Journal of biochemistry最新文献

Lysophosphatidic acid acyltransferase (LPAAT) is an enzyme responsible for the second acylation step of phospholipid biosynthesis and transforms lysophosphatidic acid to phosphatidic acid, a universal precursor of various phospholipids. In addition to the well-studied plsC-encoded LPAAT (EcPlsC), we previously found that Escherichia coli has another LPAAT that is encoded by yihG (EcYihG). EcPlsC and EcYihG are integral membrane proteins and have never been solubilized and purified in their active form. To better understand the difference in their enzymatic functions and how the two paralogs differently contribute to lipid diversity, we established a method to purify both enzymes in their active form and comparatively analysed their biochemical characteristics. Our findings illustrate that EcPlsC possesses the highest activity at pH 8.0 and 37°C with selectivity for unsaturated fatty acyl-CoAs (e.g. palmitoleoyl-CoA), whereas EcYihG works optimally at pH 7.5 and 30°C and prefers saturated fatty acyl-CoAs (e.g. myristoyl-CoA). In addition, we performed a mutational analysis based on AlphaFold2 models and revealed that one residue, which is located at the putative acyl-donor-selectivity tunnel entrance, plays a pivotal role in selecting acyl donor substrates. This provides new insights into how LPAATs recognize specific fatty acyl groups and incorporate them into membrane phospholipids.

Regulation at the level of translation is critical in the nervous system, such as for the formation of cell-type-specific proteomes or plastic changes in neural circuits. Whilst current knowledge of the translatome is relatively limited compared to transcriptome, a growing array of tools to analyse translation is becoming available. In this review, we discuss techniques for profiling translation on a genome-wide scale with a special emphasis on cell-type-specific analyses in the nervous system. This includes polysome-profiling-seq, Translating Ribosome Affinity Purification (TRAP)-seq and ribosome profiling (Ribo-seq). We review recent advances to achieve spatial resolution of translatome analysis, such as genetic labelling of the targeted cells and cell sorting, and discuss the biological implications of translational regulation in the brain and potential future extensions.

Nuclear pore complexes (NPCs) act as gateways across the nuclear envelope for molecular transport between the nucleus and the cytoplasm in eukaryotes. NPCs consist of several subcomplexes formed by multiple copies of approximately 30 different proteins known as nucleoporins (Nups). In the fission yeast Schizosaccharomyces pombe, the NPC structure is unique, particularly in its outer ring subcomplexes, where the cytoplasmic and nucleoplasmic outer rings are composed of distinct sets of proteins. However, it remains unclear how this unique outer ring structure in S. pombe is supported by interactions between subcomplexes or individual Nups. In this study, we investigated protein-protein interactions between S. pombe Nups using mass spectrometry and identified Nups that interact with each subcomplex or a specific Nup. The cytoplasmic outer ring Nups bind to both the cytoplasmic filament Nups and the inner ring Nups, while the nucleoplasmic outer ring Nups bind to the nuclear basket Nups in addition to the inner ring Nups. Among the inner ring Nups, Nup155 interacts with most of the cytoplasmic and nucleoplasmic outer ring Nups, suggesting that Nup155 may serve as a hub supporting the uniquely asymmetric outer ring structure of the S. pombe NPC.

For bidirectional replication in Escherichia coli, higher order complexes are formed at the replication origin oriC by the initiator protein DnaA, which locally unwinds the left edge of oriC to promote the loading of two molecules of DnaB helicases onto the unwound region via dynamic interactions with the helicase-loader DnaC and the oriC-bound DnaA complex. One of the two helicases must translocate rightwards through oriC-bound DnaA complex. Here, we used a synthetic forked oriC DNA, which mimics the unwound state of oriC, to examine DnaB translocation through the oriC-bound DnaA complex. We found that DnaB helicase alone cannot pass through the oriC-bound DnaA complex without the help of single-strand binding protein (SSB). In the presence of SSB, DnaB passed through this complex along with its helicase function, releasing DnaA molecules. In addition, DnaB helicase activity is known to be inhibited by oversupply of DnaC, but this inhibition was relieved by SSB. These results suggest a mechanism that when two DnaB helicases are loaded at oriC, one translocates leftwards to expand the DnaA-unwound region and allows SSB binding to the single-stranded DNA, and such SSB molecules then stimulate translocation of the other helicase rightwards through the oriC-bound DnaA complex.

Various methods have been developed to map replication initiation zones (IZs) genome-wide, often finding far fewer IZs than expected. In particular, IZs corresponding to later stages of S phase are under-represented. Here, we reanalysed IZs with respect to replication timing in mouse ES cells. These datasets identified over five times as many early IZs compared to late IZs. In addition, we have set up a polymerase-usage sequencing (Pu-seq) system in mouse ES cells to map IZs genome-wide. Pu-seq showed less bias towards early IZs, potentially indicating better sensitivity for identifying IZs in late S phase.

Sesaminol is an organic compound that shows the strong antioxidant, anti-inflammatory and neuroprotective properties. Sesaminol triglucoside (STG) is a glycosylated form of sesaminol and abundantly exists in sesame seeds. However, typical β-glucosidases could not deglycosylate STG probably due to its bulky aglycone. PSTG1 and 2 are β-glucosidases lately isolated from Paenibacillis sp. KB0459 and have the capacity to deglycosylate STG. A recent report by Yanai et al. (J. Biochem. 2023; 174:335-344) revealed the unique domain architecture of PSTG1. Apart from other β-glucosdasies in the GH3 family, PSTG1 has a novel accessary domain (domain 4) at the C-terminus. Domain 4 contributes to the dimer formation and is located close to the active site. Interestingly, several hydrophobic residues are exposed, suggesting that this domain may recognize the hydrophobic aglycone of STG. The physiological functions of the non-catalytic domains in glyco-enzymes are sometimes overlooked. This paper sheds light on the aglycone recognition by novel accessary domain.

Cellular senescence, which entails cellular dysfunction and inflammatory factor release-the senescence-associated secretory phenotype (SASP)-is a key contributor to multiple disorders, diseases and the geriatric syndromes. Targeting senescent cells using senolytics has emerged as a promising therapeutic strategy for these conditions. Among senolytics, the combination of dasatinib and quercetin (D + Q) was the earliest and one of the most successful so far. D + Q delays, prevents, alleviates or treats multiple senescence-associated diseases and disorders with improvements in healthspan across various pre-clinical models. While early senolytic therapies have demonstrated promise, ongoing research is crucial to refine them and address such challenges as off-target effects. Recent advances in senolytics include new drugs and therapies that target senescent cells more effectively. The identification of senescence-associated antigens-cell surface molecules on senescent cells-pointed to another promising means for developing novel therapies and identifying biomarkers of senescent cell abundance.

As the global population continues to age, understanding the complex role of cellular senescence and its implications in healthy lifespans has gained increasing prominence. Cellular senescence is defined as the irreversible cessation of cell proliferation, accompanied by the secretion of a range of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP), in response to various cellular stresses. While the accumulation of senescent cells has been strongly implicated in the ageing process and the pathogenesis of age-related diseases owing to their pro-inflammatory properties, recent research has also highlighted their essential roles in processes such as tumour suppression, tissue development and repair. This review provides a comprehensive examination of the dual nature of senescent cells, evaluating their deleterious contributions to chronic inflammation, tissue dysfunction and disease, as well as their beneficial roles in maintaining physiological homeostasis. Additionally, we explored the therapeutic potential of senolytic agents designed to selectively eliminate detrimental senescent cells while considering the delicate balance between transient and beneficial senescence and the persistence of pathological senescence. A deeper understanding of these dynamics is critical to develop novel interventions aimed at mitigating age-related dysfunctions and enhancing healthy life expectancies.

Sleep quality and quantity decrease with age, and sleep disturbance increases the risk of many age-associated diseases. There is a significant relationship between nutritional status and sleep outcomes, with malnutrition inducing poor sleep quality in older adults. However, it remains elusive whether, and if so how, nutritional supplementation prevents age-associated sleep problems. Here, we utilized Drosophila to investigate the effects of a malnutrition diet with restricted yeast, a primary protein source, and supplementation of 10 essential amino acids (EAAs) on sleep profiles during ageing. Compared with the standard diet containing 2.7% yeast, the malnutrition diet containing 0.27% yeast significantly decreased target of rapamycin (TOR) signalling and shortened the lifespan of male Canton-S flies. By contrast, age-associated sleep loss, sleep fragmentation and loss of rhythm strength were similarly observed under both diets. Supplementation of the malnutrition diet with EAAs in restricted yeast significantly ameliorated age-associated sleep loss and sleep fragmentation without altering loss of rhythm strength. It also rescued decreased TOR signalling activity but not the shortened lifespan, suggesting that the effects of EAAs on sleep integrity are independent of TOR activity and lifespan regulation. These results may help to develop dietary interventions that improve age-related sleep problems in humans.

Brown adipocytes are characterized by a high abundance of mitochondria, allowing them to consume fatty acids for heat production. Increasing the number of brown adipocytes is considered a promising strategy for combating obesity. However, the molecular mechanisms underlying their differentiation remain poorly understood. In this study, we demonstrate that etomoxir, an inhibitor of Carnitine Palmitoyltransferase 1 (CPT1), inhibits their differentiation through mechanisms independent of β-oxidation inhibition. In the presence of etomoxir during brown adipocyte differentiation, reduced expression of the thermogenic gene UCP1 and decreased lipid droplets formation were observed. Furthermore, a transient reduction in the expression of PPARγ2, a critical factor in adipocyte differentiation, was also observed in the presence of etomoxir. These findings suggest the presence of a regulatory mechanism that specifically enhances PPARγ2 expression during brown adipocyte differentiation, thereby modulating thermogenic gene expression.