Structure of a Glomulin-RBX1-CUL1 Complex: Inhibition of a RING E3 Ligase through Masking of Its E2-Binding Surface

The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein,?either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF(FBW7) complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.     

LDL and HDL transfer rates across peripheral microvascular endothelium agree with those predicted for passive ultrafiltration in humans

The mechanisms by which LDLs and HDLs cross the vascular endothelium from plasma into interstitial fluid are not understood, and have never been studied in humans in vivo. We determined whether the plasma-to-lymph clearance rates of LDL and HDL conform with those predicted by passive ultrafiltration through intercellular pores, or if it is necessary to invoke an active process such as receptor-mediated transcytosis. Plasma and afferent peripheral lymph were collected under steady-state conditions from 30 healthy men, and assayed for seven globular proteins of molecular radii 2.89–8.95 nm, complement C3, and apo AI, apo AII, and apo B. Plasma-to-lymph clearance rates of the seven proteins fitted the relation expected for molecules of their size when transported through two populations of pores of radius 4.95 and 20.1 nm. The same model parameters were then found to accurately predict the clearance rates of both HDL and LDL. The apparent clearance of complement C3, previously shown to be secreted by cultured endothelium, exceeded that predicted by the model. We conclude that the transport of HDL and LDL from plasma into interstitial fluid across the peripheral vascular endothelium in healthy humans can be explained by ultrafiltration without invoking an additional active process such as transcytosis.     

Socioeconomic Predictors of Cognition in Ugandan Children: Implications for Community Interventions

Background

Several interventions to improve cognition in at risk children have been suggested. Identification of key variables predicting cognition is necessary to guide these interventions. This study was conducted to identify these variables in Ugandan children and guide such interventions.

Methods

A cohort of 89 healthy children (45 females) aged 5 to 12 years old were followed over 24 months and had cognitive tests measuring visual spatial processing, memory, attention and spatial learning administered at baseline, 6 months and 24 months. Nutritional status, child''s educational level, maternal education, socioeconomic status and quality of the home environment were also measured at baseline. A multivariate, longitudinal model was then used to identify predictors of cognition over the 24 months.

Results

A higher child''s education level was associated with better memory (p = 0.03), attention (p = 0.005) and spatial learning scores over the 24 months (p = 0.05); higher nutrition scores predicted better visual spatial processing (p = 0.002) and spatial learning scores (p = 0.008); and a higher home environment score predicted a better memory score (p = 0.03).

Conclusion

Cognition in Ugandan children is predicted by child''s education, nutritional status and the home environment. Community interventions to improve cognition may be effective if they target multiple socioeconomic variables.     

The α1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells

Background

We have previously shown that c-FLIP_L is a more potent inhibitor than c-FLIP_S of Fas ligand-induced apoptosis and that c-FLIP_L physically binds to Daxx, an alternative Fas-signaling adaptor. Here we examined whether c-FLIP_S effectively inhibits TNFR1-mediated apoptosis and triggers JNK activation through its interaction with TRAF2.

Results

Some cancer cell lines, such as DU145, AGS, and PC3, have higher levels of c-FLIP_S than other cell lines, such as SNU-719 and T24. The expression of c-FLIP_S correlated with the susceptibility to TNFR1-mediated apoptosis. In contrast to DU145 and PC3, which are resistant to TNFR1-mediated apoptosis, T24 and SNU719 were sensitive to TNF-α treatment. To address the role of c-FLIP_S in TNFR1-mediated apoptosis, we examined the molecular interaction between c-FLIP_S and TRAF2. As expected, western blot analysis revealed that TRAF2 antibody immunoprecipitated a greater amount of c-FLIP_S than c-FLIP_L. Also, we measured the involvement of c-FLIP_S in TNF-α-induced JNK activation and apoptosis by comparing these in TNF-α-resistant and TNF-α-sensitive cell lines. Treatment with TNF-α increased the phosphorylated JNK level in SNU719 and T24 cells, whereas DU145 and AGS cells were resistant to TNF-α-mediated apoptosis.

