Association of stomatin with lipid bodies

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)(HA) occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/ lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2 center dot ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B - C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.

Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3' nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds.

Self-condensation of a thiazole-peptide bearing a 21-membered loop into a library of giant macrocycles with multiple orthogonal loops

Tetrapeptide analogue H-[Glu-Ser-Lys(Thz)]-OH, containing a turn-inducing thiazole constraint, was used as a template to produce a 21-membered structurally characterized loop by linking Glu and Lys side chains with a Val-Ile dipeptide. This template was oligomerized in one pot to a library (cyclo-[1](n), n = 2-10) of giant symmetrical macrocycles (up to 120-membered rings), fused to 2-10 appended loops that were carried intact through multiple oligomerization (chain extension) and cyclization (chain terminating) reactions of the template. A three-dimensional solution structure for cyclo-[1](3) shows all three appended loops projecting from the same face of the macrocycle. This is a promising approach to separating pepticle motifs over large distances.

NOD2 mutations and colorectal cancer - Where do we stand?

Due to the overwhelming burden of colorectal cancer (CRC), great effort has been placed on identifying genetic mutations that contribute to disease development and progression. One of the most studied polymorphisms that could potentially increase susceptibility to CRC involves the nucleotide-binding and oligomerization-domain containing 2 (NOD2) gene. There is growing evidence that the biological activity of NOD2 is far greater than previously thought and a link with intestinal microbiota and mucosal immunity is increasingly sought after. In fact, microbial composition may be an important contributor not only to inflammatory bowel diseases (IBD) but also to CRC. Recent studies have showed that deficient NOD2 function confers a communicable risk of colitis and CRC. Despite the evidence from experimental models, population-based studies that tried to link certain NOD2 polymorphisms and an increase in CRC risk have been described as conflicting. Significant geographic discrepancies in the frequency of such polymorphisms and different interpretations of the results may have limited the conclusions of those studies. Since being first associated to IBD and CRC, our understanding of the role of this gene has come a long way, and it is tempting to postulate that it may contribute to identify individuals with susceptible genetic background that may benefit from early CRC screening programs or in predicting response to current therapeutic tools. The aim of this review is to clarify the status quo of NOD2 mutations as genetic risk factors to chronic inflammation and ultimately to CRC. The use of NOD2 as a predictor of certain phenotypic characteristics of the disease will be analyzed as well.

NOD2 mutations and colorectal cancer - Where do we stand?

Due to the overwhelming burden of colorectal cancer (CRC), great effort has been placed on identifying genetic mutations that contribute to disease development and progression. One of the most studied polymorphisms that could potentially increase susceptibility to CRC involves the nucleotide-binding and oligomerization-domain containing 2 (NOD2) gene. There is growing evidence that the biological activity of NOD2 is far greater than previously thought and a link with intestinal microbiota and mucosal immunity is increasingly sought after. In fact, microbial composition may be an important contributor not only to inflammatory bowel diseases (IBD) but also to CRC. Recent studies have showed that deficient NOD2 function confers a communicable risk of colitis and CRC. Despite the evidence from experimental models, population-based studies that tried to link certain NOD2 polymorphisms and an increase in CRC risk have been described as conflicting. Significant geographic discrepancies in the frequency of such polymorphisms and different interpretations of the results may have limited the conclusions of those studies. Since being first associated to IBD and CRC, our understanding of the role of this gene has come a long way, and it is tempting to postulate that it may contribute to identify individuals with susceptible genetic background that may benefit from early CRC screening programs or in predicting response to current therapeutic tools. The aim of this review is to clarify the status quo of NOD2 mutations as genetic risk factors to chronic inflammation and ultimately to CRC. The use of NOD2 as a predictor of certain phenotypic characteristics of the disease will be analyzed as well.

Role of the Tryptophan Residues in the Specific interaction of the Sea Anemone Stichodacty la helianthus’s Actinoporin Sticholysin II with Biological Membranes

Actinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a eterminant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer−protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins’ Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.