice unloaded for the first time was 1.6-fold greater than the second time and nearly twofold greater than the third time. Similarly, the increase in trabecular thickness during the first RA cycle was twofold greater than during the second and third RA cycle. Together, our data demonstrate that even though multiple exposures to mechanical unloading are more detrimental than a single unloading period, bone's mechanosensitivity is reduced with consecutive unloading/reambulation cycles.     

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

Background

Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.

Methods

MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.

Results

After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.

Conclusions

These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm

Izumo, a sperm membrane protein, is essential for gamete fusion in the mouse. It has an Immunoglobulin (Ig) domain and an N-terminal domain for which neither the functions nor homologous sequences are known. In the present work we identified three novel proteins showing an N-terminal domain with significant homology to the N-terminal domain of Izumo. We named this region “Izumo domain,” and the novel proteins “Izumo 2,” “Izumo 3,” and “Izumo 4,” retaining “Izumo 1” for the first described member of the family. Izumo 1–3 are transmembrane proteins expressed specifically in the testis, and Izumo 4 is a soluble protein expressed in the testis and in other tissues. Electrophoresis under mildly denaturing conditions, followed by Western blot analysis, showed that Izumo 1, 3, and 4 formed protein complexes on sperm, Izumo 1 forming several larger complexes and Izumo 3 and 4 forming a single larger complex. Studies using different recombinant Izumo constructs suggested the Izumo domain possesses the ability to form dimers, whereas the transmembrane domain or the cytoplasmic domain or both of Izumo 1 are required for the formation of multimers of higher order. Co-immunoprecipitation studies showed the presence of other sperm proteins associated with Izumo 1, suggesting Izumo 1 forms a multiprotein membrane complex. Our results raise the possibility that Izumo 1 might be involved in organizing or stabilizing a multiprotein complex essential for the function of the membrane fusion machinery. Mol. Reprod. Dev. 76: 1188–1199, 2009. © 2009 Wiley-Liss, Inc.