Journal of biochemistry最新文献

Analogs of pyrrole alkaloid lamellarins exhibit anticancer activity by modulating multiple cellular events. Lethal doses of several lamellarins were found to enhance autophagy flux in HeLa cells, suggesting that lamellarins may modulate protein homeostasis through the interference of proteins or kinases controlling energy and nutrient metabolism. To further delineate molecular mechanisms and their targets, our results herein show that azalamellarin D (AzaD) cytotoxicity could cause translational attenuation, as indicated by a change in eIF2α phosphorylation. Intriguingly, acute AzaD treatment promoted the phosphorylation of GCN2, a kinase that transduces the integrated stress response (ISR), and prolonged exposure to AzaD could increase the levels of the phosphorylated forms of eIF2α and the other ISR kinase protein kinase R (PKR). However, the effects of AzaD on ISR signalling were marginally abrogated in cells with genetic deletion of GCN2 and PKR, and evaluation of protein target engagement by cellular thermal shift assay (CETSA) revealed no significant interaction between AzaD and ISR kinases. Further investigation revealed that acute AzaD treatment negatively affected mechanistic target of rapamycin (mTOR) phosphorylation and signalling. The analyses by CETSA and computational modelling indicated that mTOR may be a possible protein target for AzaD. These findings indicate the potential for developing lamellarins as novel agents for cancer treatment.

D-Glutamate, a novel d-amino acid found in animal tissues, exclusively exists in the male reproductive tissues of the kuruma prawn, Marsupenaeus japonicus. Herein, changes in the d-glutamate content were determined in the male reproductive tissues of M. japonicus during acclimation to breeding seawater temperatures of 18-22°C and unilateral eyestalk ablation. The d-glutamate content in the testis increased with increasing seawater temperature and with unilateral eyestalk ablation. This suggests that both stimulations induced d-glutamate synthesis in the testis. Although the d-alanine content in the testis increased after unilateral eyestalk ablation, it did not change with elevated seawater temperature. Furthermore, we determined the d-glutamate distribution in the M. japonicus spermatophore. This indicates that d-glutamate is crucial in prawn fertilization.

Senescent cells accumulate in various organs with ageing, and its accumulation induces chronic inflammation and age-related physiological dysfunctions. Several remodelling of intracellular environments have been identified in senescent cells, including enlargement of cell/nuclear size and intracellular acidification. Although these alterations of intracellular environments were reported to be involved in the unique characteristics of senescent cells, the contribution of intracellular acidification to senescence-associated cellular phenotypes is poorly understood. Here, we identified that the upregulation of TXNIP and its paralog ARRDC4 as a hallmark of intracellular acidification in addition to KGA-type GLS1. These genes were also upregulated in response to senescence-associated intracellular acidification. Neutralization of the intracellular acidic environment ameliorated not only senescence-related upregulation of TXNIP, ARRDC4 and KGA but also inflammation-related genes, possibly through suppression of PDK-dependent anaerobic glycolysis. Furthermore, we found that expression of the intracellular acidification-induced genes, TXNIP and ARRDC4, correlated with inflammatory gene expression in heterogeneous senescent cell population in vitro and even in vivo, implying that the contribution of intracellular pH to senescence-associated cellular features, such as SASP.

Glycogen debranching enzyme is a single polypeptide with distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. To allow phosphorylase to degrade the inner tiers of highly branched glycogen, 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch G-G-G-G-(G-G-G-G↔)G-G- (G: d-glucose, hyphens: α-1,4-linkages; double-headed arrow: α-1,6-linkage) into the G-G-G-G-(G↔)G-G- residue, which is then subjected to amylo-α-1,6-glucosidase to release the remaining G↔ residue. However, while the essential side-chain structure of the 4-α-glucanotransferase donor substrate has been determined to be the G-G-G-G↔ residue (Watanabe, Y., et al. (2008) J. Biochem.143, 435-440), its essential main-chain structure remains to be investigated. In this study, we probed the 4-α-glucanotransferase donor-binding region using novel fluorogenic dextrins Gm-(G4↔)G-Gn-F (F: 1-deoxy-1-[(2-pyridyl)amino]-d-glucitol) and maltohexaose (G6) as the donor and acceptor substrates, respectively. 4-α-Glucanotransferase exhibited maximum activity towards G4-(G4↔)G-F and G4-(G4↔)G-G-F, indicating that recognition of the G4-(G4↔)G-moiety was essential for full enzyme function. Notably, when the 4-α-glucanotransferase activity towards G4-(G4↔)G-G-F was taken as unity, those towards nonbranching dextrins were < 0.001. This indicated that the disproportionation activities towards maltooligosaccharides (Gm) are abnormal behaviours of 4-α-glucanotransferase. Notably, however, these activities have been traditionally measured to identify the 4-α-glucanotransferase mutations causing glycogen storage disease type III. This study provides a basis for more accurate identification.

