Extent of structural asymmetry in homodimeric proteins: prevalence and relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2:1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers.     

Training the Brain to Survive Stroke

Background

Presently, little can be done to repair brain tissue after stroke damage. We hypothesized that the mammalian brain has an intrinsic capacity to adapt to low oxygen which would improve outcome from a reversible hypoxic/ischemic episode. Acclimation to chronic hypoxia causes increased capillarity and tissue oxygen levels which may improve the capacity to survive ischemia. Identification of these adaptations will lead to protocols which high risk groups could use to improve recovery and reduce costs.

Methods and Findings

Rats were exposed to hypoxia (3 weeks living at ½ an atmosphere). After acclimation, capillary density was measured morphometrically and was increased by 30% in the cortex. Novel implantable oxygen sensors showed that partial pressure of oxygen in the brain was increased by 40% in the normal cortex. Infarcts were induced in brain with 1 h reversible middle cerebral artery occlusions. After ischemia (48 h) behavioural scores were improved and T2 weighted MRI lesion volumes were reduced by 52% in acclimated groups. There was a reduction in inflammation indicated by reduced lymphocytes (by 27–33%), and ED1 positive cells (by 35–45%).

Conclusions

It is possible to stimulate a natural adaptive mechanism in the brain which will reduce damage and improve outcome for a given ischemic event. Since these adaptations occur after factors such as HIF-1α have returned to baseline, protection is likely related more to morphological changes such as angiogenesis. Such pre-conditioning, perhaps with exercise or pharmaceuticals, would not necessarily reduce the incidence of stroke, but the severity of damage could be reduced by 50%.     

Genetic Association of the KLK4 Locus with Risk of Prostate Cancer

The Kallikrein-related peptidase, KLK4, has been shown to be significantly overexpressed in prostate tumours in numerous studies and is suggested to be a potential biomarker for prostate cancer. KLK4 may also play a role in prostate cancer progression through its involvement in epithelial-mesenchymal transition, a more aggressive phenotype, and metastases to bone. It is well known that genetic variation has the potential to affect gene expression and/or various protein characteristics and hence we sought to investigate the possible role of single nucleotide polymorphisms (SNPs) in the KLK4 gene in prostate cancer. Assessment of 61 SNPs in the KLK4 locus (±10 kb) in approximately 1300 prostate cancer cases and 1300 male controls for associations with prostate cancer risk and/or prostate tumour aggressiveness (Gleason score <7 versus ≥7) revealed 7 SNPs to be associated with a decreased risk of prostate cancer at the P(trend)<0.05 significance level. Three of these SNPs, rs268923, rs56112930 and the HapMap tagSNP rs7248321, are located several kb upstream of KLK4; rs1654551 encodes a non-synonymous serine to alanine substitution at position 22 of the long isoform of the KLK4 protein, and the remaining 3 risk-associated SNPs, rs1701927, rs1090649 and rs806019, are located downstream of KLK4 and are in high linkage disequilibrium with each other (r(2)≥0.98). Our findings provide suggestive evidence of a role for genetic variation in the KLK4 locus in prostate cancer predisposition.     

Role of biomaterials, therapeutic molecules and cells for hepatic tissue engineering

Current liver transplantation strategies face severe shortcomings owing to scarcity of donors, immunogenicity, prohibitive costs and poor survival rates. Due to the lengthy list of patients requiring transplant, high mortality rates are observed during the endless waiting period. Tissue engineering could be an alternative strategy to regenerate the damaged liver and improve the survival and quality of life of the patient. The development of an ideal scaffold for liver tissue engineering depends on the nature of the scaffold, its architecture and the presence of growth factors and recognition motifs. Biomimetic scaffolds can simulate the native extracellular matrix for the culture of hepatocytes to enable them to exhibit their functionality both in vitro and in vivo. This review highlights the physiology and pathophysiology of liver, the current treatment strategies, use of various scaffolds, incorporation of adhesion motifs, growth factors and stem cells that can stabilize and maintain hepatocyte cultures for a long period.     

