Identification of calpastatin and mu-calpain and studies of their association in pulmonary smooth muscle mitochondria

Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis.     

Oxygenase domain of Drosophila melanogaster nitric oxide synthase: unique kinetic parameters enable a more efficient NO release

Although nitric oxide (NO) is important for cell signaling and nonspecific immunity in the fruit fly Drosophila melanogaster, little is known about its single NO synthase (dNOS). We expressed the oxygenase domain of dNOS (dNOSoxy), characterized its spectroscopic, kinetic, and catalytic properties, and interpreted them in light of a global kinetic model for NO synthesis. Single turnover reactions with ferrous dNOSoxy showed it could convert Arg to N'omega-hydroxy-l-arginine (NOHA), or NOHA to citrulline and NO, when it was given 6R-tetrahydrobiopterin and O2. The dNOSoxy catalyzed Arg hydroxylation and NOHA oxidation at rates that matched or exceeded the rates catalyzed by the three mammalian NOSoxy enzymes. Consecutive heme-dioxy, ferric heme-NO, and ferric heme species were observed in the NOHA reaction of dNOSoxy, indicating that its catalytic mechanism is the same as in the mammalian NOS. However, NO dissociation from dNOSoxy was 4 to 9 times faster than that from the mammalian NOS enzymes. In contrast, the dNOSoxy ferrous heme-NO complex was relatively unreactive toward O2 and in this way was equivalent to the mammalian neuronal NOS. Our data show that dNOSoxy has unique settings for the kinetic parameters that determine its NO synthesis. Computer simulations reveal that these unique settings should enable dNOS to be a more efficient and active NO synthase than the mammalian NOS enzymes, which may allow it to function more broadly in cell signaling and immune functions in the fruit fly.     

APSY-NMR for protein backbone assignment in high-throughput structural biology

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [¹H,¹H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.     

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

Engineering cell surfaces via liposome fusion

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.     

Physicochemical Characterization of NPC 1161C,A Novel Antimalarial 8-Aminoquinoline,in Solution and Solid State

NPC 1161C is a novel antimalarial drug of interest because of its superior curative and prophylactic activity, and favorable toxicity profile against in vivo and in vitro models of malaria, pneumocystis carinii pneumonia, and leishmaniasis. The preformulation studies performed included determination of pK_as, aqueous and pH solubility, cosolvent solubility, log P, pH stability, thermal analysis, and preliminary hygroscopicity studies. The mean pK_a1, pK_a2, and pK_a3 were determined to be 10.12, 4.07, and 1.88, respectively. The aqueous solubility was found to be 2.4 × 10⁻⁴ M having a saturated solution pH of 4.3–5.0 and a low intrinsic solubility of 1.6 × 10⁻⁶ M. A mathematical model of the pH-solubility profile was derived from pH 2.2 to 8.0. An exponential decrease in solubility was observed with increasing pH. The excess solid phase in equilibrium with the solution in aqueous buffers was determined to be the free-base form of the drug. A significant increase in solubility was observed with all the cosolvents studied, in both unbuffered and buffered systems. Mean log P of the salt and the free base were estimated to be 2.18 and 3.70, respectively. The compound had poor stability at pH 7.0 at 37°C, with a t ₉₀ of 3.58 days. Thermal analysis of the drug using DSC and TGA revealed that the drug is present as a semi-crystalline powder, which transformed into the amorphous state after melting. The drug was also found to sublime at higher temperatures. Determination of physicochemical properties of NPC 1161C provided useful information for the development of a dosage form and preclinical evaluation.