Biology subjects

Huang, W.

Publications and source records attributed to Huang, W..

At least 181 records · Page 10Linked to original sources

bioRxiv2023

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

Single-cell sorting is essential to explore cellular heterogeneity in biology and medicine. Recently developed Raman-activated cell sorting (RACS) circumvents the limitations of fluorescence-activated cell sorting, such as the cytotoxicity of labels. However, the sorting throughputs of all forms of RACS are limited by the intrinsically small cross-section of spontaneous Raman scattering. Here, we report a stimulated Raman-activated cell ejection (S-RACE) platform that enables high-throughput single-cell sorting based on high-resolution multi-channel stimulated Raman chemical imaging, in situ image decomposition, and laser-induced cell ejection. The performance of this platform was illustrated by sorting a mixture of 1 m polymer beads, where 95% yield, 98% purity, and 14 events per second throughput were achieved. Notably, our platform allows live cell ejection, allowing for the growth of single colonies of bacteria and fungi after sorting. To further illustrate the chemical selectivity, lipid-rich Rhodotorula glutinis cells were successfully sorted from a mixture with Saccharomyces cerevisiae, confirmed by downstream quantitative PCR. Furthermore, by integrating a closed-loop feedback control circuit into the system, we realized real-time single-cell imaging and sorting, and applied this method to precisely eject regions of interest from a rat brain tissue section. The reported S-RACE platform opens exciting opportunities for a wide range of single-cell applications in biology and medicine. Significance statementImage-guided single-cell sorting is a potent tool in diverse biological applications. Current microfluidic cell sorting methods encounter challenges in handling smaller cells and are not applicable to tissue sections. To address these challenges, we have developed a stimulated Raman-activated cell ejection (S-RACE) platform, which is the first demonstration of single-cell ejection coupled with coherent Raman scattering. S-RACE allows label-free chemical imaging guided cell sorting through multispectral stimulated Raman scattering (SRS) imaging, on-the-fly image analysis, and laser-induced cell ejection. Versatile applications of S-RACE to a wide range of samples, such as polymer particles, single-live bacteria, single-live fungus, and tissue sections, are demonstrated.

bioengineering↗

bioRxiv2023

Anti-adipogenic properties of clock activator chlorhexidine and a new derivative

BackgroundThe circadian clock exerts temporal control of metabolic pathways to maintain homeostasis, and its disruption leads to the development of obesity and insulin resistance. In adipose tissue, key regulators of clock machinery orchestrate adipogenic processes via the Wnt signaling pathway to impact mature adipocyte development. MethodsBased on the recent finding of chlorhexidine as a new clock activator, we determined its potential anti-adipogenic activities in distinct adipogenic progenitor models. Furthermore, we report the structural optimization of chlorhexidine leading to the discovery of analogs with improved efficacy in inhibiting adipogenesis. ResultsIn adipogenic progenitors with Per2::dLuc luciferase reporter, Chlorhexidine shortened clock period length with induction of core clock components. Consistent with its clock-activating function, Chlorhexidine robustly suppressed the lineage commitment and maturation of adipogenic mesenchymal precursors, with comparable effect on inhibiting preadipocyte terminal differentiation. Mechanistically, we show that Chlorhexidine induces signaling components of the Wnt pathway resulting in activation of Wnt activity. Via modification of its chemical scaffold, we generated analogs of chlorhexidine that led to the identification of CM002 as a new clock- activating molecule with improved anti-adipogenic activity. ConclusionsCollectively, our findings uncovered the anti-adipogenic functions of a new class of small molecule clock activators. These compounds provide novel chemical probes to dissect clock function in maintaining metabolic homeostasis and may have therapeutic implications in obesity and associated metabolic disorders.

cell biology↗

bioRxiv2023

Redox Activated Substrates for Enhancing Activatable Cyclopropene Bioorthogonal Reactions

