Biochemical Journal最新文献

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.

Plants are vital to human health and well-being, as well as helping to protect the environment against the negative impacts of climate change. They are an essential part of the 'One Health' strategy that seeks to balance and optimize the health of people, animals and the environment. Crucially, plants are central to nature-based solutions to climate mitigation, not least because soil carbon storage is an attractive strategy for mitigating greenhouse gas emissions and the associated climate change. Agriculture depends on genetically pure, high-quality seeds that are free from pests and pathogens and contain a required degree of genetic purity. This themed collection addresses key questions in the field encompassing the biochemical mechanisms that underlie plant responses and adaptations to a changing climate. This collection encompasses an analysis of the biochemistry and molecular mechanisms underpinning crop and forest resilience, together with considerations of plant adaptations to climate change-associated stresses, including drought, floods and heatwaves, and the increased threats posed by pathogens and pests.

MASH is a prevalent liver disease that can progress to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death, but there are no approved therapies. Leukotriene B4 (LTB4) is a potent pro-inflammatory chemoattractant that drives macrophage and neutrophil chemotaxis, and genetic loss or inhibition of its high affinity receptor, leukotriene B4 receptor 1 (BLT1), results in improved insulin sensitivity and decreased hepatic steatosis. To validate the therapeutic efficacy of BLT1 inhibition in an inflammatory and pro-fibrotic mouse model of MASH and fibrosis, mice were challenged with a choline-deficient, L-amino acid defined high fat diet and treated with a BLT1 antagonist at 30 or 90 mg/kg for 8 weeks. Liver function, histology, and gene expression were evaluated at the end of the study. Treatment with the BLT1 antagonist significantly reduced plasma lipids and liver steatosis but had no impact on liver injury biomarkers or histological endpoints such as inflammation, ballooning, or fibrosis compared to control. Artificial intelligence-powered digital pathology analysis revealed a significant reduction in steatosis co-localized fibrosis in livers treated with the BLT1 antagonist. Liver RNA-seq and pathway analyses revealed significant changes in fatty acid, arachidonic acid, and eicosanoid metabolic pathways with BLT1 antagonist treatment, however, these changes were not sufficient to impact inflammation and fibrosis endpoints. Targeting this LTB4-BLT1 axis with a small molecule inhibitor in animal models of chronic liver disease should be considered with caution, and additional studies are warranted to understand the mechanistic nuances of BLT1 inhibition in the context of MASH and liver fibrosis.

The exploitation of a cell's natural degradation machinery for therapeutic purposes is an exciting research area in its infancy with respect to bacteria. Here, we review current strategies targeting the ClpCP system, which is a proteolytic degradation complex essential in the biology of many bacterial species of scientific interest. Strategies include using natural product antibiotics or acyldepsipeptides to initiate the up- or down-regulation of ClpCP activity. We also examine exciting recent forays into BacPROTACs to trigger the degradation of specific proteins of interest through the hijacking of the ClpCP machinery. These strategies represent an important emerging avenue for combatting antimicrobial resistance.

Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.

Mitochondrial dysfunction in skeletal muscle fibres occurs with both healthy aging and a range of neuromuscular diseases. The impact of mitochondrial dysfunction in skeletal muscle and the way muscle fibres adapt to this dysfunction is important to understand disease mechanisms and to develop therapeutic interventions. Furthermore, interactions between mitochondrial dysfunction and skeletal muscle biology, in mitochondrial myopathy, likely have important implications for normal muscle function and physiology. In this review, we will try to give an overview of what is known to date about these interactions including metabolic remodelling, mitochondrial morphology, mitochondrial turnover, cellular processes and muscle cell structure and function. Each of these topics is at a different stage of understanding, with some being well researched and understood, and others in their infancy. Furthermore, some of what we know comes from disease models. Whilst some findings are confirmed in humans, where this is not yet the case, we must be cautious in interpreting findings in the context of human muscle and disease. Here, our goal is to discuss what is known, highlight what is unknown and give a perspective on the future direction of research in this area.

There is an unmet need to classify cancer-promoting kinase mutations in a mechanistically cognizant way. The challenge is to understand how mutations stabilize different kinase configurations to alter function, and how this influences pathogenic potential of the kinase and its responses to therapeutic inhibitors. This goal is made more challenging by the complexity of the mutational landscape of diseases, and is further compounded by the conformational plasticity of each variant where multiple conformations coexist. We focus here on the human MEK1 kinase, a vital component of the RAS/MAPK pathway in which mutations cause cancers and developmental disorders called RASopathies. We sought to explore how these mutations alter the human MEK1 kinase at atomic resolution by utilizing enhanced sampling simulations and free energy calculations. We computationally mapped the different conformational stabilities of individual mutated systems by delineating the free energy landscapes, and showed how this relates directly to experimentally quantified developmental transformation potentials of the mutations. We conclude that mutations leverage variations in the hydrogen bonding network associated with the conformational plasticity to progressively stabilize the active-like conformational state of the kinase while destabilizing the inactive-like state. The mutations alter residue-level internal molecular correlations by differentially prioritizing different conformational states, delineating the various modes of MEK1 activation reminiscent of a gear-shifting mechanism. We define the molecular basis of conversion of this kinase from its inactive to its active state, connecting structure, dynamics, and function by delineating the energy landscape and conformational plasticity, thus augmenting our understanding of MEK1 regulation.

As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed.

Although Microrchidia 2 (MORC2) is widely overexpressed in human malignancies and linked to cancer cell proliferation, metabolism, and metastasis, the mechanism of action of MORC2 in cancer cell migration and invasion is yet undeciphered. Here, we identified for the first time that MORC2, a chromatin remodeler, regulates E-cadherin expression and, subsequently regulates breast cancer cell migration and invasion. We observed a negative correlation between the expression levels of MORC2 and E-cadherin in breast cancer. Furthermore, the overexpression of MORC2 resulted in decreased expression levels of E-cadherin. In addition, co-immunoprecipitation and chromatin immunoprecipitation assays revealed that MORC2 interacts with HDAC1 and gets recruited onto the E-cadherin promoter to inhibit its transcription, thereby suppress its expression. Consequently, knockdown of HDAC1 in MORC2-overexpressing cells led to reduced cancer cell migration and invasion. Interestingly, we noticed that MORC2-regulated glucose metabolism via c-Myc, and LDHA, also modulates the expression of E-cadherin. Collectively, these results demonstrate for the first time a mechanistic role for MORC2 as an upstream regulator of E-cadherin expression and its associated functions in breast cancer.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.