Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

Structural insights into the specific recognition of mitochondrial ribosome-binding factor hsRBFA and 12 S rRNA by methyltransferase METTL15.

Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m4C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m4C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli. Here, we reported a series of crystal structures of human METTL15-hsRBFA-h44-SAM analog, METTL15-hsRBFA-SAM, METTL15-SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.

Imaging the Self-Healing Dynamics of Single-Nanoparticle Electron Transfer Event Regulated by Local Electron Insertion.

The self-healing properties of nanomaterials to resist electron beam damage are of great concern, which is inspiring to improve the stability and electron transfer efficiency of nanoelectronic devices especially in an abnormal environment. However, the influence of electron beam insertion on the electron transfer efficiency of single nanoentities at a heterogeneous electrochemical interface is still in debate, which is a concern for the development of in situ liquid cell transmission electron microscopy of the next generation. Herein, we employ an electro-optical imaging technique and directly visualize the controllable recovery of electron transfer ability for single Prussian blue nanoparticle (PBNP) after electron beam insertion with different electron doses. While eliminating e-beam damage by decreasing charge accumulation, the precise control of electron insertion behaviors induces a lossless chemical reduction mechanism for metal ions on the framework structure of PBNP, which leads to static imbalance and temporarily blocks the electron transfer channels. A subsequent charge rebalance process at a sub-nanoparticle level driven by electrochemical cycling controllably rebuilds the ion migration channels on the outer layer of single PBNP to repair the electron transfer path, which is confirmed by single-nanoparticle spectral characterizations. This work provides a generic methodology to study the electron-particle interplay and mechanism of electrode materials for eliminating the heterogeneity of electrochemical activity down to a sub-nanoparticle level.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

The recognition mode between hsRBFA and mitoribosome 12S rRNA during mitoribosomal biogenesis.

Eukaryotes contain two sets of genomes: the nuclear genome and the mitochondrial genome. The mitochondrial genome transcripts 13 mRNAs that encode 13 essential proteins for the oxidative phosphorylation complex, 2 rRNAs (12s rRNA and 16s rRNA), and 22 tRNAs. The proper assembly and maturation of the mitochondrial ribosome (mitoribosome) are critical for the translation of the 13 key proteins and the function of the mitochondrion. Human ribosome-binding factor A (hsRBFA) is a mitoribosome assembly factor that binds with helix 28, helix 44 and helix 45 of 12S rRNA and facilitates the transcriptional modification of 12S rRNA during the mitoribosomal biogenesis. Previous research mentioned that the malfunction of hsRBFA will induce the instability of mitoribosomes and affect the function of mitochondria, but the mechanisms underlying the interaction between hsRBFA and 12S rRNA and its influence on mitochondrial function are still unknown. In this study, we found that hsRBFA binds with double strain RNA (dsRNA) through its whole N-terminus (Nt) instead of the KH-like domain alone, which is different from the other homologous. Furthermore, we mapped the key residues that affected the RNA binding and maturation of mitoribosomes in vitro. Finally, we investigated how these residues affect mitochondrial functions in detail and systematically.

Structural insights into the recognition of telomeric variant repeat TTGGGG by broad-complex, tramtrack and bric-à-brac - zinc finger protein ZBTB10.

Multiple proteins bind to telomeric DNA and are important for the role of telomeres in genome stability. A recent study established a broad-complex, tramtrack and bric-à-brac - zinc finger (BTB-ZF) protein, ZBTB10 (zinc finger and BTB domain-containing protein 10), as a telomeric variant repeat-binding protein at telomeres that use an alternative method for lengthening telomeres). ZBTB10 specifically interacts with the double-stranded telomeric variant repeat sequence TTGGGG by employing its tandem C2H2 zinc fingers (ZF1-2). Here, we solved the crystal structure of human ZBTB10 ZF1-2 in complex with a double-stranded DNA duplex containing the sequence TTGGGG to assess the molecular details of this interaction. Combined with calorimetric analysis, we identified the vital residues in TTGGGG recognition and determined the specific recognition mechanisms that are different from those of TZAP (telomere zinc finger-associated protein), a recently defined telomeric DNA-binding protein. Following these studies, we further identified a single amino-acid mutant (Arg767Gln) of ZBTB10 ZF1-2 that shows a preference for the telomeric DNA repeat TTAGGG sequence. We solved the cocrystal structure, providing a structural basis for telomeric DNA recognition by C2H2 ZF proteins.

Structural basis for Gemin5 decamer-mediated mRNA binding.

Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.

Molecular mechanism underlying the di-uridylation activity of Arabidopsis TUTase URT1.

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5'-AAAU-3' RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3' extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an 'open' to a 'closed' state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.

Directly Imaging Dynamic Electronic Coupling during Electrochemical Oxidation of Single Silver Nanoparticles.

The electronic coupling between a metal electrode and single nano-entities is of unfading significance which impacts the heterogeneous electron transfer. Herein, we demonstrated a simple optical technique for directly imaging the transient interfacial electronic coupling events during electrochemical oxidation of single Ag nanoparticles on Au electrode. The electronic coupling brings out a dramatic dip behavior of bright field imaging traces, and is conductive to cross the energy barrier of oxidation for single silver nanoparticles. This dip behavior is further verified by in situ vis-transmission spectroscopy, and the heterogeneity of the Au-Ag electronic coupling down to single-nanoparticle level is uncovered by unifying the morphology and size of individual silver nanoparticles. These results suggest the interfacial electronic coupling facilitates electron transfer of single nanoparticles, and provide important insight into understanding detailed mechanism of nanoelectrochemistry.

Structural insights reveal the specific recognition of meiRNA by the Mei2 protein.

In the fission yeast Schizosaccharomyces pombe, Mei2, an RNA-binding protein essential for entry into meiosis, regulates meiosis initiation. Mei2 binds to a specific non-coding RNA species, meiRNA, and accumulates at the sme2 gene locus, which encodes meiRNA. Previous research has shown that the Mei2 C-terminal RNA recognition motif (RRM3) physically interacts with the meiRNA 5' region in vitro and stimulates meiosis in vivo. However, the underlying mechanisms still remain elusive. We first employed an in vitro crosslinking and immunoprecipitation sequencing (CLIP-seq) assay and demonstrated a preference for U-rich motifs of meiRNA by Mei2 RRM3. We then solved the crystal structures of Mei2 RRM3 in the apo form and complex with an 8mer RNA fragment, derived from meiRNA, as detected by in vitro CLIP-seq. These results provide structural insights into the Mei2 RRM3-meiRNA complex and reveal that Mei2 RRM3 binds specifically to the UUC(U) sequence. Furthermore, a structure-based Mei2 mutation, Mei2F644A causes defective karyogamy, suggesting an essential role of the RNA-binding ability of Mei2 in regulating meiosis.

Analysis of World-Scale Mitochondrial DNA Reveals the Origin and Migration Route of East Asia Goats.

Despite much attention on the history of goat evolution, information on origin, demographic history, and expansion route remains controversial. To address these questions, we collected 4,189 published goat DNA sequences including 1,228 sequences from 57 breeds in China and 2,961 sequences including 193 goat breeds from 71 other countries and carried out an integrated analysis. We found goat breeds from South China had the highest genetic diversity of lineage B, and subclades B2 only were found in Southwest China, suggesting that lineage B (particularly, subclade B2) probably originated from Southwest China and its surrounding areas. In addition, in this study, we found that lineage A from South China also presented higher genetic diversity and earlier expansion time (10, 606 years ago), even earlier than breeds from the Middle East. Hence, we speculated that South China and surrounding areas were the origin of lineage B and also the transportation hub for lineage A spreading to North China and Southwest Asia. Furthermore, according to the analysis of correlation between genetic differentiation value λ1 and λ2 and geographical distance, we further confirmed two phases of migration in goat breeds of North China. These results will contribute to a better understanding of the origin and migration history of domestic goat.

