Faculty Bibliography

The mechanism of lipoprotein lipase (LPL) transport from muscle and fat cells to the luminal (apical) surface of capillaries, where it hydrolyses lipoprotein triglycerides, has remained undefined. In this issue, Davies et al. (2010) report that GPIHBP1 is required for LPL transcytosis from the basolateral to apical capillary endothelial surface

To tailor organic p/n heterojunctions with molecular-level precision, a rational design strategy using side-chain incompatibility of a covalently connected donor-acceptor (D-A) dyad has been successfully carried out. An oligothiophene-perylenediimide dyad, when modified with triethylene glycol side chains at one terminus and dodecyl side chains at the other (2(Amphi)), self-assembles into nanofibers with a long-range D/A heterojunction. In contrast, when the dyad is modified with dodecyl side chains at both termini (2(Lipo)), ill-defined microfibers result. In steady-state measurements using microgap electrodes, a cast film of the nanofiber of 2(Amphi) displays far better photoconducting properties than that of the microfiber of 2(Lipo). Flash-photolysis time-resolved microwave conductivity measurements, in conjunction with transient absorption spectroscopy, clearly indicate that the nanofiber of 2(Amphi) intrinsically allows for better carrier generation and transport properties than the microfibrous assembly of 2(Lipo).

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1alpha activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC(50), 1.9 mum) interacts selectively with the reduced, active form of ERO1alpha and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1alpha followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1alpha with small molecule inhibitors

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals.

White matter disorders can involve injury to myelin or axons but the respective contribution of each to clinical course is difficult to evaluate non-invasively. Here, to develop a paradigm for further investigations of axonal pathology by MRI, we compared two genetic mouse models exhibiting relatively selective axonal or myelin deficits using quantitative MRI relaxography of the transverse relaxation times (T2) in vivo and ultrastructural morphometry. In HM-DKO mice, which lack genes encoding the heavy (NF-H) and medium (NF-M) subunits of neurofilaments, neurofilament content of large myelinated axons of the central nervous system (CNS) is markedly reduced in the absence of changes in myelin thickness and volume. In shiverer mutant mice, which lack functional myelin basic protein, CNS myelin sheath formation is markedly reduced but neurofilament content is normal. We observed increases in T2 in nearly all white matter in shiverer mice compared to their wild type, while more subtle increases in T2 were observed in HM-DKO in the corpus callosum. White matter T2 was generally greater in shiverer mice than HM-DKO mice. Ultrastructural morphometry of the corpus callosum, which exhibited the greatest T2 differences, confirmed that total cross-sectional area occupied by axons was similar in the two mouse models and that the major ultrastructural differences, determined by morphometry, were an absence of myelin and larger unmyelinated axons in shiverer mice and absence of neurofilaments in HM-DKO mice. Our findings indicate that T2 is strongly influenced by myelination state and axonal volume, while neurofilament structure within the intra-axonal compartment has a lesser effect upon single compartment T2 estimates

The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na(+),K(+)-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na(+),K(+)-ATPase polarity depends on interactions between the beta subunits of Na(+),K(+)-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these beta subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.

OBJECTIVE: To determine 1) whether a protein interaction network exists between granulin-epithelin precursor (GEP), ADAMTS-7/ADAMTS-12, and cartilage oligomeric matrix protein (COMP); 2) whether GEP interferes with the interactions between ADAMTS-7/ADAMTS-12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects tumor necrosis factor alpha (TNFalpha)-mediated induction of ADAMTS-7/ADAMTS-12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis. METHODS: Yeast two-hybrid, in vitro glutathione S-transferase pull-down, and coimmunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS-7/ADAMTS-12, and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS-7/ADAMTS-12. Immunofluorescence cell staining was performed to visualize the subcellular localization of GEP and ADAMTS-7/ADAMTS-12. An in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS-7/ADAMTS-12-mediated digestion of COMP. The role of GEP in inhibiting TNFalpha-induced ADAMTS-7/ADAMTS-12 expression and COMP degradation in cartilage explants was also analyzed. RESULTS: GEP bound directly to ADAMTS-7 and ADAMTS-12 in vitro and in chondrocytes, and the 4 C-terminal thrombospondin motifs of ADAMTS-7/ADAMTS-12 and each granulin unit of GEP mediated their interactions. Additionally, GEP colocalized with ADAMTS-7 and ADAMTS-12 on the cell surface of chondrocytes. More importantly, GEP inhibited COMP degradation by ADAMTS-7/ADAMTS-12 in a dose-dependent manner through 1) competitive inhibition through direct protein-protein interactions with ADAMTS-7/ADAMTS-12 and COMP, and 2) inhibition of TNFalpha-induced ADAMTS-7/ADAMTS-12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis. CONCLUSION: Our observations demonstrate a novel protein-protein interaction network between GEP, ADAMTS-7/ADAMTS-12, and COMP. Furthermore, GEP is a novel specific inhibitor of ADAMTS-7/ADAMTS-12-mediated COMP degradation and may play a significant role in preventing the destruction of joint cartilage in arthritis

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest-derived frontal and paraxial mesodermal-derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest-derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal-derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest-derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3beta and increases the nuclear levels of beta-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest-derived frontal bone.

Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen.