Biochemistry Biochemistry最新文献

Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha. We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP₇ and 1/3,5-InsP₈, highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta. Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha. Collectively, this study provides insights into the regulation of specific PP-InsP messengers by DDP1-type phosphohydrolases in land plants.

Synaptotagmin 7 (SYT7), a member of the synaptotagmin family, exhibits high expression in various tumors and is closely associated with patient prognosis. The tight regulation of SYT7 expression assumes paramount significance in the progression of tumorigenesis. In this study, we detected a high GC content in the first 1000 bp of the promoter region of SYT7, suggesting a potential role of the G-quadruplex in its transcriptional regulation. Circular dichroism spectroscopy results showed that -187 to -172 bp sequence can form a typical parallel G-quadruplex structure, and site mutation revealed the critical role of the ninth guanine in its formation. Then, treatment of two ligands of G-quadruplex (TMPyP4 and Pyridostatin) reduced both the expression of SYT7 and subsequent tumor proliferation, demonstrating the potential of the G-quadruplex as a targeted therapy for tumors. By shedding light on the pivotal role of the G-quadruplex in regulating SYT7 transcription, our study not only advances our comprehension of this intricate regulatory mechanism but also emphasizes the significance of SYT7 in tumor proliferation. These findings collectively contribute to a more comprehensive understanding of the interplay between G-quadruplex regulation and SYT7 function in tumor development.

The mechanism by which small proteins fold, i.e., via intermediates or via a two-state mechanism, is a subject of intense investigation. Intermediate states in the folding pathways of these proteins are sparsely populated due to transient lifetimes under normal conditions rendering them transparent to a majority of the biophysical methods employed for structural, thermodynamic, and kinetic characterization, which attributes are essential for understanding the cooperative folding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled the characterization of folding intermediates of ubiquitin that exist in equilibrium under conditions of low pH and denaturants. At low pH, an unlocked state defined as N' is in fast exchange with an invisible state, U″, as observed by CEST NMR. Addition of urea to ubiquitin at pH 2 creates two new states F' and U', which are in slow exchange (k_F'→U' = 0.14 and k_U'→F' = 0.28 s^-1) as indicated by longitudinal ZZ-magnetization exchange spectroscopy. High-resolution solution NMR structures of F' show it to be in an "unlocked" conformation with measurable changes in rotational diffusion, translational diffusion, and rotational correlational times. U' is characterized by the presence of just the highly conserved N-terminal β1-β2 hairpin. The folding of ubiquitin is cooperative and is nucleated by the formation of an N-terminal β-hairpin followed by significant hydrophobic collapse of the protein core resulting in the formation of bulk of the secondary structural elements stabilized by extensive tertiary contacts. U' and F' may thus be described as early and late folding intermediates in the ubiquitin folding pathway.

Hematopoietic cell kinase (Hck) is a member of the Src kinase family and is a promising drug target in myeloid leukemias. Here, we report the crystal structure of human Hck in complex with the pyrrolopyrimidine inhibitor A-419259, determined at a resolution of 1.8 Å. This structure reveals the complete Hck active site in the presence of A-419259, including the αC-helix, the DFG motif, and the activation loop. A-419259 binds at the ATP-site of Hck and induces an overall closed conformation of the kinase with the regulatory SH3 and SH2 domains bound intramolecularly to their respective internal ligands. A-419259 stabilizes the DFG-in/αC-helix-out conformation observed previously with Hck and the pyrazolopyrimidine inhibitor PP1 (PDB: 1QCF). However, the activation loop conformations are distinct, with PP1 inducing a folded loop structure with the tyrosine autophosphorylation site (Tyr416) pointing into the ATP binding site, while A-419259 stabilizes an extended loop conformation with Tyr416 facing out into the solvent. Autophosphorylation also induces activation loop extension and significantly reduces the Hck sensitivity to PP1 but not A-419259. In cancer cells where Hck is constitutively active, the extended autophosphorylation loop may render Hck more sensitive to inhibitors like A-419259 which prefer this kinase conformation. More generally, these results provide additional insight into targeted kinase inhibitor design and how conformational preferences of inhibitors may impact selectivity and potency.

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may "dampen" the expression of genes for methionine import but likely does not turn them "OFF".

The human genome is tightly packed into the 3D environment of the cell nucleus. Rapidly evolving and sophisticated methods of mapping 3D genome architecture have shed light on fundamental principles of genome organization and gene regulation. The genome is physically organized on different scales, from individual genes to entire chromosomes. Nuclear landmarks such as the nuclear envelope and nucleoli have important roles in compartmentalizing the genome within the nucleus. Genome activity (for example, gene transcription) is also functionally partitioned within this 3D organization. Rather than being static, the 3D organization of the genome is tightly regulated over various time scales. These dynamic changes in genome structure over time represent the fourth dimension of the genome. Innovative methods have been used to map the dynamic regulation of genome structure during important cellular processes including organism development, responses to stimuli, cell division and senescence. Furthermore, disruptions to the 4D genome have been linked to various diseases, including of the kidney. As tools and approaches to studying the 4D genome become more readily available, future studies that apply these methods to study kidney biology will provide insights into kidney function in health and disease.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Human isocitrate dehydrogenase 1 (IDH1) is an enzyme that is found in humans that plays a critical role in aerobic metabolism. As a part of the citric acid cycle, IDH1 becomes responsible for catalyzing the oxidative decarboxylation of isocitrate to form α-ketoglutarate (αKG), with nicotinamide adenine dinucleotide phosphate (NADP⁺) as a cofactor. Strikingly, mutations of the IDH1 enzyme have been discovered in several cancers including glioblastoma multiforme (GBM), a highly aggressive form of brain cancer. It has been experimentally determined that single-residue IDH1 mutations occur at a very high frequency in GBM. Specifically, the IDH1 R132H mutation is known to produce (D)2-hydroxyglutarate (2HG), a recognized oncometabolite. Using the previously determined catalytic mechanism of IDH1, a DFT QM model was developed to study the mechanistic properties of IDH1 R132H compared to wild type enzyme. Validating these insights, biochemical in vitro assays of metabolites produced by mutant vs wild type enzymes were measured and compared. From the results discussed herein, we discuss the mechanistic impact of mutations in IDH1 on its ability to catalyze the formation of αKG and 2HG.