Biochemical Journal最新文献

There is an increasing interest in the role of fructose as a major driver of non-alcoholic fatty liver disease (NAFLD), and it is linked closely with the intake of sugar. However, there has also been the recognition that fructose can be produced directly from intracellular glucose via the evolutionarily conserved polyol pathway whose access is governed by aldose reductase (AR). The purpose of this article is to review the biochemistry of AR and the role of the polyol pathway in opening fructose metabolism. This article provides a new perspective about AR and the other key enzymes surrounding the decision to divert glucose into the polyol pathway which suggests that the production of endogenous fructose may be of much greater significance than historically viewed. There are important aspects of the regulation of the polyol pathway and its committal step catalyzed by AR, which supports the notion that fructose-uric acid pathway is activated by elevated glucose with the downstream consequence of NAFLD and perhaps other chronic metabolic diseases.

Three-prime repair exonuclease 1 (TREX1) is a 3'-5' exonuclease that plays an important role in clearing cytoplasmic DNA. Additionally, TREX1 is translocated to the nucleus after DNA damage and assists in DNA repair. In this work, we evaluated the activity of TREX1 in the context of the removal of methyl DNA adducts. We observed that TREX1 was less efficient at degrading methyl methanesulfonate (MMS)-treated methylated DNA compared with normal DNA. Two methyl DNA adducts, N1-methyladenine and N3-methylcytosine, were found to block TREX1 exonuclease activity. To understand the mechanism of limited TREX1-mediated degradation of MMS-damaged DNA, stem-loop substrates containing solitary methyl adducts were prepared. We found that when the solitary methyl adducts were present at the 3'-terminal single-stranded overhang, it prevented degradation by TREX1. However, TREX1 could efficiently process internally located duplex DNA methyl adducts when the 3'-terminal of the scissile strand was damage-free. Broadly, these observations suggest that TREX1 may be capable of resecting methyl adducts containing DNA, but it might be less proficient of removing 3'-terminal DNA methyl adducts.

The concept of higher-order assembly signalling or signalling by co-operative assembly formation (SCAF) was proposed based on the structures of signalling assemblies formed by proteins featuring domains from the death-fold family and the Toll/interleukin-1 receptor domain family. Because these domains form filamentous assemblies upon stimulation and activate downstream pathways through induced proximity, they were envisioned to sharpen response thresholds through the extreme co-operativity of higher-order assembly. Recent findings demonstrate that a central feature of the SCAF mechanism is the nucleation barrier that allows a switch-like, digital or 'all-or-none' response to minute stimuli. In agreement, this signalling mechanism features in cell-death and innate immunity activation pathways where a binary decision is required. Here, we broaden the concept of SCAF to encapsulate the essential kinetic properties of open-ended assembly in signalling, compare properties of filamentous assemblies and other co-operative assemblies such as biomolecular condensates, and review how this concept operates in cells.

Family 1 Coenzyme A transferases (CtfAB) from the extremely thermophilic bacterium, Thermosipho melanesiensis, has been used for in vivo acetone production up to 70°C. This enzyme has tentatively been identified as the rate-limiting step, due to its relatively low-binding affinity for acetate. However, existing kinetic and mechanistic studies on this enzyme are insufficient to evaluate this hypothesis. Here, kinetic analysis of purified recombinant T. melanesiensis CtfAB showed that it has a ping-pong bi-bi mechanism typical of Coenzyme A (CoA) transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 μM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wild-type and mutant T. melanesiensis CtfAB were solved in the presence of acetate and in the presence or absence of acetyl-CoA. These structures led to a proposed structural basis for the competitive and non-competitive inhibition of acetyl-CoA: acetate binds independently of acetyl-CoA in an apparent low-affinity binding pocket in CtfA that is directly adjacent to a catalytic glutamate in CtfB. Similar to other CoA transferases, acetyl-CoA is bound in an apparent high-affinity binding site in CtfB with most interactions occurring between the phospho-ADP of CoA and CtfB residues far from the acetate binding pocket. This structural-based mechanism also explains the organic acid promiscuity of CtfAB. High-affinity interactions are predominantly between the conserved phospho-ADP of CoA, and the variable organic acid binding site is a low-affinity binding site with few specific interactions.

Both mouse double-minute 2 (MDM2), an E3 ubiquitin ligase, and its closely related paralog, MDM4, which lacks E3 activity, play central roles in cellular homeostasis. MDM-linked dysfunction is associated with an increased risk of oncogenesis, primarily through targeting the tumor suppressor protein p53 for ubiquitination and degradation. Recent studies have revealed multifaceted roles of MDM proteins that are p53 independent with implications for their oncogenic properties. This review aims to provide an overview of MDM2 and MDM4, by assessing gene and protein structure and implications for protein-protein interactions and functions in cell and animal physiology. We also explore MDM2 and MDM4 role(s) in angiogenesis, a critical feature of solid tumor growth and progression. Finally, we discuss the current landscape in the development of MDM2 and MDM4 inhibitors for cancer therapy.

The maturation of the RNA cap involving guanosine N-7 methylation, catalyzsed by the HsRNMT (RNA guanine-7 methyltransferase (HsRNMT)-RAM (RNA guanine-N7 methyltransferase activating subunit (RAM) complex, is currently under investigation as a novel strategy to combat PIK3CA -mutant breast cancer. However, the development of effective drugs is hindered by a limited understanding of the enzyme's mechanism and a lack of small molecule inhibitors. Following the elucidation of the HsRNMT-RAM molecular mechanism, we report the biophysical characterizsation of two small molecule hits. Biophysics, biochemistry and structural biology confirm that both compounds bind competitively with cap and bind effectively to HsRNMT-RAM in the presence of the co-product SAH, with a binding affinity (KD) of approximately 1 μM. This stabilisation of the enzyme--product complex results in uncompetitive inhibition. Finally, we describe the properties of the cap pocket and provided suggestions for further development of the tool compounds.