Bioorthogonal chemistry has become a mainstay in chemical biology and is making inroads in the clinic with recent advances in protein targeting and drug release. Since the fields beginning, a major focus has been on designing bioorthogonal reagents with good selectivity, reactivity, and stability in complex biological environments. More recently, chemists have imbued reagents with new functionalities like click-and-release or light/enzyme-controllable reactivity. We have previously developed a controllable cyclopropene-based bioorthogonal ligation, which has excellent stability in physiological conditions and can be triggered to react with tetrazines by exposure to enzymes, biologically significant small molecules, or light spanning the visual spectrum. Here, to improve reactivity and gain a better understanding of this system, we screened diene reaction partners for the cyclopropene. We found that a cyclopropene-quinone pair is 26 times faster than reactions with 1,2,4,5-tetrazines. Additionally, we showed that the reaction of the cyclopropene-quinone pair can be activated by two orthogonal mechanisms, caging group removal on the cyclopropene and oxidation/reduction of the quinone. Finally, we demonstrated that this caged cyclopropene-quinone can be used as a bioorthogonal imaging tool to label the membranes of cultured cells.

biochemistry↗

bioRxiv2023

Clonal fitness decline in somatic differentiation hierarchies

The concept of clonal fitness is fundamental to describe the evolutionary dynamics in somatic tissues. It is now well established that otherwise healthy somatic tissues become increasingly populated by expanding clones with age. However, the dynamic properties and respective fitnesses of these clones are less well understood. Here we show, that in somatic tissues organised as a differentiation hierarchy, theory predicts a natural decline of effective clonal fitness over time in the absence of additional driver events. This decline is intrinsic to the tissue organisation and can be captured quantitatively by a simple heuristic equation that is proportional to 1/time. We also show that the expected fitness decline is directly observable in human haematopoiesis. The predicted short and long term dynamics agree with in vivo observations using data of Neutrophil recovery after bone marrow transplants and naturally progressing Chronic Lymphocyte Leukemia (CLL). We further show that theory predicts the existence of a long term equilibrium fitness. All CLL patients transition into a stable equilibrium fitness eventually. We find significant inter-patient variation of long term fitness and a strong correlation with disease aggressiveness. Interestingly, CLL long term fitness can be forecast based on the early stages of disease progression, suggesting a Big Bang like model for CLL evolution.

cancer biology↗

bioRxiv2023

CellPolaris: Decoding Cell Fate through Generalization Transfer Learning of Gene Regulatory Networks

Cell fate changes are determined by gene regulatory network (GRN), a sophisticated system regulating gene expression in precise spatial and temporal patterns. However, existing methods for reconstructing GRNs suffer from inherent limitations, leading to compromised accuracy and application generalizability. In this study, we introduce CellPolaris, a computational system that leverages transfer learning algorithms to generate high-quality, cell-type-specific GRNs. Diverging from conventional GRN inference models, which heavily rely on integrating epigenomic data with transcriptomic information or adopt causal strategies through gene co-expression networks, CellPolaris employs high-confidence GRN sources for model training, relying exclusively on transcriptomic data to generate previously unknown cell-type-specific GRNs. Applications of CellPolaris demonstrate remarkable efficacy in predicting master regulatory factors and simulating in-silico perturbations of transcription factors during cell fate transition, attaining state-of-the-art performance in accurately predicting candidate key factors and outcomes in cell reprogramming and spermatogenesis with validated datasets. It is worth noting that, with a transfer learning framework, CellPolaris can perform GRN based predictions in all cell types even across species. Together, CellPolaris represents a significant advancement in deciphering the mechanisms of cell fate regulation, thereby enhancing the precision and efficiency of cell fate manipulation at high resolution.

bioinformatics↗

bioRxiv2023

Spatial and temporal pattern of structure-function coupling of human brain connectome with development

