FEBS Open Bio最新文献

Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.

Viral infections are a major global health concern, affecting millions of people each year. Viral entry is one of the crucial stages in the infection process, but its details remain elusive. Enveloped viruses are enclosed by a lipid membrane that protects their genetic material and these viruses are linked to various human illnesses, including influenza, and COVID-19. Due to the advancements made in the field of molecular simulation, significant progress has been made in unraveling the dynamic processes involved in viral entry of enveloped viruses. Simulation studies have provided deep insight into the function of the proteins responsible for attaching to the host receptors and promoting membrane fusion (fusion proteins), deciphering interactions between these proteins and receptors, and shedding light on the functional significance of key regions, such as the fusion peptide. These studies have already significantly contributed to our understanding of this critical aspect of viral infection and assisted the development of effective strategies to combat viral diseases and improve global health. This review focuses on the vital role of fusion proteins in facilitating the entry process of enveloped viruses and highlights the contributions of molecular simulation studies to uncover the molecular details underlying their mechanisms of action.

Alzheimer's disease (AD) is characterized by significant alterations in hippocampal function and structure, but the molecular mechanisms underlying the hippocampal region remain elusive. We integrated multiple transcriptome datasets including human or rat hippocampus (GSE173955, GSE129051, GSE84422) to identify candidate genes. Subsequent analyses including gene ontology analysis and protein–protein interaction mapping were performed to identify key genes and pathways. We found that glutamate ionotropic receptor NMDA-type subunit 3A (GRIN3A) and glutamate metabotropic receptor 8 (GRM8), which are related to the glutamatergic system, were the top two annotated genes and directly related to MAPT, which encodes a tau protein. Since there is no direct evidence of interaction between tauopathy and these genes in AD, further transcriptomic data (GSE125957, GSE56772) from tau transgenic mice and experimental validations through primary rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived brain organoids were performed. Interestingly, we identified that decreased NR3A (encoded by GRIN3A) and mGluR8 (encoded by GRM8) are correlated with tauopathy and loss of postsynaptic function in AD. Taken together, our results identified a novel tauopathy biomarker GRIN3A in AD. Furthermore, our findings suggest that an integrated approach combining public databases and diverse experimental validations can contribute to the advancement of precision medicine therapies.

The budding yeast Saccharomyces cerevisiae is commonly used as an expression platform for the production of valuable compounds. Yeast-based genetic research can uniquely utilize auxotrophy in transformant selection: auxotrophic complementation by an auxotrophic marker gene on exogenous DNA (such as plasmids). However, the number of required auxotrophic nutrients restricts the number of plasmids maintained by the cells. We, therefore, developed novel Δ10 strains that are auxotrophic for 10 different nutrients and new plasmids with two multiple cloning sites and auxotrophic markers for use in Δ10 strains. We confirmed that Δ10 strains were able to maintain 10 types of plasmids. Using plasmids encoding model proteins, we detected the co-expression of 17 different genes in Δ10 cell lines. We also constructed Δ9 strains that exhibited auxotrophy for nine nutrients and increased growth compared to Δ10. This study opens a new avenue for the co-expression of a large number of genes in eukaryotic cells.

Tyrosinases (TYRs) are type-3 copper proteins that are widely distributed in nature. They can hydroxylate and oxidize phenolic molecules and are mostly known for producing melanins that confer protection against photo induced damage. TYRs are also thought to play an important role in the ‘latch mechanism’, where high concentrations of phenolic compounds inhibit oxidative decomposition of organic biomass and subsequent CO₂ release, especially relevant in wetland environments. In the present study, we describe two TYRs, HcTyr1 and HcTyr2, from halophilic bacterium Hahella sp. CCB MM4 previously isolated at Matang mangrove forest in Perak, Malaysia. The structure of HcTyr1 was determined by X-ray crystallography at a resolution of 1.9 Å and represents an uncharacterized group of prokaryotic TYRs as demonstrated by a sequence similarity network analysis. The genes encoding the enzymes were cloned, expressed, purified and thoroughly characterized by biochemical methods. HcTyr1 was able to self-cleave its lid-domain (LID) in a protease independent manner, whereas the LID of HcTyr2 was essential for activity and stability. Both enzymes showed variable activity in the presence of different metals, surfactants and NaCl, and were able to oxidize lignin constituents. The high salinity tolerance of HcTyr1 indicates that the enzyme can be an efficient catalyst in the habitat of the host.

The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.

Mushrooms are the fruiting bodies of fungi and are important reproductive structures that produce and disseminate spores. The Pri3 gene was originally reported to be specifically expressed in the primordia (a precursor to the mature fruiting body) of the edible mushroom Cyclocybe aegerita. Here, we cloned a Pri3-related cDNA from Cyclocybe cylindracea, another species in the same genus, and showed that the gene is specifically expressed at the pileus surface of the immature fruiting body but not in the primordia. Immunohistochemistry showed that the translated protein is secreted into a polysaccharide layer of the pileus surface. The recombinant C-terminal Cys-rich domain of the protein showed antifungal activity against three filamentous fungi and inhibited hyphal growth and conidiogenesis. These results suggest that the PRI3-related protein of C. cylindracea, named cylindracin, plays an important role in the defense against pathogens.

The domain-swapping mechanism involves the exchange of structural elements within a secondary or supersecondary structure between two (or more) proteins. The present paper proposes to interpret the domain-swapping mechanism using a model that assesses the structure of proteins (and complexes) based on building the structure of a common hydrophobic core in a micelle-like arrangement (a central hydrophobic core with a polar shell in contact with polar water), which has a considerable impact on the stabilisation of the domain structure built by domain swapping. Domains with a hydrophobicity system that is incompatible with the micelle-like structure have also been identified. This incompatibility is the form of structural codes related to biological function.

Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of proteins, which are synthesized in the cytosol and imported into mitochondria via different targeting routes. In addition, a small number of proteins are encoded by the organellar genome and synthesized by mitochondrial ribosomes. In this ‘In the Limelight’ special issue of FEBS Open Bio, five review articles describe these different biogenesis routes of mitochondrial proteins and provide a comprehensive overview of the structures and mechanisms by which mitochondrial proteins are synthesized and transported to their respective location within the organelle. These reviews, written by leading experts, provide a general overview, but also highlight current developments in the field of mitochondrial biogenesis.

The activity of Hippo signaling is commonly dysregulated in various human malignancies, including hepatocellular carcinoma (HCC). YAP, the key effector of Hippo pathway, is regulated through several posttranslational modifications. However, the mechanism by which YAP is regulated by arginine methylation remains unknown. In this study, immunoprecipitation and mass spectrometry were used to identify the arginine methylation site of YAP in HCC cells. The transcriptional activity of YAP and TEAD were further characterized by real-time qPCR and immunofluorescence assay, and a subcutaneous and orthotopic tumor mouse model was used to assess the effect of PRMT1-knockdown on HCC tumor growth. The result of mass spectrometry analysis identified that YAP was methylated at arginine 124. Moreover, we found that arginine methyltransferase PRMT1 interacted with YAP to mediate its arginine methylation, thus inhibited YAP phosphorylation and promoted YAP activity in the nucleus. PRMT1 was up-regulated in HCC tissues and positively associated with the expressions of YAP target genes. Silencing PRMT1 in HCC cells inhibited cell proliferation and tumor growth, while PRMT1-overexpression promoted HCC growth through YAP methylation. Our study reveals that PRMT1-mediated arginine methylation at R124 is mutually exclusive with YAP S127 phosphorylation, thereby facilitating YAP activity in the nucleus and promoting tumorigenesis in HCC.