Journal of biochemistry最新文献

Brown adipocytes dissipate chemical energy as heat and confer protection against type 2 diabetes and obesity. Nuclear factor I-A (NFIA) is a transcription factor that orchestrates the brown fat gene programme by activating cell type-specific enhancers and facilitating the genomic binding of PPARγ, the master regulator of adipogenesis, to these enhancers. NFIA promotes mitochondrial oxidative phosphorylation and thermogenesis, while reciprocally suppressing adipose tissue inflammation, thereby contributing to the maintenance of glucose and body weight homeostasis in mice. Here the author provides an overview of the identification of NFIA as a pivotal regulator of brown adipocyte biology, elucidates its underlying mechanisms of action, examines its implications for systemic metabolism and outlines future perspectives for research in this field.

In higher plants, ferredoxin (Fd) and Fd-NADP+ reductase (FNR) are each present as distinct isoproteins of photosynthetic type and non-photosynthetic type, which exhibit differential function despite their similarity in the 3D structures. In this study, we addressed differential regulation of Fd/FNR reaction between the two types from two perspectives and investigated the amino acid residues of Fd responsible for the differences. Firstly, pH-dependent profile of Fd/FNR electron transfer activity varied among the combinations of the two types of Fd and FNR; non-photosynthetic type FNR showed similar pattern for the two types of Fds while photosynthetic type FNR was previously shown to exhibit opposite pattern, which was explained by the different pH-dependent profile of Km for the two Fds. Secondly, the extent of the suppression of the affinity (in terms of Km value) between Fd and FNR by NADPH significantly varied among the combinations of the two types of Fd:FNR. The difference was shown to be mainly due to the different property of Fd between the two types. Kinetic analyses using site-directed mutants of Fd showed the contribution of C-terminal residues, together with that of 78th residue of Fd, on the differential profile of Fd/FNR reaction by pH and NADPH.

The α-amylase from Klebsiella pneumoniae (KpAmy13), which belongs to glycoside hydrolase family 13 subfamily 19, produces maltohexaose as an initial product when acting on starch and has been characterized as a maltohexaose-producing α-amylase. The crystal structure of KpAmy13 was determined at a resolution of 1.9 Å, revealing the structures of all its domains: N, A, B and C. Domain N resembles the starch-binding domain known as carbohydrate-binding module family 69, found in α-glucan-related proteins. Although domain N does not conserve the starch-binding residues observed in other proteins, it has several hydrophobic residues on its surface, which might be involved in promoting catalysis. The catalytic cleft is located at the bottom of a circular depression. The domain N-truncated mutant of KpAmy13 in complex with maltohexaose showed that its non-reducing end glucose docks at subsite -6. The long and complex structure of domain B contributes to forming a cleft of the right size for six glucose moieties, demonstrating the exo-acting mechanism.

Structural variations of N-glycans critically regulate glycoprotein functions and are involved in various human diseases. N-Acetylglucosaminyltransferase-III (GnT-III or MGAT3) is highly expressed in the brain and kidney and is an N-glycan branching enzyme that biosynthesizes the unique N-glycan branch designated as bisecting GlcNAc. Its roles in Alzheimer's disease and cancer have been revealed, but the functions of bisecting GlcNAc in the kidney are poorly understood. Here, we show that kidneys in the GnT-III-knockout (KO) mouse exhibit impaired body fluid balance and present interstitial edema. To understand the molecular mechanisms further, we biochemically purified the glycoproteins modified by GnT-III in the mouse kidney and identified these proteins using proteomics. We found that the proteins involved in the pathway for angiotensin II (Ang II) metabolism are modified by GnT-III, and that the subcellular localization of angiotensin-converting enzyme was altered in GnT-III-KO cells. Furthermore, the pathology in models of Ang II-related disease was slightly more severe in GnT-III-KO than in wild-type mice. Our data indicate a protective role for bisecting GlcNAc in the mouse kidney, highlighting a newly described link between specific N-glycan structures and renal functions.

Glucuronyltransferase GlcAT-P is a rate-limiting enzyme involved in the biosynthesis of the Human Natural Killer-1 carbohydrate and is essential for acquiring higher brain functions. Alternative splicing produces two isoforms, short-form GlcAT-P (sGlcAT-P) and long-form GlcAT-P (lGlcAT-P), which share identical peptide sequences except for an additional 13 amino acids (AA) in the cytoplasmic N-terminal tail of lGlcAT-P. Although sGlcAT-P localizes to the Golgi apparatus (GA), where many glycosyltransferases reside, lGlcAT-P is distributed in both the GA and endoplasmic reticulum (ER). However, the mechanisms responsible for this distinct intracellular distribution remain poorly understood. In this study, we explored the role of the 13 AA in the cytoplasmic N-tail of lGlcAT-P in trafficking between the GA and the ER using the Retention Using Selective Hooks system. Our findings revealed that lGlcAT-P undergoes enhanced retrograde trafficking from the GA to the ER, whereas its anterograde trafficking from the ER to the GA remains largely unaffected. In addition, three glutamic acid residues within the 13 AA of lGlcAT-P were identified as crucial for promoting retrograde trafficking. These results suggest that the ER distribution of lGlcAT-P is primarily governed by Golgi-to-ER trafficking regulated by specific sequences in its cytoplasmic N-tail.