Schistosoma japonicum glutathione-S-transferase (SjGST), the so-called GST-tag, is one of the most widely used protein tags for the purification of recombinant proteins by affinity chromatography. Attachment of SjGST enables the purification of a protein of interest (POI) using commercially available glutathione-immobilizing resins. Here we produced an SjGST mutant pair that forms heterodimers by adjusting the salt bridge pairs in the homodimer interface of SjGST. An MD study confirmed that the SjGST mutant pair did not disrupt the heterodimer formation. The modified SjGST protein pair coexpressed in Escherichia coli was purified by glutathione-immobilized resin. The stability of the heterodimeric form of the SjGST mutant pair was further confirmed by size exclusion chromatography. Surface plasmon resonance measurements unveiled the selective formation of heterodimers within the pair, accompanied by a significant suppression of homodimerization. The heterodimeric SjGST exhibited enzymatic activity in assays employing a commercially available fluorescent substrate. By fusing one member of the heterodimeric SjGST pair with a fluorescent protein and the other with the POI, we were able to conveniently and sensitively detect protein-protein interactions using fluorescence spectroscopy in the pull-down assays. Thus, utilization of the heterodimeric SjGST would be a useful tag for protein science.

Protection against oxidative stress is a vital defense mechanism for Mycobacterium tuberculosis within the host. However, few transcription factors that control bacterial antioxidant defense are known. Here, we present evidence that SdrR, encoded by the MSMEG_5712 (Ms5712) gene, functions as an oxidative stress response regulator in Mycobacterium smegmatis. SdrR recognizes an 11-bp motif sequence in the operon's upstream regulatory region and negatively regulates the expression of short-chain dehydrogenases/reductases (SDR). Overexpressing sdrR inhibited SDR expression, which rendered the strain oxidative more stress-sensitive. Conversely, sdrR knockout alleviates SDR repression, which increases its oxidative stress tolerance. Thus, SdrR responds to oxidative stress by negatively regulating sdr expression. Therefore, this study elucidated an underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

The small GTPase Ras plays an important role in intracellular signal transduction and functions as a molecular switch. In this study, we used a photoresponsive protein as the molecular regulatory device to photoregulate Ras GTPase activity. Photo zipper (PZ), a variant of the photoresponsive protein Aureochrome1 developed by Hisatomi et al. was incorporated into the C-terminus of Ras as a fusion protein. The three constructs of the Ras-PZ fusion protein had spacers of different lengths between Ras and PZ. They were designed using an Escherichia coli expression system. The Ras-PZ fusion proteins exhibited photoisomerization upon blue light irradiation and in the dark. Ras-PZ dimerized upon light irradiation. Moreover, Ras GTPase activity, which is accelerated by the Ras regulators guanine nucleotide exchange factors and GTPase-activating proteins, is controlled by photoisomerization. It has been suggested that light-responsive proteins are applicable to the photoswitching of the enzymatic activity of small GTPases as photoregulatory molecular devices.

G protein-coupled receptor 84 (Gpr84) is reportedly activated by medium-chain fatty acids and is involved in the pathology of liver fibrosis. Inflammatory stimulants, such as lipopolysaccharide and tumor necrosis factor-α, upregulate Gpr84 expression. However, the detailed molecular mechanism by which Gpr84 is induced remains unknown. Inflammatory stimulation also evokes endoplasmic reticulum (ER) stress, but there has been no direct evidence to link Gpr84 expression and the ER stress response. Administration of tunicamycin (Tm) provokes ER stress and acute steatosis in the liver tissue of mice. Here, in situ hybridization analysis revealed that induction of Gpr84 expression occurred in parenchymal cells in the liver tissue following Tm administration. Gene expression analysis using a reporter assay showed that the intron 1 region of Gpr84 was involved in induction of the gene under ER stress conditions. Furthermore, Tm-dependent upregulation of Gpr84 was blocked by the small chemical compound AEBSF, an inhibitor of ER stress transducers, in vitro and in vivo. In conclusion, the current study marks the discovery that the ER stress agent Tm induces the expression of Gpr84.

G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans and are categorized into six classes (A-F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) on the structural determinations of GPCR-G-protein complexes. Specific technologies, such as nanobodies and mini-G-proteins, stabilize complexes and facilitate structural determination. We discuss the structural alterations upon receptor activation in different GPCR classes, revealing their diverse mechanisms. This review highlights the robust foundation for comprehending GPCR function and pave the way for future breakthroughs in drug discovery and therapeutic targeting.