Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.     

Protein folding in the cytoplasm and the heat shock response

Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.     

Reactivity and reaction order in acylhomoserine lactone formation by Pseudomonas aeruginosa RhlI

The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.0 +/- 0.2 s (-1) and decayed with a rate constant of 3.7 +/- 0.2 s (-1). No other intermediates were detected, demonstrating that the RhlI reaction proceeds via acylation of S-adenosylmethionine, followed by lactonization. S-Adenosylhomocysteine acted as a pseudosubstrate, in that it did not undergo either acylation or lactonization but did induce the deacylation of butyryl-acyl carrier protein. The K m for S-adenosylhomocysteine was approximately 15-fold higher than the K m for S-adenosylmethionine. The reactivities of acylated and unacylated sulfonium ions that were analogues of S-adenosylmethionine were investigated by computational methods. The calculations indicated that acylation of the substrate amino group activated the substrate for lactonization, by allowing the carboxyl group oxygen to approach more closely the methylene carbon to which it adds. This observation provides a satisfying chemical rationale for the order of the individual reactions in the catalytic cycle.     

Monte Carlo study of single molecule diffusion can elucidate the mechanism of B cell synapse formation

B cell receptors have been shown to cluster at the intercellular junction between a B cell and an antigen-presenting cell in the form of a segregated pattern of B cell receptor/antigen complexes known as an immunological synapse. We use random walk-based theoretical arguments and Monte Carlo simulations to study the effect of diffusion of surface-bound molecules on B cell synapse formation. Our results show that B cell synapse formation is optimal for a limited range of receptor-ligand complex diffusion coefficient values, typically one-to-two orders of magnitude lower than the diffusion coefficient of free receptors. Such lower mobility of receptor-ligand complexes can significantly affect the diffusion of a tagged receptor or ligand in an affinity dependent manner, as the binding/unbinding of such receptor or ligand molecules crucially depends on affinity. Our work shows how single molecule tracking experiments can be used to estimate the order of magnitude of the diffusion coefficient of receptor-ligand complexes, which is difficult to measure directly in experiments due to the finite lifetime of receptor-ligand bonds. We also show how such antigen movement data at the single molecule level can provide insight into the B cell synapse formation mechanism. Thus, our results can guide further single molecule tracking experiments to elucidate the synapse formation mechanism in B cells, and potentially in other immune cells.     

A double blind,randomized, placebo controlled study of the efficacy and safety of 5-Loxin<Superscript>®</Superscript>for treatment of osteoarthritis of the knee

Introduction

5-Loxin^® is a novel Boswellia serrata extract enriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid (AKBA), which exhibits potential anti-inflammatory properties by inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind, randomized, placebo-controlled study was conducted to evaluate the efficacy and safety of 5-Loxin^® in the treatment of osteoarthritis (OA) of the knee.

Methods

Seventy-five OA patients were included in the study. The patients received either 100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin^® daily or a placebo (n = 25) for 90 days. Each patient was evaluated for pain and physical functions by using the standard tools (visual analog scale, Lequesne''s Functional Index, and Western Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0), and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix metalloproteinase-3 was also evaluated in synovial fluid from OA patients. Measurement of a battery of biochemical parameters in serum and haematological parameters, and urine analysis were performed to evaluate the safety of 5-Loxin^® in OA patients.

Results

Seventy patients completed the study. At the end of the study, both doses of 5-Loxin^® conferred clinically and statistically significant improvements in pain scores and physical function scores in OA patients. Interestingly, significant improvements in pain score and functional ability were recorded in the treatment group supplemented with 250 mg 5-Loxin^® as early as 7 days after the start of treatment. Corroborating the improvements in pain scores in treatment groups, we also noted significant reduction in synovial fluid matrix metalloproteinase-3. In comparison with placebo, the safety parameters were almost unchanged in the treatment groups.

Conclusion

5-Loxin^® reduces pain and improves physical functioning significantly in OA patients; and it is safe for human consumption. 5-Loxin^® may exert its beneficial effects by controlling inflammatory responses through reducing proinflammatory modulators, and it may improve joint health by reducing the enzymatic degradation of cartilage in OA patients.

Trail Registration

(Clinical trial registration number: ISRCTN05212803.)     

DDB2 induces nuclear accumulation of the hepatitis B virus X protein independently of binding to DDB1

The hepatitis B virus (HBV) X protein (HBx) is critical for the life cycle of the virus. HBx associates with several host cell proteins including the DDB1 subunit of the damaged-DNA binding protein DDB. Recent studies on the X protein encoded by the woodchuck hepadnavirus have provided correlative evidence indicating that the interaction with DDB1 is important for establishment of infection by the virus. In addition, the interaction with DDB1 has been implicated in the nuclear localization of HBx. Because the DDB2 subunit of DDB is required for the nuclear accumulation of DDB1, we investigated the role of DDB2 in the nuclear accumulation of HBx. Here we show that expression of DDB2 increases the nuclear levels of HBx. Several C-terminal deletion mutants of DDB2 that fail to bind DDB1 are able to associate with HBx, suggesting that DDB2 may associate with HBx independently of binding to DDB1. We also show that DDB2 enhances the nuclear accumulation of HBx independently of binding to DDB1, since a mutant that does not bind DDB1 is able to enhance the nuclear accumulation of HBx. HBV infection is associated with liver pathogenesis. We show that the nuclear levels of DDB1 and DDB2 are tightly regulated in hepatocytes. Studies with regenerating mouse liver indicate that during late G1 phase the nuclear levels of both subunits of DDB are transiently increased, followed by a sharp decrease in S phase. Taken together, these results suggest that DDB1 and DDB2 would participate in the nuclear functions of HBx effectively only during the late-G1 phase of the cell cycle.     

The computational analysis of scientific literature to define and recognize gene expression clusters

A limitation of many gene expression analytic approaches is that they do not incorporate comprehensive background knowledge about the genes into the analysis. We present a computational method that leverages the peer-reviewed literature in the automatic analysis of gene expression data sets. Including the literature in the analysis of gene expression data offers an opportunity to incorporate functional information about the genes when defining expression clusters. We have created a method that associates gene expression profiles with known biological functions. Our method has two steps. First, we apply hierarchical clustering to the given gene expression data set. Secondly, we use text from abstracts about genes to (i) resolve hierarchical cluster boundaries to optimize the functional coherence of the clusters and (ii) recognize those clusters that are most functionally coherent. In the case where a gene has not been investigated and therefore lacks primary literature, articles about well-studied homologous genes are added as references. We apply our method to two large gene expression data sets with different properties. The first contains measurements for a subset of well-studied Saccharomyces cerevisiae genes with multiple literature references, and the second contains newly discovered genes in Drosophila melanogaster; many have no literature references at all. In both cases, we are able to rapidly define and identify the biologically relevant gene expression profiles without manual intervention. In both cases, we identified novel clusters that were not noted by the original investigators.     

A literature-based method for assessing the functional coherence of a gene group

MOTIVATION: Many experimental and algorithmic approaches in biology generate groups of genes that need to be examined for related functional properties. For example, gene expression profiles are frequently organized into clusters of genes that may share functional properties. We evaluate a method, neighbor divergence per gene (NDPG), that uses scientific literature to assess whether a group of genes are functionally related. The method requires only a corpus of documents and an index connecting the documents to genes. RESULTS: We evaluate NDPG on 2796 functional groups generated by the Gene Ontology consortium in four organisms: mouse, fly, worm and yeast. NDPG finds functional coherence in 96, 92, 82 and 45% of the groups (at 99.9% specificity) in yeast, mouse, fly and worm respectively.     

PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.