Mediation of a GDSL Esterase/Lipase in Carotenoid Esterification in Tritordeum Suggests a Common Mechanism of Carotenoid Esterification in Triticeae Species

Carotenoids are essential in human diet, so that the development of programs toward carotenoid enhancement has been promoted in several crops. The cereal tritordeum, the amphiploid derived from the cross between Hordeum chilense Roem. et Schulz. and durum wheat has a remarkable carotenoid content in the endosperm. Besides, a high proportion of these carotenoids are esterified with fatty acids. The identification of the gene(s) responsible for xanthophyll esterification would be useful for breeding as esterified carotenoids show an increased ability to accumulate within plant cells and have a higher stability during post-harvest storage. In this work, we analyzed five genes identified as candidates for coding the xanthophyll acyltransferase (XAT) enzyme responsible for lutein esterification in H. chilense genome. All these genes were expressed during grain development in tritordeum, but only HORCH7HG021460 was highly upregulated. Sequence analysis of HORCH7HG021460 revealed a G-to-T transversion, causing a Glycine to Cysteine substitution in the protein of H290 (the only accession not producing quantifiable amounts of lutein esters, hereinafter referred as zero-ester) of H. chilense compared to the esterifying genotypes. An allele-specific marker was designed for the SNP detection in the H. chilense diversity panel. From the 93 accessions, only H290 showed the T allele and the zero-ester phenotype. Furthermore, HORCH7HG021460 is the orthologue of XAT-7D, which encodes a XAT enzyme responsible for carotenoid esterification in wheat. Thus, HORCH7HG021460 (XAT-7Hch) is a strong candidate for lutein esterification in H. chilense and tritordeum, suggesting a common mechanism of carotenoid esterification in Triticeae species. The transference of XAT-7Hch to wheat may be useful for the enhancement of lutein esters in biofortification programs.

Crash landing of Vibrio cholerae by MSHA pili-assisted braking and anchoring in a viscous environment

Mannose-sensitive hemagglutinin (MSHA) pili and flagellum are critical for the surface attachment of Vibrio cholerae. However, the cell landing mechanism remains largely unknown. Here, combining the cysteine-substitution-based labelling method with single-cell tracking techniques, we quantitatively characterized the landing of V. cholerae by directly observing both pili and flagellum of cells in viscous solutions. MSHA pili are evenly distributed along the cell length and can stick to surfaces at any point along the filament. With such properties, MSHA pili are observed to act as a brake and anchor during cell landing which include three phases: running, lingering, and attaching. Resistive-force-theory based models are proposed to describe near-surface motion. Importantly, the role of MSHA pili during cell landing is more apparent in viscous solutions. Our work provides a detailed picture of the landing dynamics of V. cholerae under viscous conditions, which can provide insights into ways to better control V. cholerae infections.

Mitotic spindle disassembly in human cells relies on CRIPT-bearing hierarchical redox signals

Swift and complete spindle disassembly is essential for cell survival, yet how it happens is largely unknown. Here we used real-time live-cell microscopy and biochemical assays to show that a cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization during interphase but was distributed to spindles and underwent redox modifications to form disulfides within CXXC pairs during mitosis. Then, it interacts with and transfers redox response to tubulin subunits to induce microtubule depolymerization. The mutants with any of cysteine substitution completely block the spindle disassembly generating two cell populations with long-lasting metaphase spindles or spindle remnants. The live cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase may share a common CRIPT-bearing redox-controlled mechanism.

Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study

Background:Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment.Objective:This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation.Method:In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed.Results:According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity.Conclusion:SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.

TRPV5, TRPV6, TRPM6, and TRPM7 do not contribute to hair-cell mechanotransduction

The hair-cell mechanotransduction channel remains unidentified. We tested whether four transient receptor channel (TRP) family members, TRPV5, TRPV6, TRPM6, and TRPM7, participated in transduction. Using cysteine-substitution mouse knock-ins and methanethiosulfonate reagents selective for those alleles, we found that inhibition of TRPV5 or TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they could participate in transduction. Cochlear hair cell transduction was insensitive to shRNA knockdown ofTrpm6orTrpm7, however, and was not affected by manipulations of Mg2+, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV5, TRPV6, TRPM6, and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.

Abstract 049: Phosphodiesterase-3a Catalytic-domain Mutation and Hypertension

We recently discovered phosphodiesterase-3A (PDE3A) mutations causing a 50 mm Hg increase in blood pressure and stroke >50 years, as the first non-salt form of Mendelian genetic hypertension, autosomal-dominant hypertension with brachydactyly (HTNB). The mutations cause increased PDE3A phosphorylation and higher cAMP affinity. We now have found a completely different PDE3A mutation causing a similar syndrome in a New Zealand pedigree. The mutation resides in the enzyme’s catalytic domain, results in an arginine-to-cysteine substitution, and represents a more direct mechanism of PDE3A activation. For Michaelis-Menten kinetics of cAMP hydrolysis, we transfected HEK293 cells transiently expressing Flag-tagged versions of PDE3A1, PDE3A2, or PDE3A3 mutant vs. wildtype and stimulated with forskolin and phorbol-12-myristate-13-acetate (PMA) to enhance intrinsic phosphorylation. Vmax and Km (Michaelis constant) were calculated using GraphPad Prism software to reveal the maximum cAMP turnover rate at saturated substrate concentration and the affinity of cAMP to wildtype and mutated PDE3A1, PDE3A2 and PDE3A3. For PDE3A1 hydrolytic activity (triplicate), we observed: Vmax Km Wildtype 7.5 340 Wildtype+forskolin/PMA 7.2 203 Mutant 6.6 116 Mutant+forskolin/PMA 6.3 81 The dramatically lower Km of mutant PDE3A indicates a substantially greater affinity for cAMP consistent with gain-of-function. These data underscore the importance of PDE3A to high blood pressure by means of a different, novel genetic mechanism directly implicating the catalytic domain.