Biochemistry Biochemistry最新文献

The capacity to effectively adapt metabolism to environmental demands is crucial for cell viability, proliferation, and function. Recent discoveries in Cell Research have highlighted the role of the lysosomal pool of AMPK in promoting glutaminolysis during glucose shortage through the activation of a PDZD8-GLS1 axis.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1–C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1–C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.

Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues ₅₅₅DEE₅₅₇ (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2^ΔDEE and CNM-linked DNM2^A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2^WT). However, DNM2^A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2^WT or DNM2^ΔDEE, suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

Hematological disorders result in significant health consequences, and traditional therapies frequently entail adverse reactions without addressing the root cause. A potential solution for hematological disorders characterized by gain-of-function mutations lies in the emergence of small interfering RNA (siRNA) molecules as a therapeutic option. siRNAs are a class of RNA molecules composed of double-stranded RNAs that can degrade specific mRNAs, thereby inhibiting the synthesis of underlying disease proteins. Therapeutic interventions utilizing siRNA can be tailored to selectively target genes implicated in diverse hematological disorders, including sickle cell anemia, β-thalassemia, and malignancies such as lymphoma, myeloma, and leukemia. The development of efficient siRNA silencers necessitates meticulous contemplation of variables such as the RNA backbone, stability, and specificity. Transportation of siRNA to specific cells poses a significant hurdle, prompting investigations of diverse delivery approaches, including chemically modified forms of siRNA and nanoparticle formulations with various biocompatible carriers. This review delves into the crucial role of siRNA technology in targeting and treating hematological malignancies and disorders. It sheds light on the latest research, development, and clinical trials, detailing how various pharmaceutical approaches leverage siRNA against blood disorders, mainly concentrating on cancers. It outlines the preferred molecular targets and physiological barriers to delivery while emphasizing the growing potential of various therapeutic delivery methods. The need for further research is articulated in the context of overcoming the shortcomings of siRNA in order to enrich discussions around siRNA's role in managing blood disorders and aiding the scientific community in advancing more targeted and effective treatments.

KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.