The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia

Carbon, nitrogen and O(2) fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N2 fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N2-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N2 fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O2 h−1 in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O2 h−1, and it reached the limit set by O2 diffusion from the surrounding water to colonies (>1 mm). N2 fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N2 fixation in the bulk water. Net NH4+ release in colonies equaled 8–33% of the estimated gross N2 fixation during photosynthesis. NH4+ concentrations within light-exposed colonies, modeled from measured net NH4+ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH4+ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth

Carbon and nitrogen fluxes associated with the cyanobacterium Aphanizomenon sp. in the Baltic Sea

Carbon and nitrogen fluxes in Aphanizomenon sp. colonies in the Baltic Sea were measured using a combination of microsensors, stable isotopes, mass spectrometry, and nanoscale secondary ion mass spectrometry (nanoSIMS). Cell numbers varied between 956 and 33 000 in colonies ranging in volume between 1.4 × 10−4 and 230 × 10−4 mm−3. The high cell content and their productivity resulted in steep O2 gradients at the colony–water interface as measured with an O2 microsensor. Colonies were highly autotrophic communities with few heterotrophic bacteria attached to the filaments. Volumetric gross photosynthesis in colonies was 78 nmol O2 mm−3 h−1. Net photosynthesis was 64 nmol O2 mm−3 h−1, and dark respiration was on average 15 nmol O2 mm−3 h−1 or 16% of gross photosynthesis. These volumetric photosynthesis rates belong to the highest measured in aquatic systems. The average cell-specific net carbon-fixation rate was 38 and 40 fmol C cell−1 h−1 measured by microsensors and by using stable isotopes in combination with mass spectrometry and nanoSIMS, respectively. In light, the net C:N fixation ratio of individual cells was 7.3±3.4. Transfer of fixed N2 from heterocysts to vegetative cells was fast, but up to 35% of the gross N2 fixation in light was released as ammonium into the surrounding water. Calculations based on a daily cycle showed a net C:N fixation ratio of 5.3. Only 16% of the bulk N2 fixation in dark was detected in Aphanizomenon sp. Hence, other organisms appeared to dominate N2 fixation and NH4+ release during darkness

4-Nitro­anilinium triiodide monohydrate

In the title compound, C6H7N2O2 +·I3 −·H2O, the triiodide anions form two-dimensional sheets along the a and c axes. These sheets are separated by the 4-nitro­anilinium cations and water mol­ecules, which form part of an extended hydrogen-bonded chain with the triiodide along the c axis, represented by the graph set C 3 3(14). The second important hydrogen-bonding inter­action is between the nitro group, the water mol­ecule and the anilinium group, which forms an R 2 2(6) ring and may be the reason for the deviation of the torsion angle between the benzene ring and the nitro group from 180 to 163.2 (4)°. These two strong hydrogen-bonding inter­actions also cause the benzene rings to pack off-centre from one another, with an edge-on-edge π–π stacking distance of 3.634 (6) Å and a centroid–centroid separation of 4.843 (2) Å

Evolution and Medical Significance of LU Domain-Containing Proteins

Proteins containing Ly6/uPAR (LU) domains exhibit very diverse biological functions and have broad taxonomic distributions in eukaryotes. In general, they adopt a characteristic three-fingered folding topology with three long loops projecting from a disulfide-rich globular core. The majority of the members of this protein domain family contain only a single LU domain, which can be secreted, glycolipid anchored, or constitute the extracellular ligand binding domain of type-I membrane proteins. Nonetheless, a few proteins contain multiple LU domains, for example, the urokinase receptor uPAR, C4.4A, and Haldisin. In the current review, we will discuss evolutionary aspects of this protein domain family with special emphasis on variations in their consensus disulfide bond patterns. Furthermore, we will present selected cases where missense mutations in LU domain-containing proteins leads to dysfunctional proteins that are causally linked to genesis of human disease

Structure of the lipoprotein lipase-GPIHBP1 complex that mediates plasma triglyceride hydrolysis

The intravascular processing of triglyceride-rich lipoproteins by the lipoprotein lipase (LPL)–GPIHBP1 complex is crucial for clearing triglycerides from the bloodstream and for the delivery of lipid nutrients to vital tissues. A deficiency of either LPL or GPIHBP1 impairs triglyceride processing, resulting in severe hypertriglyceridemia (chylomicronemia). Despite intensive investigation by biochemists worldwide, the structures for LPL and GPIHBP1 have remained elusive. Inspired by the recent discovery that GPIHBP1 stabilizes LPL structure and activity, we crystallized the LPL–GPIHBP1 complex and solved its structure. The structure provides insights into the ability of GPIHBP1 to preserve LPL structure and activity and also reveals how inherited defects in these proteins impair triglyceride hydrolysis and cause chylomicronemia

Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies

The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume. © 1999 Cancer Research Campaig

Lipoprotein lipase is active as a monomer

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state