Biology subjects

Sakamoto, S.

Publications and source records attributed to Sakamoto, S..

23 records · Page 2Linked to original sources

bioRxiv2020

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hours. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a "programmed" process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP. (148 words)

cell biology↗

bioRxiv2020

The linear ubiquitin chain assembly complex generates heterotypic ubiquitin chains

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

biochemistry↗

bioRxiv2020

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121

Enzymes acting on -L-arabinofuranosides have been extensively studied; however, the structures and functions of {beta}-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of {beta}-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular {beta}-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing {beta}-1,2-linked arabinofuranose disaccharide ({beta}-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 [A] resolution. The HypBA2 structure consists of a central catalytic (/)6 barrel domain and two flanking (N- and C-terminal) {beta}-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the {beta}-Ara2 disaccharide; this pocket is highly conserved among GH121 proteins. The three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all ~270 members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of GH63 -glycosidase, GH94 chitobiose phosphorylase, GH142 {beta}-L-arabinofuranosidase, GH78 -L-rhamnosidase, and GH37 ,-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. {beta}-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the {beta}-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

biochemistry↗

bioRxiv2020

NF90 Modulates Processing of a Subset of Human Pri-miRNAs

MicroRNAs are predicted to regulate the expression of more than 60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The dsRNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of pri-miRNAs. Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA EMSA. NF90-bound and modulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly modulated. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

molecular biology↗

bioRxiv2018

High-throughput Analysis of Arabidopsis Stem Vibrations to Identify Mutants with Altered Mechanical Properties

Mechanical properties are rarely used as quantitative indices for the large-scale mutant screening of plants, even in the model plant Arabidopsis thaliana. The mechanical properties of plant stems generally influence their vibrational characteristics. Here, we developed Python-based software, named AraVib, for the high-throughput analysis of free vibrations of plant stems, focusing specifically on Arabidopsis stem vibrations, and its extended version, named AraVibS, to identify mutants with altered mechanical properties. These programs can be used without knowledge of Python and require only an inexpensive handmade setting stand and an iPhone/iPad with a high-speed shooting function for data acquisition. Using our system, we identified an nst1 nst3 double-mutant lacking secondary cell walls in fiber cells and a wrky12 mutant displaying ectopic formation of secondary cell wall compared with wild type by employing only two growth traits (stem height and fresh weight) in addition to videos of stem vibrations. Furthermore, we calculated the logarithmic decrement, the damping ratio, the natural frequency and the stiffness based on the spring-mass-damper model from the video data using AraVib. The stiffness was estimated to be drastically decreased in nst1 nst3, which agreed with previous tensile test results. However, in wrky12, the stiffness was significantly increased. These results demonstrate the effectiveness of our new system. Because our method can be applied in a high-throughput manner, it can be used to screen for mutants with altered mechanical properties.

plant biology↗