Conclusion

We now report that the short form of c-FLIP_S is a more efficient inhibitor of TNF-receptor 1-mediated apoptosis signaling than the long form of the protein.     

Nestedness, niche metrics and temporal dynamics of a metacommunity in a dynamic natural model system

Prediction of extinction and colonization rates for whole species assemblages emerges as an urgent task for ecology. We hypothesized that nestedness of species assemblage reflects differential ability of species to occupy sites and of sites to support species. If correct, a nested ordering of species and sites should condense long‐term dynamics of metacommunities. To test this we characterized the differential ability of species to use habitat (niche position and niche breadth) using eight surveys of invertebrate communities inhabiting 49 tropical rock pools. We examined temporal consistency of the nested rank of species and pools, and related them to species and pool characteristics to infer temporal dynamics of species composition. Invertebrate assemblages in the rock‐pools were significantly nested and species ranks were generally preserved over time. By contrast, pool ranks were usually conserved between adjacent years only but their similarity declined with time separating surveys. The nested species‐by‐pool matrix of the first survey served as a benchmark to assess individual species and local community changed in subsequent years. As hypothesized, benchmark cells with high state occupancy probability had low extinction rates in subsequent years. Moreover, species high in the nested matrix (also with high regional occupancy probability) were better survivors and colonizers relative to species that ranked low. The year‐to‐year dynamics were similar. Species with non‐marginal niche position retained high ranks in the matrix. Yet, niche position predicted only colonization rate of species. Niche breadth and species’ nested ranking, extinction risk, or ability to colonize a pool showed no relationship. Counter to the expectation, pool ranks did not predict species extinction and colonization rates. Apparently, even in dynamic systems, regional nested pattern remains consistent and the underlying extinction and colonization dynamics appear to be largely determined by the hierarchical order among species and much less by that among sites.     

Two highly thermostable paralogous single-stranded DNA-binding proteins from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.     

Functional annotation of proteomic sequences based on consensus of sequence and structural analysis

To maximise the assignment of function of the proteins encoded by a genome and to aid the search for novel drug targets, there is an emerging need for sensitive methods of predicting protein function on a genome-wide basis. GeneAtlas is an automated, high-throughput pipeline for the prediction of protein structure and function using sequence similarity detection, homology modelling and fold recognition methods. GeneAtlas is described in detail here. To test GeneAtlas, a 'virtual' genome was used, a subset of PDB structures from the SCOP database, in which the functional relationships are known. GeneAtlas detects additional relationships by building 3D models in comparison with the sequence searching method PSI-BLAST. Functionally related proteins with sequence identity below the twilight zone can be recognised correctly.     

Infertility in CFW/R1 mice associated with cystic dilatation of the bulbourethral gland

A third of male inbred CFW/R1 mice in a breeding colony developed subcutaneous, bilateral, perineal masses determined to be cystic bulbourethral glands. The masses developed in mice between 4 and 15 months of age. After development of these perineal masses, diseased males were unable to produce offspring. Gross examination revealed the masses impinging on the scrotum and displacing the testes into the inguinal canal. The perineal masses were paired, membranous, translucent cysts, 6 to 10 mm3, attached to the bulbocavernosus muscle and connected to the pelvic urethra by way of a non-patent duct. The cysts contained a clear to tan, minimally cellular, viscous fluid with high mucus content, as documented by examination of Wright Giemsa-stained cytologic preparations. Histologic examination of hematoxylin and eosin-stained sections revealed cystic tubuloalveolar glands surrounded by striated muscle and lined by a single layer of pyramidal cuboidal to columnar epithelial cells with pale, basophilic, lacy cytoplasm and round, basal, condensed nuclei. These gross and histopathologic findings were consistent with cystic dilatation of the bulbourethral gland.     

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting.