Bioscience Reports最新文献

Targeting AXL receptor kinase with a highly selective antibody presents a promising approach for inhibiting AXL and potentially improving cancer treatment. An essential step in antibody optimisation is the mapping of paratope residues to epitope residues. In the present study, we identify the residues of tilvestamab, a function-blocking anti-AXL monoclonal antibody, that are essential for its binding to the extracellular domain of AXL. A single-chain variable fragment (scFv) fused to osmotically inducible protein Y (osmY) was designed to enable the secretion of soluble scFv-osmY mutants, which could be directly subjected to high-throughput biolayer interferometry screening for binding to the AXL Ig1 domain. Each complementarity-determining region residue of scFv was mutated to Ala, while additional mutations were made on the basis of predicted contribution to binding. We generated AlphaFold3 predictions for the scFv (tilvestamab)-AXL Ig1 complex to gain insights into the molecular interactions of the essential residues, as determined by the experimental data. Our study reveals that tilvestamab binds to the Ig1 domain of AXL, with twelve residues on scFv (tilvestamab) contributing most to binding, likely being situated at the binding interface. Glu2 near the N-terminus of AXL is essential for binding. The data give a structural view into the AXL-tilvestamab complex and allow for further optimisation of the binding interface.

Brain-expressed voltage-gated sodium (Nav) and potassium (Kv) channels are essential for maintaining the balance of neuronal excitability, each having opposing effects on membrane potential and neuronal firing. Genetic alterations in these channels can disrupt this balance, leading to epilepsy and/or developmental impairments through gain-of-function (GoF) or loss-of-function (LoF) mechanisms. This review catalogs 48 transgenic mouse models involving sodium channels (SCN1A, SCN2A, SCN3A, SCN8A) and potassium channels (KCNQ2, KCNQ3, KCNT1, KCNA1, KCNB1, KCND2), detailing the effects of genetic alterations in terms of channel function, affected cell types, and phenotypic manifestations. Mechanistic insights from these models reveal that initial channel dysfunction triggers cascading pathological processes including glutamate excitotoxicity, oxidative stress, gliosis, neuroinflammation, and blood-brain barrier disruption. Therapeutic approaches include antisense oligonucleotides to enhance functional allele expression or reduce pathogenic channel expression, viral-mediated gene therapy, gene editing, and small molecule modulators that target persistent sodium currents or that stabilize channel inactivation. The timing of intervention appears to be critical, with early treatment showing greater efficacy in preventing pathological cascades. Strain-specific background effects and compensatory ion channel expression affect phenotypic severity and treatment response, complicating translation of model results. Importantly, transgenic models offer opportunities to better understand mechanisms underlying comorbidities commonly suffered by patients, including behavioral disorders, motor impairments, and sleep disturbances. The integration of these findings suggests that effective treatment strategies may require combinations of channel-directed therapies and interventions targeting downstream pathological processes, particularly for established disease. This comprehensive examination of channelopathy models provides a framework for developing transformative therapeutics for genetic epilepsies.

Ever since its discovery more than 70 years ago, the enzyme polynucleotide phosphorylase (PNPase) has been the subject of intensive research that has highlighted its key functional roles. The enzyme was first described in 1955 for its ability to synthesise RNA from nucleoside diphosphates. This discovery led to a Nobel Prize in Physiology or Medicine in 1959 for using PNPase to synthesise artificial RNA. However, it soon became evident that the primary function of this enzyme, conserved across diverse species, is 3'-5' RNA phosphorolysis rather than polymerisation. Remarkably, over 60 years later, it was discovered that PNPase has an even broader range of functions as it was shown to act as a conditional RNA chaperone in bacteria. In humans, PNPase (hPNPase) is located in mitochondria, where it plays a role in mitochondrial RNA (mtRNA) metabolism, thereby regulating mitochondrial function and the overall cell fitness. In this review, we present the current scope of knowledge of hPNPase, including its structure, subcellular localisation, metabolic activity, roles in mtRNA transport, processing and degradation, and its involvement in apoptosis.

Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries. Early detection remains challenging, with no established plasma-based biomarkers for clinical use. This study aimed to evaluate plasma adipokines and their receptor expression as diagnostic biomarkers for EC. Plasma levels of leptin, soluble leptin receptor, visfatin and asprosin were quantified in EC and control patients using ELISA. The free leptin index (FLI) was calculated as a ratio of leptin to soluble leptin receptor. Gene expression of corresponding receptors, including leptin receptor (Ob-R), insulin receptor (INSR), glucagon-like peptide-1 receptor [GLP-1 receptor (GLP-1R)], and asprosin-associated receptors, toll-like receptor 4 (TLR4), protein tyrosine phosphatase receptor type D (PTPRD), and olfactory receptor family 4 subfamily M member 1, was assessed by RT-qPCR from total blood. Plasma leptin levels were significantly elevated in EC patients, with the FLI over four times higher than controls (P=0.008). Soluble leptin receptor levels trended lower in EC, though non-significantly. Visfatin and asprosin plasma levels showed non-significant elevations. Gene expression analyses revealed significantly increased levels of GLP-1R, TLR4 and PTPRD in EC patients, suggestive of a diagnostic potential. Notably, plasma biomarker levels were not independently correlated with body mass index (BMI). Elevated FLI and up-regulation of adipokine receptor expression highlight the potential of combining plasma-based and molecular biomarkers for EC diagnosis. However, the lack of independence from BMI and conflicting literature underscores the need for larger, standardised studies to validate these findings and determine clinical applicability.

