A flipped ion pair at the dynein–microtubule interface is critical for dynein motility and ATPase activation

Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein–MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.     

S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO?, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.     

Lipoprotein lipase is a novel amyloid beta (Abeta)-binding protein that promotes glycosaminoglycan-dependent cellular uptake of Abeta in astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.     

Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.     

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.     

Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury.     

Cloning and sequencing of inulinase and β-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes

An inulinase-producing Microbulbifer sp. strain, JAM-3301, was isolated from a deep-sea sediment. An inulin operon that contained three open reading frames was cloned and sequenced. Two of the three genes were expressed. One product was an endo-inulinase, and the other was a β-fructofuranosidase. Both enzymes worked together to effectively degrade inulin.     

Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis

Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II.     

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.Microtubules (MTs) are important cytoskeletal polymers that are conserved in eukaryotic cells and are assembled from α- and β-tubulin heterodimers (Desai and Mitchison, 1997). In plants, MTs have important functions in essential cellular processes, such as cell division, and in cell morphogenesis. MTs in plant cells adopt several distinct higher order arrays and are remodeled in response to the cell cycle, developmental programs, and environmental cues (Hashimoto, 2015). Genetic, molecular, and cell biological approaches have been used to identify cellular factors that regulate the organization and dynamics of plant MTs. Considerable effort has been devoted to simulating the organization of cortical MT arrays by computational modeling.Cell-free in vitro studies are essential for the biochemical characterization of various MT regulators and for elucidating the mechanistic principles underlying the versatility of this dynamic polymer in cellular functions. The purification of sufficient amounts of assembly-competent tubulin is a prerequisite for these in vitro studies. Tubulin is traditionally purified from mammalian brains, since these tissues contain sufficiently high concentrations of tubulin to allow MT assembly in crude cell extracts. Polymerized MTs and their associated MT-binding proteins are separated from other cellular proteins by sedimentation. Pelleted MTs are then depolymerized upon drug washout under MT-destabilizing conditions, such as high concentrations of salt and calcium and low temperature. A few rounds of assembly-disassembly cycles highly enrich for tubulin and copurify MT-associated proteins, which can subsequently be removed by column chromatography (Borisy et al., 1975). Tubulin has also been purified from several plant sources (Morejohn and Fosket, 1982; Mizuno, 1985; Jiang et al., 1992; Bokros et al., 1993; Moore et al., 1997). However, since tubulin concentrations are low in plant cells, taxol, which stabilizes MTs, is generally included in the polymerization buffer, and cytoplasm-rich miniprotoplasts, which lack vacuoles, are sometimes used as starting material (Hamada et al., 2013). Since it is technically challenging to isolate assembly-competent pure tubulin from nonneural sources (Sackett et al., 2010), general plant science laboratories may hesitate to prepare plant tubulin themselves.Although the primary amino acid sequences of eukaryotic tubulins are fairly well conserved and the biophysical mechanisms of MT assembly and disassembly are thought to be similar for all MTs, the kinetics of MT dynamic instability differ for MTs assembled from animal and plant tubulin (Moore et al., 1997). Interactions with MT-interacting proteins may differ for tubulins isolated from different biological sources, as reported for the MT-dependent activation of kinesin (Alonso et al., 2007). Posttranslational modifications of tubulin, which generate distinct tubulin signatures and may modulate the functions of MT-interacting proteins, such as kinesin (Sirajuddin et al., 2014), are extensive in brain tubulin (Janke, 2014) but may be quantitatively and qualitatively different in plant tubulin. Furthermore, MT nucleation by the γ-tubulin ring complex shows a strong preference for tubulin from the same species (Kollman et al., 2015). Thus, it is important to use plant tubulin, and not brain tubulin, for in vitro studies of plant MTs.Tubulin is folded by a series of molecular chaperones to form an αβ-tubulin heterodimer in which one structural GTP is embedded in the interdimer interface (Lundin et al., 2010). The requirement of these eukaryote-specific chaperones precludes the use of prokaryotic expression systems for synthesizing properly folded and functional tubulin. Bacterially synthesized tubulin can be folded in rabbit reticulocyte lysate to produce functional tubulin, but with moderate yields (Shah et al., 2001). A yeast (Saccharomyces cerevisiae) expression system has been developed to produce modified yeast tubulin (Uchimura et al., 2006; Johnson et al., 2011), but this system is not suitable for the synthesis of animal (Sirajuddin et al., 2014) and plant (our unpublished data) tubulin. A baculovirus-insect cell expression system was recently reported to yield functional human tubulin (Minoura et al., 2013).Tubulin-binding proteins have been used to develop affinity-purification columns. The TOG domains (named after the human MT regulator, colonic and hepatic Tumor Overexpressed Gene [ch-TOG]) are among the best-characterized tubulin-binding domains. ch-TOG and orthologs from other eukaryotes bind to the growing plus ends of MTs and accelerate MT growth (Al-Bassam and Chang, 2011). TOG domains from the yeast ortholog Stu2 were recently used to affinity purify assembly-competent tubulin from fungal and animal sources (Widlund et al., 2012). In this study, we demonstrate that a TOG-based affinity column can be used to purify functional tubulin from tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We examined the posttranslational modifications of the isolated tubulins by mass spectrometry and immunoblot analysis and showed that a His-tagged Arabidopsis tubulin isotype could be purified using this column. These results show that wild-type and recombinant functional tubulin from plant sources can be isolated efficiently.