The sodium phosphate cotransporter-2A (NPT2A) mediates basal and parathyroid hormone (PTH)- and fibroblast growth factor-23 (FGF23)-regulated phosphate transport in proximal tubule cells of the kidney. Both basal and hormone-sensitive transport require sodium hydrogen exchanger regulatory factor-1 (NHERF1), a scaffold protein with tandem PDZ domains, PDZ1 and PDZ2. NPT2A binds to PDZ1. RGS14 persistently represses hormone action by binding to PDZ2. The RGS14 canonical RGS domain, Ras/Rap-binding domains, and G protein regulatory motif cannot explain its regulatory effects on hormone-sensitive phosphate transport because these actions are mediated not only by the PTH receptor, a G protein-coupled receptor (GPCR), but also by the fibroblast growth factor receptor-1, a receptor tyrosine kinase that is not governed by G protein activity. Here, we identify the structural elements of RGS14 that mutually control the action of PTH and FGF23. RGS14 truncation constructs lacking upstream sequence and the RGS domain were fully functional. Removing the linker sequence between the RGS and RBD1 domains abolished RGS14 action. Examination of the α-helical linker region suggested candidate serine residues that might facilitate regulatory activities. RGS14 Ser266 and Ser269 are phosphorylated in response to PTH and FGF23, and replacement of these residues by Ala eliminated the actions of RGS14 on hormone-sensitive phosphate transport. PTH and FGF23 stimulated the phosphorylation of a peptide construct harboring the sites of purported phosphorylation and full-length RGS14. Mutating Ser266Ala and Ser269Ala abolished phosphorylation. The results establish that RGS14 regulation of phosphate transport requires targeted phosphorylation within the linker and an intact PDZ ligand.

Telomeres, essential for maintaining genomic stability, are typically preserved through the action of telomerase, a ribonucleoprotein complex that synthesizes telomeric DNA. One of its two core components, telomerase RNA (TR), serves as the template for this synthesis, and its evolution across different species is both complex and diverse. This review discusses recent advancements in understanding TR evolution, with a focus on plants (Viridiplantae). Utilizing novel bioinformatic tools and accumulating genomic and transcriptomic data, combined with corresponding experimental validation, researchers have begun to unravel the intricate pathways of TR evolution and telomere maintenance mechanisms. Contrary to previous beliefs, a monophyletic origin of TR has been demonstrated first in land plants and subsequently across the broader phylogenetic megagroup Diaphoretickes. Conversely, the discovery of plant-type TRs in insects challenges assumptions about the monophyletic origin of TRs in animals, suggesting evolutionary innovations coinciding with arthropod divergence. The review also highlights key challenges in TR identification and provides examples of how these have been addressed. Overall, this work underscores the importance of expanding beyond model organisms to comprehend the full complexity of telomerase evolution, with potential applications in agriculture and biotechnology.

Translation is a highly regulated process that includes three steps: initiation, elongation, and termination. Tremendous efforts have been spent to study the regulation of each translation step. In the last two decades, researchers have begun to investigate translation by tracking it in its native and live intracellular environment with high spatiotemporal resolution. To achieve this goal, a handful of tagging tools have been developed that can distinguish nascent chains from previously synthesized mature proteins. In this review, we will focus on these tagging tools and describe their development, working mechanisms, and advantages and drawbacks in tracking translation in live mammalian cells and organisms. In the second part of the review, we will summarize novel discoveries in translation by a recently developed nascent polypeptide tracking technology using tandem epitope tag array tagging tools. The superior spatiotemporal resolution of this technology enables us to directly and continuously track nascent chains live and thus reveal preferred translation location and timing, as well as the kinetics of canonical and noncanonical translation, translation bursts, ribosome quality control, and nonsense-mediated mRNA decay. In the future, we expect more tagging tools to be developed that allow us to track other regulation processes of a protein, such as folding, modifications, and degradation. With the expanding tagging toolbox, there is potential that we can track a protein from translation to degradation to fully understand its regulation in a native live cell environment.

The Stenotrophomonas maltophilia L2 cephalosporinase is one of two beta-lactamases that afford S. maltophilia beta-lactam resistance. With the overuse of beta-lactams, selective pressures have contributed to the evolution of these proteins, generating proteins with an extended spectrum of activity. Variant L2 cephalosporinases have been detected, as has their distribution into two main clades (clades 1 and 2). Comprehensive analysis of six L2 variants, cloned into pET41a(+) and expressed in Escherichia coli BL21(DE3) cells, revealed that clade 1 variants exhibited higher ceftazidime resistance compared to clade 2. Notably, the Sm5341 L2 variant, carrying a Phe72Ile variation, displayed a significantly reduced resistance profile across all substrates tested, suggesting a key role of Phe72 in enzymatic activity. An Ile72Phe substitution in the pET41a(+) based Sm5341_L2 variant resulted in a gain-of-function for this protein, confirming the role of Phe72 in the activity of L2. Furthermore, residue interaction network analysis elucidated a pi-cation interaction between Tyr272 and Arg244, which may potentially be stabilizing the enzyme and its binding site. The presence of Tyr272 in clade 1 variants correlates with higher ceftazidime affinity, contrasting Asp272 in clade 2 variants. Displaying lower Km values and higher kcat/Km ratios, clade 1 L2 enzymes demonstrated a higher binding efficiency and greater catalytic efficiency for most of the substrates assessed. These results indicate that L2 enzymes are continuing to evolve and adapt to a selective environment fuelled by the overuse of beta-lactams. This adaptation may signal the beginning of an evolutionary process yielding variant L2 cephalosporinases with extended substrate profiles.