Lipocalin 2 deficiency dysregulates iron homeostasis and exacerbates endotoxin-induced sepsis

Various states of inflammation, including sepsis, are associated with hypoferremia, which limits iron availability to pathogens and reduces iron-mediated oxidative stress. Lipocalin 2 (Lcn2; siderocalin, 24p3) plays a central role in iron transport. Accordingly, Lcn2-deficient (Lcn2KO) mice exhibit elevated intracellular labile iron. In this study, we report that LPS induced systemic Lcn2 by 150-fold in wild-type mice at 24 h. Relative to wild-type littermates, Lcn2KO mice were markedly more sensitive to endotoxemia, exhibiting elevated indices of organ damage (transaminasemia, lactate dehydrogenase) and increased mortality. Such exacerbated endotoxemia was associated with substantially increased caspase-3 cleavage and concomitantly elevated immune cell apoptosis. Furthermore, cells from Lcn2KO mice were hyperresponsive to LPS ex vivo, exhibiting elevated cytokine secretion. Additionally, Lcn2KO mice exhibited delayed LPS-induced hypoferremia despite normal hepatic hepcidin expression and displayed decreased levels of the tissue redox state indicators cysteine and glutathione in liver and plasma. Desferroxamine, an iron chelator, significantly protects Lcn2KO mice from LPS-induced toxicity, including mortality, suggesting that Lcn2 may act as an antioxidant in vivo by regulating iron homeostasis. Thus, Lcn2-mediated regulation of labile iron protects the host against sepsis. Its small size and simple structure may make Lcn2 a deployable treatment for sepsis.     

Targeted Metabolomics Reveals a Male Pheromone and Sex-Specific Ascaroside Biosynthesis in Caenorhabditis elegans

In the model organism Caenorhabditis elegans, a class of small molecule signals called ascarosides regulate development, mating, and social behaviors. Ascaroside production has been studied in the predominant sex, the hermaphrodite, but not in males, which account for less than 1% of wild-type worms grown under typical laboratory conditions. Using HPLC-MS-based targeted metabolomics, we show that males also produce ascarosides and that their ascaroside profile differs markedly from that of hermaphrodites. Whereas hermaphrodite ascaroside profiles are dominated by ascr#3, containing an α,β-unsaturated fatty acid, males predominantly produce the corresponding dihydro-derivative ascr#10. This small structural modification profoundly affects signaling properties: hermaphrodites are retained by attomole-amounts of male-produced ascr#10, whereas hermaphrodite-produced ascr#3 repels hermaphrodites and attracts males. Male production of ascr#10 is population density-dependent, indicating sensory regulation of ascaroside biosynthesis. Analysis of gene expression data supports a model in which sex-specific regulation of peroxisomal β-oxidation produces functionally different ascaroside profiles.     

Actin reorganization as the molecular basis for the regulation of apoptosis in gastrointestinal epithelial cells

The gastrointestinal (GI) epithelium is a rapidly renewing tissue in which apoptosis represents part of the overall homeostatic process. Regulation of apoptosis in the GI epithelium is complex with a precise relationship between cell position and apoptosis. Apoptosis occurs spontaneously and in response to radiation and cytotoxic drugs at the base of the crypts. By contrast, the villus epithelial cells are extremely resistant to apoptosis. The molecular mechanism underlying this loss of function of villus epithelial cells to undergo apoptosis shortly after their exit from the crypt is unknown. In this study we demonstrate for the first time, that deletion of two homologous actin-binding proteins, villin and gelsolin renders villus epithelial cells extremely sensitive to apoptosis. Ultrastructural analysis of the villin-gelsolin(-/-) double-knockout mice shows an abnormal accumulation of damaged mitochondria demonstrating that villin and gelsolin function on an early step in the apoptotic signaling at the level of the mitochondria. A characterization of functional and ligand-binding mutants demonstrate that regulated changes in actin dynamics determined by the actin severing activities of villin and gelsolin are required to maintain cellular homeostasis. Our study provides a molecular basis for the regulation of apoptosis in the GI epithelium and identifies cell biological mechanisms that couple changes in actin dynamics to apoptotic cell death.     

Quasi-harmonic method for studying very low frequency modes in proteins

A quasi-harmonic approximation is described for studying very low frequency vibrations and flexible paths in proteins. The force constants of the empirical potential function are quadratic approximations to the potentials of mean force; they are evaluated from a molecular dynamics simulation of a protein based on a detailed anharmonic potential. The method is used to identify very low frequency (～1 cm^?1) normal modes for the protein pancreatic trypsin inhibitor. A simplified model for the protein is used, for which each residue is represented by a single interaction center. The quasi-harmonic force constants of the virtual internal coordinates are evaluated and the normal-mode frequencies and eigenvectors are obtained. Conformations corresponding to distortions along selected low-frequency modes are analyzed.