Determining the depth of surface charging layer of single Prussian blue nanoparticles with pseudocapacitive behaviors.

Understanding the hybrid charge-storage mechanisms of pseudocapacitive nanomaterials holds promising keys to further improve the performance of energy storage devices. Based on the dependence of the light scattering intensity of single Prussian blue nanoparticles (PBNPs) on their oxidation state during sinusoidal potential modulation at varying frequencies, we present an electro-optical microscopic imaging approach to optically acquire the Faradaic electrochemical impedance spectroscopy (oEIS) of single PBNPs. Here we reveal typical pseudocapacitive behavior with hybrid charge-storage mechanisms depending on the modulation frequency. In the low-frequency range, the optical amplitude is inversely proportional to the square root of the frequency (∆I ∝ f-0.5; diffusion-limited process), while in the high-frequency range, it is inversely proportional to the frequency (∆I ∝ f-1; surface charging process). Because the geometry of single cuboid-shaped PBNPs can be precisely determined by scanning electron microscopy and atomic force microscopy, oEIS of single PBNPs allows the determination of the depth of the surface charging layer, revealing it to be ~2 unit cells regardless of the nanoparticle size.

Optical Imaging of the Molecular Mobility of Single Polystyrene Nanospheres.

An ultrathin surface layer with extraordinary molecular mobility has been discovered and intensively investigated on thin-film polymer materials for decades. However, because of the lack of suitable characterization techniques, it remains largely unexplored whether such a surface mobile layer also exists on individual polymeric nanospheres. Here, we propose a thermal-optical imaging technique to determine the glass transition (Tg) and rubber-fluid transition (Tf) temperatures of single isolated polystyrene nanospheres (PSNS) in a high-throughput and nonintrusive manner for the first time. Two distinct steps, corresponding to the glass transition and rubber-fluid transition, respectively, were clearly observed in the optical trace of single PSNS during temperature ramping. Because the transition temperature and size of the same individuals were both determined, single nanoparticle measurements revealed the reduced apparent Tf and increased Tg of single PSNS on the gold substrate with a decreasing radius from 130 to 70 nm. Further experiments revealed that the substrate effect played an important role in the increased Tg. More importantly, a gradual decrease in the optical signal was detected prior to the glass transition, which was consistent with a surface layer with enhanced molecular mobility. Quantitative analysis further revealed the thickness of this layer to be ∼8 nm. This work not only uncovered the existence and thickness of a surface mobile layer in single isolated nanospheres but also demonstrated a general bottom-up strategy to investigate the structure-property relationship of polymeric nanomaterials by correlating the thermal property (Tg and Tf) and structural features (size) at single nanoparticle level.

The design and synthesis of transient receptor potential vanilloid 3 inhibitors with novel skeleton.

Transient receptor potential vanilloid 3 (TRPV3) channel as a member of thermo TRPV subfamily is primarily expressed in the keratinocytes of the skin and sensory neurons, and plays critical roles in various physiological and pathological processes such as inflammation, pain sensation and skin disorders. However, TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Recently, we synthesized a series of cinnamate ester derivatives and evaluated their inhibitory activities on human TRPV3 channels expressed in HEK293 cells using whole-cell patch clamp recordings. And, we identified two potent TRPV3 antagonists 7c and 8c which IC50 values were 1.05 µM and 86 nM, respectively. It also showed good selectivity to other subfamily members of TRPV, such as TRPV1 and TRPV4.

Minimalist Design for a Hand-Held SARS-Cov-2 Sensor: Peptide-Induced Covalent Assembly of Hydrogel Enabling Facile Fiber-Optic Detection of a Virus Marker Protein.

