Crystal structure of a BCL-W domain-swapped dimer: implications for the function of BCL-2 family proteins

The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.     

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7 days to 8 weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1–2 days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.     

Inhibition of colony formation by mouse mammary cancer cells co-cultured with early embryonic cells.

Mammary cancer cells from C3H/HeOUJ mice fail to form colonies when grown on monolayers of cells from 9- and 10-d mouse embryos but not on those from 11- and 12-d-old embryos.     

Progressive Injury in Chronic Multiple Sclerosis Lesions Is Gender-Specific: A DTI Study

Objective

To evaluate the longitudinal integrity of white matter tracts in patients with relapsing remitting multiple sclerosis (RRMS) as determined by changes in diffusivity indices of lesional and non-lesional white matter in the optic radiation over 12 months.

Methods

The optic radiation (OR) was identified in sixty RRMS patients using probabilistic tractography. MS lesions were segmented on FLAIR T2 images and a lesion mask was intersected with the co-registered OR. Lesions within the OR were identified in 39 patients. Voxel-based analysis of axial diffusivity (AD) and radial diffusivity (RD) within OR lesions and non-lesional normal appearing white matter (NAWM) was performed at baseline and 12 months in 34 patients (five patients excluded due to new OR lesions).

Results

Both RD and AD demonstrated much higher values within the lesions compared with non-lesional NAWM. There was a significant (p<0.001) increase of lesional AD and RD during the follow-up period. This increase, however, was driven almost entirely by the male cohort, in which a significantly greater change in both AD (M-2.7%, F-0.9%) and RD (M-4.6%, F-0.7%) was observed during the follow-up period. Non-lesional NAWM also demonstrated an increase in both AD and RD, albeit on a much lesser scale (1.0% and 0.6% respectively). In contradistinction to lesions, the diffusivity change in non-lesional NAWM was similar between sexes.

Conclusions

The evolution of AD and RD in chronic MS lesions over 12 months suggests ongoing inflammatory demyelinating activity accompanied by axonal loss. In addition, our findings are consistent with the recently observed trend of more rapid clinical progression in males and establish a potential in vivo biomarker of gender dichotomy by demonstrating a significantly faster rate of microstructural change in the chronic lesions of male patients with MS.     

Signaling by Toll-like receptors 8 and 9 requires Bruton's tyrosine kinase

Toll-like receptors (TLRs) are a primary surveillance system for the detection of pathogens and are crucial to the activation of host defense. TLR7 and TLR8 sense single-stranded RNA from viruses or host ribonucleoproteins and synthetic imidazoquinolines such as R848, whereas TLR9 senses unmethylated CpG motifs in viral and bacterial DNA and in host DNA. Here we report the endogenous interaction between Brutons's tyrosine kinase (Btk) and human TLR8 and TLR9 in the monocytic cell line THP1. We also show that R848, single-stranded RNA, and CpGB-DNA activate Btk in THP1 cells as shown by phosphorylation of the tyrosine 223 residue of Btk and also by increased autokinase activity. We demonstrate that Btk is required for NFkappaB activation, participating in the pathway to increased phosphorylation of p65 on serine 536 activated by TLR8 and TLR9. Finally we demonstrate that peripheral blood mononuclear cells from patients with X-linked agammaglobulinaemia (XLA) that have dysfunctional Btk are impaired in the induction of interleukin-6 by CpGB-DNA. This study therefore establishes Btk as a key signaling molecule that interacts with and acts downstream of TLR8 and TLR9. Lack of functioning Btk in XLA patients downstream of TLR8 and TLR9 might explain the susceptibility of XLA patients to viral infections.     

Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 Å) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 Ų) increase in buried surface area at the tetramerization interface of the pyruvate-bound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an “Achilles heel” that can be exploited in structure-based drug design.     

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex

Background information. Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat‐shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. Results. The localization of Hsp20 was further analysed using high‐resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding‐associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate phospholipids could be observed. Conclusions. Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped‐arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.     

The role of the Eph-ephrin signalling system in the regulation of developmental patterning

The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

Daughter cell assembly in the protozoan parasite Toxoplasma gondii

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii. Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.