Skeletal muscle myosin is generally considered insoluble under physiological, low ionic strength, or salt-free conditions due to its tendency to self-assemble into filamentous polymers in vitro. Our previous study showed that myosin can be solubilized in low ionic strength solutions containing l-histidine. However, another report suggested that 1-methylhistidine could not solubilize myosin, and the factors essential for myosin solubilization remain unclear. To elucidate the role of l-histidine in the water solubilization of myosin, we examined myosin solubility and the molecular properties of its rod domain, l-meromyosin, using structurally related buffer compounds. Under salt-free conditions, solubility depended heavily on the acid dissociation constant of buffer, indicating that maintaining a neutral pH is critical. The rod domain showed molecular elongation regardless of the buffer type, yet surface charge and hydrophobicity remained comparable to conditions with high ionic strength. These results suggest that myosin is inherently soluble and maintains its structural integrity under neutral, salt-free conditions. The apparent insolubility under such conditions is likely to result from hydrochloric acid used for pH adjustment. Since l-histidine and imidazole achieve neutrality without acid addition, they are ideal buffers for myosin solubilization.

Nucleic acids (NAs) are recognized by endosomal Toll-like receptors (TLRs) and cytoplasmic sensors in innate immune cells. NAs accumulate within lysosomes due to either excessive NA influx or defective lysosomal degradation. The resultant storage of NAs and/or NA metabolites in the lysosome, referred to here as lysosomal NA stress, elicits a spectrum of responses, ranging from inflammation to tissue repair, through NA sensor activation. Although these responses contribute to host defence against infection, they may also drive diseases. For instance, loss of function of the lysosomal nucleoside transporter SLC29A3 drives lysosomal nucleoside stress, which activates TLR8 in macrophages to cause histiocytic diseases collectively called SLC29A3 disorders. Similarly, DNase II deficiency promotes lysosomal DNA stress, leading to activation of cytoplasmic double-stranded DNA sensors, such as cGAS-STING and AIM2, and thereby autoinflammatory and autoimmune diseases. Thus, lysosomal NA stress is viewed as a pivotal environmental signal that initiates innate immune responses.

Histones bind directly to DNA and play a role in regulating gene expression in part by influencing chromatin structure. The DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this study, we performed a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity. Our results revealed that the H2bc27 gene exhibited brain-specific expression in mice at E14.5. We generated mice lacking the H2bc27 gene using the CRISPR/Cas9 system. While the phenotype of H2bc27 knockout mouse brains was not different from that of wild-type mouse brains, transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.

Autophagy suppresses tumourigenesis in normal cells, but in established tumours, it can promote tumour progression, particularly by enhancing resistance to stress. However, the mechanism underlying this tumour-promoting function remains unclear. To investigate this, we adopted an interdisciplinary approach combining database analysis with experimental validation. Specifically, by classifying the autophagy-related genes using AutoML analysis on their expression patterns in the COSMIC database, we identified an autophagy subnetwork that correlated with the PLK1-RAD9A axis, a pathway we had previously linked to genotoxic resistance. Cell-based experiments confirmed that autophagy enhanced polo-like-kinase1 (PLK1) expression at both the transcriptional and translational levels, facilitating genotoxic resistance. Notably, in stressed S-phase cells, we found that PLK1 expression levels varied among individual cells, yet overall cell population acquired genotoxin resistance. The genotoxin resistance in the cell population with heterogeneous PLK1 expression was driven by autophagy by facilitating the secretion of currently unidentified factors, likely by switching function of RAD9A from DNA checkpoint to substance secretion. Together our data demonstrate that intra-tumour heterogeneity contributes to the malignant features of tumours through an autophagy-PLK-RAD9A axis that promotes intercellular communication via secretion.

Heme oxygenase-1 (HO-1) is unique to be directly regulated by diverse stress-responsible transcription factors; however, the cross-talk between oxidative stress and heat shock stress has not been completely elucidated. It is widely accepted that HO activity is not induced by heat shock in cultured cells derived from humans and mice but from rats. Previously, we reported that the discrepancies in heat shock-induced HO-1 expression in different animal species were caused by the access of heat shock factor 1 (HSF1) to heat shock element (HSE) in the different regions of the HO-1 gene. Recently, we found that the human monocyte-derived cell line THP-1, which has been extensively used to study monocyte/macrophage functions, represents the heat shock induction of HO-1 after differentiation to macrophage-like cells, although not responsible before differentiation. In this study, we demonstrated that heat shock loading to macrophage-like cells derived from THP-1 specifically activated HSF1 to bind to HSE in the promotor region in the HO-1 gene, resulting in the induction of HO-1. Our finding is significant in understanding the regulation system by macrophages for inflammation caused by oxidative insults and associated with hyperthermia in vivo.