Biochemistry Biochemistry最新文献

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gα_i·GDP, Pins has been found to associate with both Drosophila Gα_i (dGα_i) and Gα_o (dGα_o) isoforms. Moreover, dGα_o was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of dGα_o on the conformational states of Pins remains unclear, despite extensive investigations into the Gα_i·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and dGα_o in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded dGα_o. Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of dGα_o·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between dGα_i and Pins-GLs, thus implying the pivotal role of dGα_o as the principal participant in the ACD pathway associated with Pins.

The use of next-generation sequencing technologies such as exome and genome sequencing in research and clinical care has transformed our understanding of the molecular architecture of genetic kidney diseases. Although the capability to identify and rigorously assess genetic variants and their relationship to disease has advanced considerably in the past decade, the curation of clinically relevant relationships between genes and specific phenotypes has received less attention, despite it underpinning accurate interpretation of genomic tests. Here, we discuss the need to accurately define gene–disease relationships in nephrology and provide a framework for appraising genetic and experimental evidence critically. We describe existing international programmes that provide expert curation of gene–disease relationships and discuss sources of discrepancy as well as efforts at harmonization. Further, we highlight the need for alignment of disease and phenotype terminology to ensure robust and reproducible curation of knowledge. These collective efforts to support evidence-based translation of genomic sequencing into practice across clinical, diagnostic and research settings are crucial for delivering the promise of precision medicine in nephrology, providing more patients with timely diagnoses, accurate prognostic information and access to targeted treatments.

An emerging NMR method, MA’AT analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The MA’AT model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2′-fucosyllactoside (Me2′FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2′,3-difucosyllactoside (Me2′,3DFL). MA’AT analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages. α-Fucosylation at both O2′Gal and O3Glc of MeL to give Me2′,3DFL significantly constrained librational motion about ψ (70% reduction in the CSD) and shifted its mean value by ∼18°. α-Fucosylation at the O3Glc of MeL to give Me3FL constrained ψ more than α-fucosylation at the O2Gal to give Me2′FL. These effects can be explained by the expected solution conformation of Me3FL, which closely resembles the Lewis^x trisaccharide. Comparisons of MA’AT models of ψ to those obtained by 1 μs aqueous molecular dynamics simulation (GLYCAM06) revealed identical trends, that is, MA’AT analysis was able to recapitulate molecular behavior in solution that was heretofore only available from MD simulation. The results highlight the capabilities of MA’AT analysis to determine probability distributions of molecular torsion angles in solution as well as degrees of librational averaging of these angles.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ¹-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. N-propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH. This compound consists of a recognition module (glycine) that directs the inactivator to the active site and an alkyne warhead that reacts with the FAD after oxidative activation, leading to covalent modification of the FAD N5 atom. Here we report structural and kinetic data on analogs of N-propargylglycine with the goals of understanding the initial docking step of the inactivation mechanism and to test the allyl group as a warhead. The crystal structures of PRODH complexed with unreactive analogs in which N is replaced by S show how the recognition module mimics the substrate proline by forming ion pairs with conserved arginine and lysine residues. Further, the C atom adjacent to the alkyne warhead is optimally positioned for hydride transfer to the FAD, providing the structural basis for the first bond-breaking step of the inactivation mechanism. The structures also suggest new strategies for designing improved N-propargylglycine analogs. N-allylglycine, which consists of a glycine recognition module and allyl warhead, is shown to be a covalent inactivator; however, it is less efficient than N-propargylglycine in both enzyme inactivation and cellular assays. Crystal structures of the N-allylglycine-inactivated enzyme are consistent with covalent modification of the N5 by propanal.

Congenital nephrogenic diabetes insipidus (NDI; also known as arginine vasopressin resistance) is a rare inherited disorder of water homeostasis, caused by insensitivity of the distal nephron to arginine vasopressin. Consequently, the kidney loses its ability to concentrate urine, which leads to polyuria, polydipsia and the risk of hypertonic dehydration. The diagnosis and management of NDI are very challenging and require an integrated, multidisciplinary approach. Here, we present 36 recommendations for diagnosis, treatment and follow-up in both children and adults, as well as emergency management, genetic counselling and family planning, for patients with NDI. These recommendations were formulated and graded by an international group of experts in NDI from paediatric and adult nephrology, urology and clinical genetics from the European Rare Kidney Disease Reference Network and the European Society of Paediatric Nephrology, as well as patient advocates, and were validated by a voting panel in a Delphi process. The goal of these recommendations is to provide guidance to health care professionals who care for patients with NDI and to patients and their families. In addition, we emphasize the need for further research on different aspects of this potentially life-threatening disorder to support the development of evidence-based guidelines in the future.

In a recent study in Cell, Lascaux et al. revealed a novel pathway to repair toxic DNA lesions, providing a direct link between nucleophagy, a type of selective autophagy, and the resolution of damaged DNA.

Some species of the Nocardia genus harbor a highly conserved biosynthetic gene cluster designated as the NOCardiosis-Associated Polyketide (NOCAP) synthase that produces a unique glycolipid natural product. The NOCAP glycolipid is composed of a fully substituted benzaldehyde headgroup linked to a polyfunctional alkyl tail and an O-linked disaccharide composed of 3-α-epimycarose and 2-O-methyl-α-rhamnose. Incorporation of the disaccharide unit is preceded by a critical step involving hydroxylation by NocapM, a flavin monooxygenase. In this study, we employed biochemical, spectroscopic, and kinetic analyses to explore the substrate scope of NocapM. Our findings indicate that NocapM catalyzes hydroxylation of diverse aromatic substrates, although the observed coupling between NADPH oxidation and substrate hydroxylation varies widely from substrate to substrate. Our in-depth biochemical characterization of NocapM provides a solid foundation for future mechanistic studies of this enzyme as well as its utilization as a practical biocatalyst.

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis. Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.