Brain structural circuitry shapes a richly patterned functional synchronization, supporting for complex cognitive and behavioural abilities. However, how coupling of structural connectome (SC) and functional connectome (FC) develops and its relationships with cognitive functions and transcriptomic architecture remain unclear. We used multimodal magnetic resonance imaging data from 439 participants aged 5.7 to 21.9 years to predict functional connectivity by incorporating intracortical and extracortical structural connectivity, characterizing SC-FC coupling. Our findings revealed that SC-FC coupling was strongest in the visual and somatomotor networks, consistent with evolutionary expansion, myelin content, and functional principal gradient. As development progressed, SC-FC coupling exhibited heterogeneous alterations dominated by an increase in cortical regions, broadly distributed across the somatomotor, frontoparietal, dorsal attention, and default mode networks. Moreover, we discovered that SC-FC coupling significantly predicted individual variability in general intelligence, mainly influencing frontoparietal and default mode networks. Finally, our results demonstrated that the heterogeneous development of SC-FC coupling is positively associated with genes in oligodendrocyte-related pathways and negatively associated with astrocyte-related genes. This study offers insight into the maturational principles of SC-FC coupling in typical development.

neuroscience↗

bioRxiv2023

Negative allosteric modulation of the μ-opioid receptor

The {micro}-opioid receptor ({micro}OR) is a well-established target for analgesia, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression, which have led to the present opioid overdose epidemic. {micro}OR negative allosteric modulators (NAMs) may serve as powerful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. We screened a large DNA-encoded chemical library against inactive {micro}OR, counter-screening with active, G-protein and agonist bound receptor to "steer" selections toward functional negative allosteric modulators. We discovered a NAM compound with high and selective enrichment to inactive {micro}OR; the molecule potently blocks the activity of orthosteric agonists and enhances the affinity of the key opioid overdose reversal molecule, naloxone. It accomplishes this by binding to a site on the extracellular vestibule proximal to naloxone, stabilizing a unique inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM perturbs orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone in vivo to inhibit morphine-induced antinociception, respiratory depression and conditioned place preference while minimizing withdrawal behaviors. Our results provide detailed structural insights into the mechanism of a negative allosteric modulator for the {micro}OR and demonstrate how it can be exploited in vivo.

biochemistry↗

bioRxiv2023

CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

Macrophage efferocytosis, the process by which phagocytes engulf and remove apoptotic cells (ACs), plays a critical role in maintaining tissue homeostasis. Efficient efferocytosis prevents secondary necrosis, mitigates chronic inflammation, and impedes atherosclerosis progression. However, the regulatory mechanisms of efferocytosis under atherogenic conditions remain poorly understood. We previously demonstrated that oxidized LDL (oxLDL), an atherogenic lipoprotein, induces mitochondrial reactive oxygen species (mtROS) in macrophages via CD36. In this study, we demonstrate that macrophage mtROS facilitate continual efferocytosis through a positive feedback mechanism. However, oxLDL disrupts continual efferocytosis by dysregulating the internalization of ACs. This disruption is mediated by an overproduction of mtROS. Mechanistically, oxLDL/CD36 signaling promotes the translocation of cytosolic PKM2 to mitochondria, facilitated by the chaperone GRP75. Mitochondrial PKM2 then binds to Complex III of the electron transport chain, inducing mtROS production. This study elucidates a novel regulatory mechanism of efferocytosis in atherosclerosis, providing potential therapeutic targets for intervention. SUMMARYMacrophages clear apoptotic cells through a process called efferocytosis, which involves mitochondrial ROS. However, the atherogenic oxidized LDL overstimulates mitochondrial ROS via the CD36-PKM2 pathway, disrupting continual efferocytosis. This finding elucidates a novel molecular mechanism that explains defects in efferocytosis, driving atherosclerosis progression.

