FEBS Open Bio最新文献

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.

Aminoacyl-tRNA synthetases (AARSs) are fundamental enzymes that pair amino acids and tRNAs for protein synthesis. Aminoacylation occurs in two discrete steps. The amino acid is first activated by ATP, leading to an aminoacyl-adenylate intermediate and pyrophosphate (PP_i) formation. In a subsequent step, the aminoacyl moiety is transferred to the tRNA. Kinetic assays were developed to follow each of these steps independently, as well as cumulative two-step aminoacylation. The main advantage of following the activation step over two-step aminoacylation is that most AARSs can activate amino acids in the absence of the tRNA, the production of which is laborious. Hence, the activation step is often tested first in the kinetic analysis, including large screens exploring AARS-targeting inhibitors. Since the 1960s, the activation reaction has been routinely followed by the standard ATP/[³²P]PP_i exchange assay, which relies on the equilibrium exchange of radiolabel between PP_i and ATP using [³²P]PP_i as a labelled compound. However, this method became much less convenient when [³²P]PP_i was discontinued in 2022. As a solution, we developed a modified assay that uses easily attainable γ-[³²P]ATP as a labelled compound in the equilibrium-based assay. Using this assay, herein named the [³²P]ATP/PP_i assay, we followed the activation step of several AARSs. The obtained data are in good agreement with the previously published kinetic constants obtained with the standard ATP/[³²P]PP_i exchange assay.

β-barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β-barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β-barrel proteins. The core subunit Sam50, which has been conserved from bacteria to humans, is itself a β-barrel protein. The β-strands of β-barrel precursor proteins are assembled at the Sam50 lateral gate forming a Sam50-preprotein hybrid barrel. The assembled precursor β-barrel is finally released into the outer mitochondrial membrane by displacement of the nascent β-barrel, termed the β-barrel switching mechanism. SAM forms supercomplexes with TOM and forms a mitochondrial outer-to-inner membrane contact site with the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. SAM shares subunits with the ER-mitochondria encounter structure (ERMES), which forms a membrane contact site between the mitochondrial outer membrane and the endoplasmic reticulum. Therefore, β-barrel membrane protein biogenesis is closely connected to general mitochondrial protein and lipid biogenesis and plays a central role in mitochondrial maintenance.

The switch/sucrose non-fermenting (SWI/SNF) complex family includes important chromatin-remodeling factors that are frequently mutated in lung adenocarcinoma (LUAD). However, the role of one family member, SMARCA4, in LUAD prognosis and immunotherapy sensitivity remains unclear. In the present study, 6745 LUAD samples from the cBioPortal database were used to analyze the relationships between SMARCA4 mutations and patient prognoses and clinical characteristics. Additionally, we examined the correlation between SMARCA4 mutations and prognosis in patients treated with immunotherapy using two immune-related datasets. SMARCA4 mutations and low expression were associated with shorter survival, and mutations were associated with a high tumor mutational burden and high microsatellite instability. SMARCA4 mutations were accompanied by KRAS, KEAP1, TP53 and STK11 mutations. No significant difference was observed in the immunotherapy response between patients with and without SMARCA4 mutations. When KRAS or STK11 mutations were present, immunotherapy effectiveness was poorer; however, when both SMARCA4 and TP53 mutations were present, immunotherapy was more effective. Furthermore, low SMARCA4 expression predicted a higher immunophenoscore, and SMARCA4 expression was correlated with certain immune microenvironment features. Taken together, our results suggest that SMARCA4 mutations and low expression might be associated with poor LUAD prognosis. Additionally, immunotherapy efficacy in patients with SMARCA4 mutations depended on the co-mutant genes. Thus, SMARCA4 could be an important factor to be considered for LUAD diagnosis and treatment.

Over the past few decades, VEGF-targeted antiangiogenic therapy for cancers has gained increasing attention. Nevertheless, there are still several limitations such as the potential resistance mechanisms arising in cancer cells against these therapies and their potential adverse effects. These limitations highlight the need for novel anti-angiogenesis molecules and better understanding of the mechanisms of tumor angiogenesis. In the present study, we investigated the antiangiogenic properties of a novel 14-mer antiangiogenic peptide (14-MAP) derived from N-terminal 14 kDa buffalo prolactin and characterized its mode of action. 14-MAP at the picomolar concentration inhibited VEGF- and bradykinin (an autacoid peptide expressed in vascular tissues in pathophysiology, BK)-stimulated endothelial nitric oxide (eNO) production, cell migration, and proliferation in endothelial cells and vessel development in the chick embryo. Although this peptide inhibited both VEGF- and BK-dependent angiogenic processes, its action was more pronounced in the latter. Moreover, the interference of 14-MAP with the eNO synthase (eNOS)-cyclic GMP pathway was also identified. A combination of a low dose of Avastin, a widely used drug targeting VEGF-dependent angiogenesis, and 14-MAP significantly reduced tumor size in an in vivo model of human colon cancer. Taken together, our results suggest that 14-MAP, a BK- and eNOS-dependent antiangiogenic peptide, might be useful for overcoming the limitation of VEGF-targeted antiangiogenic therapy in cancer patients. However, further studies will be required to further characterize its mode of action and therapeutic potential.