Characterization of surface binding sites in glycoside hydrolases: A computational study

Structural properties of carbohydrate surface binding sites (SBSs) were investigated with computational methods. Eighty‐five SBSs of 44 enzymes in 119 Protein Data Bank (PDB) files were collected as a dataset. On the basis of SBSs shape, they were divided into 3 categories: flat surfaces, clefts, and cavities (types A, B, and C, respectively). Ligand varieties showed the correlation between shape of SBSs and ligands size. To reduce cut‐off differences in each SBSs with different ligand size, molecular docking were performed. Molecular docking results were used to refine SBSs classification and binding sites cut‐off. Docking results predicted putative ligands positions and displayed dependence of the ligands binding mode to the structural type of SBSs. Physicochemical properties of SBSs were calculated for all docking results with YASARA Structure. The results showed that all SBSs are hydrophilic, while their charges could vary and depended to ligand size and defined cut‐off. Surface binding sites type B had highest average values of solvent accessible surface area. Analysis of interactions showed that hydrophobic interactions occur more than hydrogen bonds, which is related to the presence of aromatic residues and carbohydrates interactions.     

Energetics of swelling in isolated hepatocytes: A comprehensive study

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Immunosuppression induced by Salmonella involves inhibition of tyrosine phosphorylation in murine T lymphocytes

Abstract It is well known that facultative intracellular pathogens such as Salmonella suppress the host immune system. In the present study we attempted to clarify the mechanism responsible for the suppression of T-cell proliferation in mice infected with Salmonella typhimurium . The proliferation of murine spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were infected with S. typhimurium , but not with Eschirichia coli . The suppression of T-cell proliferation did not necessarily parallel the level of interleukin-2 (IL-2) secretion, and was not restored by treatment with a calcium ionophore, indomethacin or IL-2. Only phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C (PKC), caused a slight recovery of cell proliferation with an augmentation of IL-2 secretion. Furthermore, Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited by S. typhimurium infection. Also, the inhibition of tyrosine phosphorylation was not restored by treatment with PMA. These results suggest that the suppression of T-cell proliferation induced by Salmonella infection may be regulated by inhibition of tyrosine phosphorylation in T-cells, although the inhibition is not associated with PKC activation and subsequent IL-2 secretion of T cells.     

Discovery of novel phenoxazinone derivatives as DKK1/LRP6 interaction inhibitors: Synthesis,biological evaluation and structure–activity relationships

Amino derivatives of NCI8642 were synthesized and evaluated as inhibitors of DKK1/LRP6 interactions. The new inhibitors were able to activate the Wnt signaling pathway as indicated by the increased levels of β-catenin, and decrease the DKK1-induced Tau phosphorylation at serine 396.     

L-DOPA Inhibits In Vitro Phosphorylation of Melanoma Glycoproteins

L-DOPA had no effect on the endogenous phosphorylation of proteins after extraction with 1% Triton X-100 from hamster melanoma. When proteins were purified further by wheat germ-agglutinin chromatography, however, a dramatic and dose-dependent inhibitory effect of DOPA on glycoprotein phosphorylation was observed in the presence of Mn ⁺².     

In vivo phosphorylation of proteins in Clostridium sphenoides

Abstract Cell contents of Clostridium sphenoides , labeled with [³²P]orthophosphate under strict anaerobic conditions, were analyzed by two-dimensional gel electrophoresis. Autoradiography of these gels demonstrated the presence of at least 15 ³²P-labeled protein species, of which M _r and iso-electric point were determined. Treatment of the radioactively labeled cell contents with alkaline phosphatase and acid phosphatase showed that all these proteins were modified by phosphorylation. These findings demonstrate for the first time the presence of phosphorproteins in a strictly anaerobic bacterium.     

PKC phosphorylates GluA1-Ser831 to enhance AMPA receptor conductance

AMPA receptors mediate fast excitatory synaptic transmission in the brain, and are dynamically regulated by phosphorylation of multiple residues within the C-terminal domain. CaMKII phosphorylates Ser831 within the AMPA receptor GluA1 subunit to increase single channel conductance, and biochemical studies show that PKC can also phosphorylate this residue. In light of the discovery of additional PKC phosphorylation sites within the GluA1 C-terminus, it remains unclear whether PKC phosphorylation of Ser831 increases GluA1 conductance in intact receptors. Here, we report that the purified, catalytic subunit of PKC significantly increases the conductance of wild-type GluA1 AMPA receptors expressed in the presence of stargazin in HEK293T cells. Furthermore, the mutation GluA1-S831A blocks the functional effect of PKC. These findings suggest that GluA1 AMPA receptor conductance can be increased by activated CaMKII or PKC, and that phosphorylation at this site provides a mechanism for channel modulation via a variety of protein signaling cascades.