Diaminopimelate decarboxylase (DAPDC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the decarboxylation of diaminopimelate (DAP) to yield L-lysine, a key step in lysine biosynthesis. This present study presents a preliminary characterization of DAPDC encoded by the cce1351 gene in Cyanothece sp. ATCC 51142 (CsDAPDC), focusing on its biochemical properties and model structure characteristics. The enzyme exhibited a peak activity at 30°C and pH 8.0, and the catalytic constant (kcat) and substrate binding affinity Michaelis constant (KM) were determined as 1.68 s-1 and 1.20 mM at the above-mentioned condition, respectively. Homology modeling and molecular docking analysis revealed that Gly286, Gly330, Tyr428, and Asp118 interacted with the PLP cofactor, and Ser249, Tyr372, and Tyr428 interacted with the DAP substrate. Additionally, Cys399, Glu400, and Tyr436 from the other monomer were also involved in binding DAP and PLP. Site-directed mutagenesis confirmed the functional roles of these key residues in catalysis. This work provides valuable insights into the catalytic mechanism of CsDAPDC and highlights the enzyme's potential for applications in metabolic engineering of cyanobacteria for enhanced lysine production.

The process of selecting indigenous Saccharomyces cerevisiae strains as a starter culture for specific fermentation has several requisites, including the assessment of distinct parameters based on desirable and traditional enological criteria. In most wineries, commercial S. cerevisiae strains are used for wine fermentation. However, it is rare to find indigenous S. cerevisiae strains used in wine production, even though these isolates are better adapted to specific regions and are often preferred for producing local fruit wines. Here, the identification and characterization of indigenous S. cerevisiae were carried out by 28S rRNA sequencing, followed by Fourier transform infrared spectroscopy analysis for further confirmation. The strain improvement technique of genome shuffling was incorporated to ameliorate sugar tolerance and enhance alcohol production in the S. cerevisiae RHTD10 strain. As a result, it was observed that the improved strain from the third round of shuffling tolerated sugar stress of 30% and produced 10.14 ± 0.21% of alcohol, which is higher than the wild strain of 7.11 ± 0.22% alcohol.

Intracellular protein crystallization represents an intriguing form of biomolecular assembly. While the list of intracellularly crystallizing proteins is growing and their physiological roles are being elucidated, the underlying requirements and processes for intracellular crystallogenesis remain largely unknown. To reveal cellular capacity and morphological plasticity to accommodate protein crystals and crystal-like inclusion bodies, this study examines how simultaneously co-expressed phase-separating proteins influence each other's behavior in the endoplasmic reticulum (ER) lumen. To this end, four cargoes were selected based on their ability to produce distinctive inclusion body types and morphologies irrespective of originating species, function, or sequence homology. The co-expressed model proteins independently phase-separated into distinctive inclusions and coexisted in the ER without losing their signature morphologic characteristics. The continued growth of intra-ER protein crystals and droplets suggested that co-expressed cargo proteins were continuously synthesized and folded in the ER, thereby fueling the growth of the corresponding inclusion bodies. Thus, even in the crowded ER environment, each of the four overexpressed cargo proteins can find their mates through self-association and assemble into four unique structures in the ER. This study demonstrates that cells can accommodate up to four distinct types of mesoscale inclusion bodies in the ER lumen simultaneously, and the respective phase-separation events proceed without interfering with each other and without morphological mixing.

Phosphopantetheine adenylyltransferase (PPAT) (PPAT; EC 2.7.3.3) is a key enzyme in coenzyme A (CoA) biosynthesis. It catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), producing pyrophosphate and 3'-dephospho-CoA (dPCoA). Although the crystal structures of PPATs with various ligands have been studied, the specific contributions of residues to catalytic efficiency remain unclear. Here, we present the crystal structures of Helicobacter pylori PPAT (HpPPAT) in its apo form and complexes with Ppant and ATP. Additionally, we report the structure of the HpPPAT P8A mutant bound to dPCoA, providing the first complete occupancy structure of a PPAT complex across the hexamer. In the HpPPAT:ATP complex structure, critical active site residues Thr10, His18, Arg88, and Arg91, conserved in Escherichia coli PPAT (EcPPAT), are identified. HpPPAT utilizes Pro8, Lys42, and Arg133 for ATP binding. This differs from the binding pattern observed in other bacterial PPATs. Mutations of these residues, except for Thr10 and Lys42, resulted in a complete loss of enzymatic activity. This result highlights their critical roles. Mutating Thr10 and Lys42 to alanine reduced catalytic efficiency compared to WT HpPPAT but retained substantial activity. These residues are expected to orient the nucleophile for an in-line displacement mechanism. Based on structural studies and mutagenesis data with kinetic measurements and insights from other bacterial PPATs, we propose a refined catalytic mechanism for HpPPAT that emphasizes species-specific active-site interactions. This mechanism provides a foundation structure-based drugs H. pylori infections.