immunology↗

bioRxiv2023

KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health. Highlights of the workO_LIKat2a and Kat2b double knockout in the murine intestinal epithelium triggers activation of the interferon signaling pathway C_LIO_LIKat2a/Kat2b knockout leads to intestinal stem cell loss and other mucosal phenotypes consistent with interferon activation C_LIO_LIHistone PTM mass spec profiling reveals the first in vivo study showing H3K9ac-specific loss with Kat2a and Kat2b double knockout, yet without correlation to interferon signaling pathway genes C_LIO_LIComprehensive proteomic analysis identifies non-histone acetyl-lysine targets of KAT2 in the mouse intestine in vivo, including mitochondrial proteins C_LIO_LIMitochondrial function is compromised upon Kat2 loss C_LIO_LIdsRIP-seq identifies double-stranded RNA from the mitochondria as a trigger for the intrinsic immune response upon Kat2 double knockout C_LI

molecular biology↗

bioRxiv2023

In-situ Single-Molecule Investigations of the Impacts of Biochemical Perturbations on Conformational Intermediates of Monomeric α-Synuclein

-Synuclein aggregation is a common trait in synucleinopathies, including Parkinsons disease. Being an unstructured protein, -synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in-situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric -synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-{beta} component (NAC) region of monomeric -synuclein, which is regulated by preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing -synucleins aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in -synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order -synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins.

biophysics↗

bioRxiv2023

CrWRKY42 positively regulates chlorophyll degradation and carotenoid biosynthesis in citrus

Chlorophyll degradation and carotenoid biosynthesis, which occur almost simultaneously during fruit ripening, are essential for coloration and nutritional value of fruits. However, the synergistic regulation of these two processes at transcriptional level remains largely unknown. Here, we identified a novel WRKY transcription factor CrWRKY42 from the transcriptome data of the yellowish bud mutant Jinlegan tangor (MT) and its wild type Shiranuhi tangor (WT), which was involved in the transcriptional regulation of both chlorophyll degradation and carotenoid biosynthesis pathways. CrWRKY42 activated the expression of {beta}-carotene hydroxylase 1 (CrBCH1) by directly binding to its promoter. Overexpression and interference of CrWRKY42 in citrus calli demonstrated that CrWRKY42 promoted carotenoid accumulation by inducing the expression of multiple carotenoid biosynthetic genes. Further assays confirmed that CrWRKY42 also directly bound to and activated the promoters of the genes involved in the carotenoid biosynthesis, including phytoene desaturase (CrPDS) and lycopene {beta}-cyclase 2 (CrLCYB2). In addition, CrWRKY42 could also bind to the promoter of STAY-GREEN (CrSGR) and activated its expression, thus promoting chlorophyll degradation. Overexpression and silencing of CrWRKY42 in citrus fruits indicated that CrWRKY42 positively regulated chlorophyll degradation and carotenoid biosynthesis by synergistically activating the expressions of genes involved in both pathways. In conclusion, our data revealed that CrWRKY42 acted as a positive regulator of chlorophyll degradation and carotenoid biosynthesis to alter the conversion of citrus fruit color. Our findings provide insight into the complex transcriptional regulation of chlorophyll and carotenoid metabolism during fruit ripening. One Sentence SummaryThe CrWRKY42 transcription factor coordinates chlorophyll degradation and carotenoid biosynthesis by directly regulating genes involved in these pathways to alter the conversion of citrus fruit color.

molecular biology↗

bioRxiv2023

Adaptive therapy achieves long-term control of chemotherapy resistance in high grade ovarian cancer