The rampaging COVID-19 needs bioassaying methods of low cost and high robustness for those living in the poorly developed regions. Here, we propose such a method that does not need expensive and complicated equipment. Only a set of hand-held small devices is sufficient. A section along an optic fiber cable is stripped, so that laser light travelling through it will leak outside, while biosensing process taken place on this stripped section can form a new cladding layer of hydrogel, restoring the laser output of the fiber. A short peptide probe immobilized on the stripped section of the fiber can covalently capture a biomarker protein of SARS-Cov-2 from the serum sample. Through the cross-linking of the target protein with the interfering proteins in the serum sample, a hydrogel is covalently immobilized around the stripped section, highly resistant to detergent rinsing that is indispensable for removing nonspecific interference from the clinical sample. Using this "covalent biosensing" strategy, only one peptide probe is sufficient to simultaneously achieve ultrahigh affinity toward the biomarker protein of SARS-Cov-2 and effective signal amplification.

B-cell acute lymphoblastic leukemia-related microRNAs: uncovering their diverse and special roles.

B-cell acute lymphoblastic leukemia (B-ALL) is a common type of hematologic malignancy characterized by the uncontrolled growth of immature B lymphocytes. Genomics, transcriptomics, and proteomics at different levels contribute to early diagnosis and can thereby provide better treatment for cancer. MicroRNAs (miRNAs) are conducive to the diagnosis and treatment of patients with B-ALL. Moreover, evidence suggests that runaway miRNAs and exosomes containing miRNA may be involved in the occurrence of B-ALL, which can then be used as potential biomarkers. This review summarizes the role of miRNAs in the pathogenesis, diagnosis, prognosis, and treatment of B-ALL.

Multi-photon absorption organotin complex for bioimaging and promoting ROS generation.

Compared to general fluorescent dyes, multi-photon fluorescent dyes exhibit deeper tissue penetration and lower auto-fluorescence in the bio-imaging field. Therefore, it is necessary to develop an efficient multiphoton imaging agent for deep tissue imaging. In this work, an organotin derivative (HSnBu3) has been designed and synthesized, which shows multiphoton absorption activity. In constrast to the ignorable three-photon activity of the ligand, the complex (HSnBu3) exhibits three-photon activity under NIR excitation (1500 nm). Results of chemical and biological tests confirmed that HSnBu3 was more easily activated by oxygen resulting in a higher level of 1O2, which could induce a decrease in mitochondrial membrane potential in HepG2 cells. It suggests that HSnBu3 has potential in photodynamic therapy.

Structural and biochemical insights into the recognition of RNA helicase CGH-1 by CAR-1 in C. elegans.

A protein-RNA complex containing the RNA helicase CGH-1 and a germline specific RNA-binding protein CAR-1 is involved in various aspects of function in C. elegans. However, the structural basis for the assembly of this protein complex remains unclear. Here, we elucidate the molecular basis of the recognition of CGH-1 by CAR-1. Additionally, we found that the ATPase activity of CGH-1 is stimulated by NTL-1a MIF4G domain in vitro. Furthermore, we determined the structures of the two RecA-like domains of CGH-1 by X-ray crystallography at resolutions of 1.85 and 2.40 Å, respectively. Structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1. NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR-1, assessed by ITC and GST-pulldown in vitro. These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1. Together, our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C. elegans.

Conformational Selection in Ligand Recognition by the First Tudor Domain of PHF20L1.

The first Tudor domain (Tudor1) of PHF20L1 recognizes (non)histone methylation to play versatile roles. However, the underlying ligand-recognition mechanism remains unknown as a closed state revealed in the free-form structure. NMR relaxation dispersion and molecular dynamics simulations suggest a pre-existing low-population conformation with a remarkable rearrangement of aromatic cage residues of PHF20L1 Tudor1. Such an open-form conformation is utilized to recognize lysine 142 methylated DNMT1, a cosolvent, and an NMR fragment screening hit, as revealed by the complex crystal structures. Intriguingly, the ligand binding capacity was enhanced by mutation that tunes up the open-state population only. The recognition of DNMT1 by PHF20L1 was further validated in cancer cells. This conformational selection mechanism will enable the discovery of small molecule inhibitors against the seemingly "undruggable" PHF20L1 Tudor1.