Journal of Molecular Biology最新文献

Nonsense-mediated mRNA decay (NMD) is one of the most extensively studied pathways of cytoplasmic mRNA degradation. It plays a critical role in diverse cellular processes by eliminating aberrant transcripts containing premature stop codons and by regulating the stability of physiological mRNAs. NMD factors were initially identified through genetic screens in S. cerevisiae (UPF1, 2, 3) and C. elegans (SMG-1, SMG5-7). Subsequent biochemical and genetic studies revealed the composition of NMD complexes and identified additional factors. A major protein hub for NMD is Upf1, an ATP-dependent RNA helicase that is part of two mutually exclusive NMD assemblies, the Upf1-Upf2-Upf3 complex and the Upf1-decapping complex, which contains the decapping enzyme and its co-factors. Here, we discuss recent findings, primarily from budding yeast, on the protein-protein interactions driving NMD complexes dynamics and their similarities to human NMD. Together, the N-terminal cysteine and histidine rich (CH) and helicase domains (HD) of Upf1 act as a hub for binding multiple partners. Upf1 is required for binding to NMD substrates and for the initiation of RNA degradation through decapping (yeast) or endonucleolytic hydrolysis (humans). We focus on the interplay between Upf2, Dcp2 and Nmd4 (yeast SMG6), which ensures the mutually exclusive formation of Upf1-bound subcomplexes modulating Upf1's affinity for RNA. Thus, the study of NMD factors interactions in different organisms sheds new light on the remarkable conservation of NMD molecular mechanisms.

Preparation of high-quality nucleosomal DNA substrates in milligram quantities remains a major bottleneck for mechanistic studies of chromatin-associated processes. Here, we present an optimized large-scale PCR workflow that enables rapid, low-cost production of diverse nucleosomal DNAs suitable for biochemical assays and high-resolution cryo-EM. Systematic optimization of amplification conditions yields milligram quantities of homogeneous DNA that can be fluorescently or biotin-labeled and enzymatically modified to introduce site-specific single-strand breaks (SSBs) or epigenetic marks. We also engineered an improved Nt.BsmAI nickase variant (R386D) that minimizes undesired double-strand cleavage while maintaining robust nicking activity. Using nucleosomes reconstituted with these engineered DNAs, we demonstrate the versatility of this platform across EMSA, biolayer interferometry, and cryo-EM. Structural analysis reveals how the PARP2 WGR domain engages an SSB within the nucleosome and uncovers associated shifts in H2B tail conformation that facilitate access to lesions positioned near the tail. Overall, this workflow provides a robust and scalable method for generating precisely modified nucleosomal substrates, enabling quantitative and structural dissection of PARP2-mediated DNA damage recognition and the coupled histone H2B tail rearrangements that facilitate lesion accessibility in chromatin.

Fragment-based drug design is a proven strategy for discovering high-quality leads. We present FDB&FragLinker, an integrated database and covalent optimization tool that enables users to explore, modify, and reassemble molecular fragments from DrugBank and a large subset of the ZINC database (800 M molecules). FragLinker is the first 3D complex generation tool based on fragement-level, allowing precise attachment of selected fragments onto small molecules at designated connection atoms via covalent docking, generating high-quality 3D protein-optimized ligand complex structures. This approach yields greater structural fidelity than traditional docking and is markedly faster than recent diffusion-based generative models. It can be widely applied to the optimization and modification of small molecules, PROTAC, small molecule polypeptides, etc. FDB&FragLinker is open-source and will be freely available via Webserver (https://hsadab.suat-sz.edu.cn/fdb/) and GitHub (https://github.com/guchengwanrenshan/FDB-FDBlinker).

Tetranychus urticae (T. urticae), commonly known as two spotted spider mites (TSSM), is a major agricultural pest worldwide that feeds on all major crops and has developed resistance to most chemical compounds used for its control. Genome sequence analysis of T. urticae revealed an expansion in gene families that play a role in digestion, detoxification, and transport of xenobiotics. This large detoxifying machinery, when paired with high transcriptional plasticity, has been linked to the unprecedented xenobiotic responsiveness of this pest. To better understand how T. urticae has evolved the extensive enzymes for xenobiotic detoxification, two closely related T. urticae Mu-class GSTs, TuGSTm06 (Tetur05g05220) and TuGSTm12 (Tetur05g05300), were structurally and functionally characterized. Enzymatic characterization of these two recombinant enzymes demonstrated different activity towards model substrates 1-chloro-2,4,-dinitrobenzene (CDNB) and isothiocyanates (ITCs). Some ITCs that we used in the studies are generated by plants and serve as defense compounds. We determined the crystal structures of TuGSTm06 and TuGSTm12 which revealed that the active sites of these enzymes differed only in three residues in the H-site. Single amino acid substitution suggested that these differences in the catalytic pocket may contribute to the specific catalytic attributes of each enzyme. Additionally, complementary molecular dynamics simulations predicted differences in the overall dynamic behavior of TuGSTm06 and TuGSTm12 and a correlation between the active site residues and protein dynamics in distant residues was determined. Our work highlights the complexity of the molecular basis underlying the activity of Mu-class TuGSTs and suggests that they may play a role in overcoming plant defenses using ITCs. Elucidating these molecular details is an essential step towards finding effective ways to manage this pest.

