Biochemical Journal最新文献

This study reveals striking differences in the content and composition of hydrophilic and lipophilic compounds in blackcurrant buds (Ribes nigrum L., cv. Ben Klibreck) resulting from winter chill or chemical dormancy release following treatment with ERGER, a biostimulant used to promote uniform bud break. Buds exposed to high winter chill exhibited widespread shifts in metabolite profiles relative to buds that experience winter chill by growth under plastic. Specifically, extensive chilling resulted in significant reductions in storage lipids and phospholipids, and increases in galactolipids relative to buds that experienced lower chill. Similarly, buds exposed to greater chill exhibited higher levels of many amino acids and dipeptides, and nucleotides and nucleotide phosphates than those exposed to lower chilling hours. Low chill buds (IN) subjected to ERGER treatment exhibited shifts in metabolite profiles similar to those resembling high chill buds that were evident as soon as 3 days after treatment. We hypothesise that chilling induces a metabolic shift which primes bud outgrowth by mobilising lipophilic energy reserves, enhancing phosphate availability by switching from membrane phospholipids to galactolipids and enhancing the availability of free amino acids for de novo protein synthesis by increasing protein turnover. Our results additionally suggest that ERGER acts at least in part by priming metabolism for bud outgrowth. Finally, the metabolic differences presented highlight the potential for developing biochemical markers for dormancy status providing an alternative to time-consuming forcing experiments.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

Pancreatic cancer is a malignancy arising from the endocrine or exocrine compartment of this organ. Tumors from exocrine origin comprise over 90% of all pancreatic cancers diagnosed. Of these, pancreatic ductal adenocarcinoma (PDAC) is the most common histological subtype. The five-year survival rate for PDAC ranged between 5 and 9% for over four decades, and only recently saw a modest increase to ∼12-13%, making this a severe and lethal disease. Like other cancers, PDAC initiation stems from genetic changes. However, therapeutic targeting of PDAC genetic drivers has remained relatively unsuccessful, thus the focus in recent years has expanded to the non-genetic factors underlying the disease pathogenesis. Specifically, it has been proposed that dynamic changes in the epigenetic landscape promote tumor growth and metastasis. Emphasis has been given to the re-organization of enhancers, essential regulatory elements controlling oncogenic gene expression, commonly marked my histone 3 lysine 4 monomethylation (H3K4me1). H3K4me1 is typically deposited by histone lysine methyltransferases (KMTs). While well characterized as oncogenes in other cancer types, recent work has expanded the role of KMTs as tumor suppressor in pancreatic cancer. Here, we review the role and translational significance for PDAC development and therapeutics of KMTs.

Temperature-dependent alternative splicing (AS) is a crucial mechanism for organisms to adapt to varying environmental temperatures. In mammals, even slight fluctuations in body temperature are sufficient to drive significant AS changes in a concerted manner. This dynamic regulation allows organisms to finely tune gene expression and protein isoform diversity in response to temperature cues, ensuring proper cellular function and physiological adaptation. Understanding the molecular mechanisms underlying temperature-dependent AS thus provides valuable insights into the intricate interplay between environmental stimuli and gene expression regulation. In this review, we provide an overview of recent advances in understanding temperature-regulated AS across various biological processes and systems. We will discuss the machinery sensing and translating temperature cues into changed AS patterns, the adaptation of the splicing regulatory machinery to extreme temperatures, the role of temperature-dependent AS in shaping the transcriptome, functional implications and the development of potential therapeutics targeting temperature-sensitive AS pathways.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Across eukaryotes, most genes required for mitochondrial function have been transferred to, or otherwise acquired by, the nucleus. Encoding genes in the nucleus has many advantages. So why do mitochondria retain any genes at all? Why does the set of mtDNA genes vary so much across different species? And how do species maintain functionality in the mtDNA genes they do retain? In this review, we will discuss some possible answers to these questions, attempting a broad perspective across eukaryotes. We hope to cover some interesting features which may be less familiar from the perspective of particular species, including the ubiquity of recombination outside bilaterian animals, encrypted chainmail-like mtDNA, single genes split over multiple mtDNA chromosomes, triparental inheritance, gene transfer by grafting, gain of mtDNA recombination factors, social networks of mitochondria, and the role of mtDNA dysfunction in feeding the world. We will discuss a unifying picture where organismal ecology and gene-specific features together influence whether organism X retains mtDNA gene Y, and where ecology and development together determine which strategies, importantly including recombination, are used to maintain the mtDNA genes that are retained.

Maintenance of genome stability is of paramount importance for the survival of an organism. However, genomic integrity is constantly being challenged by various endogenous and exogenous processes that damage DNA. Therefore, cells are heavily reliant on DNA repair pathways that have evolved to deal with every type of genotoxic insult that threatens to compromise genome stability. Notably, inherited mutations in genes encoding proteins involved in these protective pathways trigger the onset of disease that is driven by chromosome instability e.g. neurodevelopmental abnormalities, neurodegeneration, premature ageing, immunodeficiency and cancer development. The ability of cells to regulate the recruitment of specific DNA repair proteins to sites of DNA damage is extremely complex but is primarily mediated by protein post-translational modifications (PTMs). Ubiquitylation is one such PTM, which controls genome stability by regulating protein localisation, protein turnover, protein-protein interactions and intra-cellular signalling. Over the past two decades, numerous ubiquitin (Ub) E3 ligases have been identified to play a crucial role not only in the initiation of DNA replication and DNA damage repair but also in the efficient termination of these processes. In this review, we discuss our current understanding of how different Ub E3 ligases (RNF168, TRAIP, HUWE1, TRIP12, FANCL, BRCA1, RFWD3) function to regulate DNA repair and replication and the pathological consequences arising from inheriting deleterious mutations that compromise the Ub-dependent DNA damage response.

The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP.

Filamin A is an essential protein in the cell cytoskeleton because of its actin binding properties and unique homodimer rod-shaped structure, which organises actin into three-dimensional orthogonal networks imperative to cell motility, spreading and adhesion. Filamin A is subject to extensive posttranslational modification (PTM) which serves to co-ordinate cellular architecture and to modulate its large protein-protein interaction network which is key to the protein's role as a cellular signalling hub. Characterised PTMs include phosphorylation, irreversible cleavage, ubiquitin mediated degradation, hydroxylation and O-GlcNAcylation, with preliminary evidence of tyrosylation, carbonylation and acetylation. Each modification and its relation to filamin A function will be described here. These modifications are often aberrantly applied in a range of diseases including, but not limited to, cancer, cardiovascular disease and neurological disease and we discuss the concept of target specific PTMs with novel therapeutic modalities. In summary, our review represents a topical 'one-stop-shop' that enables understanding of filamin A function in cell homeostasis and provides insight into how a variety of modifications add an extra level of Filamin A control.