Drug resistance results in poor outcomes for most patients with metastatic cancer. Adaptive Therapy (AT) proposes to address this by exploiting presumed fitness costs incurred by drug-resistant cells when drug is absent, and prescribing dose reductions to allow fitter, sensitive cells to re-grow and re- sensitise the tumour. However, empirical evidence for treatment-induced fitness change is lacking. We show that fitness costs in chemotherapy-resistant ovarian cancer cause selective decline and apoptosis of resistant populations in low-resource conditions. Moreover, carboplatin AT caused fluctuations in sensitive/resistant tumour population size in vitro and significantly extended survival of tumour-bearing mice. In sequential blood-derived cell-free DNA and tumour samples obtained longitudinally from ovarian cancer patients during treatment, we inferred resistant cancer cell population size through therapy and observed it correlated strongly with disease burden. These data have enabled us to launch a multicentre, phase 2 randomised controlled trial (ACTOv) to evaluate AT in ovarian cancer.

cancer biology↗

bioRxiv2023

Modeling Human Spine-Spinal Cord Organogenesis by hPSC-Derived Neuromesodermal Progenitors

Human trunk development, including spine and spinal cord organogenesis, is a coordinated, orderly, and interdependent process with spatiotemporal tissue patterning. However, the underlying cellular and molecular mechanisms remain largely unclear due to the lack of an effective model that can simulate the early development of human body axis. Here, we reported the long-term patterning and dynamic morphogenesis of human trunk through the formation of spine-spinal cord organoids (SSCOs) self-organized from three-dimensional culture of human PSC-derived neuromesodermal progenitors (NMPs). The SSCOs resembled the morphogenetic features of spine and spinal cord along the anterior-posterior axis, and showed the chondro-osteogenic and neural trajectories consistent with developmental dynamics of spine and spinal cord in gestational embryo through single-cell RNA sequencing (scRNA-seq). In addition, we identified a new HMMR+ bipotent cell population with self-renewal ability and neural/mesodermal competence but distinct from NMPs, which may be involved in trunk development and represent an invaluable tool for disease modeling of spine- and spinal cord-related disorders. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/549829v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@14bfd30org.highwire.dtl.DTLVardef@1db7fe8org.highwire.dtl.DTLVardef@1aa66a1org.highwire.dtl.DTLVardef@380932_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Coordinated inheritance of extrachromosomal DNA species in human cancer cells

The chromosomal theory of inheritance has dominated human genetics, including cancer genetics. Genes on the same chromosome segregate together while genes on different chromosomes assort independently, providing a fundamental tenet of Mendelian inheritance. Extrachromosomal DNA (ecDNA) is a frequent event in cancer that drives oncogene amplification, dysregulated gene expression and intratumoral heterogeneity, including through random segregation during cell division. Distinct ecDNA sequences, herein termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells. However, how multiple ecDNA species within a tumor cell are assorted and maintained across somatic cell generations to drive cancer cell evolution is not known. Here we show that cooperative ecDNA species can be coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. EcDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy number gains in multiple ecDNA species prior to any selection. Computational modeling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Finally, we show that coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

cancer biology↗

bioRxiv2023

Tandemly duplicated MYB genes specifically in the Phaseoleae lineage are functionally diverged in the regulation of anthocyanin biosynthesis

Gene duplications have long been recognized as a driving force in the evolution of genes, giving rise to novel functions. The soybean genome is characterized by a large extent of duplicated genes. However, the extent and mechanisms of functional divergence among these duplicated genes in soybean remain poorly understood. In this study, we revealed that tandem duplication of MYB genes, which occurred specifically in the Phaseoleae lineage, exhibited a stronger purifying selection in soybean compared to common bean. To gain insights into the diverse functions of these MYB genes in anthocyanin biosynthesis, we examined the expression, transcriptional activity, metabolite, and evolutionary history of four MYB genes (GmMYBA5, GmMYBA2, GmMYBA1 and Glyma.09g235000), which were presumably generated by tandem duplication in soybean. Our data revealed that Glyma.09g235000 had become a pseudogene, while the remaining three MYB genes exhibited strong transcriptional activation activity and promoted anthocyanin biosynthesis in different soybean tissues. Furthermore, GmMYBA5 produced distinct compounds in Nicotiana benthamiana leaves compared to GmMYBA2 and GmMYBA1 due to variations in their DNA binding domains. The lower expression of anthocyanin related genes in GmMYBA5 resulted in lower levels of anthocyanins compared to GmMYBA2 and GmMYBA1. Metabolomics analysis further demonstrated the diverse and differential downstream metabolites, suggesting their functional divergence in metabolites following gene duplication. Together, our data provided evidence of functional divergence within the MYB gene cluster following tandem duplication, which shed light on the potential evolutionary direction of gene duplications during legume evolution.