I am currently the Choh-Ming Li Professor of Life Sciences at the School of Life Sciences (SLS) in The Chinese University of Hong Kong (CUHK). I obtained my Ph.D. in Plant Molecular Biology from Simon Fraser University in 1996 under the supervision of Dr. Allison Kermode, and then postdoc training in Plant Cell Biology at Washington State University in 1996-2000 under the supervision of Dr. John C. Rogers. I joined the Department of Biology at CUHK as an Assistant Professor in 2000 and was promoted full professor in 2007. Since 2000, I have trained and graduated 40 Ph.D. students, 18 M.Phil. students and 34 postdocs. Since 2015, with the supported of competitive grants from the Research Grants Council (RGC) of Hong Kong and CUHK matching fund, we have established several advanced electron microscopy (EM)-based and live cell-based imaging shared platforms with in-house technical support and training to promote collaborative research and research excellence with excellent track record nationally and internationally. Our research program at CUHK has been focused on illustrating the molecular mechanisms underlying membrane trafficking, organelle biogenesis and function in the plant endomembrane system as well as its crosstalk with the autophagic pathway with many important findings and discoveries in the field using a combination of cellular, molecular and genetic approaches. More recently, we have also developed and used whole-cell electron tomography (ET) and Cryo-ET/FIB (focused ion beam) technologies with nanometer resolution and close-to-native membrane structures to illustrate the membrane biology of transport vesicles, extracellular vesicles and vacuoles in plants. Here I will first highlight our major findings and contributions in the endomembrane system and then take a journal towards our adventure on vacuole biology research as an example for discussion and reflection on future research development, education and training the next generation of young scientists.

Resolving the three-dimensional structures and dynamic functions of biomacromolecules in near-native environments to uncover life activity mechanisms is a core objective of structural biology. Over the past two decades, the Sun lab has focused on innovating cryo-electron tomography (cryo-ET) technology-a cornerstone of in situ structural biology. They have advanced key techniques across the workflow: developing software for tilt-series alignment and 3D reconstruction, creating algorithms to mitigate the "missing wedge" issue, optimizing sample preparation for cells and tissues, upgrading cryo-correlative light and electron microscopy (cryo-CLEM) systems, and designing AI-assisted particle picking methods. These innovations enabled them to elucidate in situ structures and functions of diverse biomacromolecules. Looking forward, they will continue to break bottlenecks in sample fidelity, data automation, and clinical application to advance in situ structural biology, facilitating investigation of more core mysteries of life activities in the future.

RNA G-quadruplexes (rG4s) are remarkably stable secondary structures with critical regulatory roles in gene expression, RNA metabolism, and telomere maintenance. However, their behavior within cells remains controversial, partly due to challenges in detecting rG4s in complex environments. Here, we use solution NMR spectroscopy to investigate how condensates formed by the low-complexity and RGG domains of the RNA-binding protein FUS affect the structure of TERRA, a highly stable model rG4. We show that FUS LC-RGG1 interacts with TERRA in dilute solution and that binding perturbs, but does not disrupt, the G-quadruplex structure. When co-phase separated with FUS LC-RGG1, however, NMR signatures of TERRA's folded state disappear, and the remaining observable resonances indicate an unfolded conformation, even in buffer containing potassium where TERRA rG4 is exceptionally stable when outside a condensate. Quantitative comparisons with a mutant form of TERRA, used as a baseline for fully unfolded RNA, suggest that at minimum a third of TERRA RNA becomes unfolded in the condensed phase. Thus, our results demonstrate that condensates can shift the structural ensemble of rG4 towards unfolded species, offering a potential mechanistic explanation for their apparent lack of stability in vivo and revealing how phase-separated environments may actively modulate RNA structure and function.

The protein tau can undergo two types of phase transitions, amyloid fibrillar aggregation leading to neurodegenerative disease, as well as liquid-liquid phase separation (LLPS) leading to the formation of protein condensates. The link between these two processes has yet to be understood fully. In this study, the tau construct, tau (243-386), was found to undergo LLPS only below a NaCl concentration of 135 mM. Hence, fibril formation was studied in 100 and 150 mM NaCl, on either side of the phase boundary established by the salt. Protein molecules inside the condensates lost their dynamicity, as measured by the extent of fluorescence recovery after photobleaching, with a characteristic time similar to that for the formation of amyloid aggregates. Thioflavin T fluorescence intensity increased homogeneously throughout the condensate interior, indicating that amyloid aggregate formation was not restricted to the interface, and with kinetics identical to that observed in a bulk measurement of amyloid fibril formation. Fibrils were seen to be emerging from aged condensates. Hydrogen-deuterium exchange studies coupled to mass spectrometry showed that tau undergoes heterogeneous fibril formation, with structurally different populations of fibrils forming under the same conditions. The structures and local stabilities of the protein molecules assembled inside the fibrils differed when formed under the LLPS and non-LLPS conditions. Consequently, the structural heterogeneity of fibrils formed under LLPS conditions was distinct from that of fibrils formed under non-LLPS conditions. The results indicate that LLPS might facilitate the selective formation of a particular structural polymorph in a heterogeneous fibril population.

The biological activities of many membrane proteins are pH-regulated, yet mapping their pH dependence experimentally is slow and expensive. In this work, we present MPKaDB (http://computbiophys.com/DeepKa/mpkadb), a comprehensive pK_adatabase for membrane proteins that instantly decode the protonation states of ionizable residues under a specified pH. Leveraging MPKaDB, we performed pH-coupled electrostatic characterization of transmembrane proteins. To facilitate use, a user-friendly search engine was developed to retrieve proteins of interest and return residue-specific pK_avalues, isoelectric points (pI) at cytoplasmic and extra-cytoplasmic faces, and automated screenings of active site residues. In the end, two case studies were proposed to demonstrate how pK_a's from MPKaDB could be applied to exploring the pH-dependent relationship between membrane protein structure and function.