evolutionary biology↗

bioRxiv2023

Fusion Neural Network (FusNet) for predicting protein-mediated loops

The organization of the three-dimensional (3D) genome is a complex, and requires a plethora of proteins to ensure the proper formation and regulation of chromatin loops as well as higher order structures. Studying protein-mediated loop regulation can help unravel the intricate interplay between these loops and their crucial roles in modulating gene expression across different cellular contexts. However, current targeted chromatin conformation capture experiments face limitations in capturing protein-mediated loops across various cell types, and existing computational methods fail to predict diverse protein-mediated loops. To address these issues, we propose a fusion neural network (FusNet) designed for predicting protein-mediated loops. FusNet leverages genome sequence information, open chromatin, and ChIP-seq data to efficiently represent and analyze the positions of loop anchors. To extract informative features and reduce the complexity of FusNet, we constructed a convolutional neural network, which compresses the dimensionality of the features while also preserving the most significant ones. To enhance the accuracy and generalization capacity of FusNet, we built a fusion layer by stacking the prediction of fundamental models with a meta-model. FusNet demonstrated its effectiveness in predicting protein-mediated loops, exhibiting high consistency with Hi-C data. Moreover, we find that the loops output from FusNet are highly associated with regulatory functions. Through association analysis with genetic risk variants, FusNet further revealed its potential for unraveling disease-related mechanisms. In conclusion, our study offers a novel computational approach for predicting various protein-mediated chromatin loops, which could substantially enhance research on the functional significance of protein-mediated loop structures in diverse cellular contexts. Significance StatementThe intricate spatial organization of the three-dimensional (3D) genome involves functional proteins critically contributing to chromatin loop formation and regulation. Understanding these protein-mediated loops is vital for elucidating their influence on 3D genome architecture and gene regulation across different cellular types and disease-related contexts. In this study, we propose a Fusion Neural Network (FusNet) for predicting protein-mediated loops. FusNet can concurrently capture and analyze multiple protein-mediated loops in various cell types to advance our understanding of the multitude of protein-mediated loop structures and their functional significance. Importantly, through association analysis with risk variants, FusNet manifests potential in revealing disease-related mechanisms.

bioinformatics↗

bioRxiv2023

Structural insights into Rad18 targeting by the SLF1 BRCT domains

Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1tBRCT) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1tBRCT interacts with two phosphoserines and adjacent residues in Rad18 for high affinity and specificity Rad18 recognition. We found that SLF1tBRCT utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an -helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.

biochemistry↗

bioRxiv2023

Structural Basis for Regulated Assembly of the Mitochondrial Fission GTPase Drp1

Mitochondrial fission is crucial for distributing cellular energy throughout cells and for isolating damaged regions of the organelle that are targeted for degradation. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. De novo missense mutations in the Drp1 gene, DNM1L, are associated with severe neurodevelopmental diseases in patients, and no effective treatments are available. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the regulatory mechanisms that determine when and where Drp1 self-assembles, and proper mitochondrial dynamics requires correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length, native Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intermolecular interactions to achieve the assembly competent state previously observed in crystal and filament structures. Specifically, the G domain is closed against the stalk domain and occludes intermolecular interactions necessary for self-assembly beyond a dimer. Similarly, adjacent stalks in the dimer form a more continuous interface that further occludes conserved intermolecular contact sites. This structural insight provides a novel mechanism for regulated self-assembly of the mitochondrial fission machinery.

cell biology↗