Biology subjects

Hong, L.

Publications and source records attributed to Hong, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Thymidine Phosphorylase Promotes the Formation of Abdominal Aortic Aneurysm in Mice Fed a Western Diet

AimsThe precise molecular drivers of abdominal aortic aneurysm (AAA) remain unclear. Thymidine phosphorylase (TYMP) contributes to increased platelet activation, thrombosis, and inflammation, all of which are key factors in AAA development. Additionally, TYMP suppresses the proliferation of vascular smooth muscle cells (VSMCs), which are central to the development and progression of AAA. We hypothesize that TYMP plays a key role in AAA development. Methods and ResultsWe conducted a histological study using human AAA samples and normal abdominal aortas, revealing heightened levels of TYMP in human AAA vessel walls. To validate this observation, we utilized an Ang II perfusion-induced AAA model in wild-type C57BL/6J (WT) and Tymp-/-mice, feeding them a Western diet (TD.88137) starting from 4 weeks of age. We found that Tymp-/-mice were protected from Ang II perfusion-induced AAA formation. Furthermore, by using TYMP-expressing VSMCs as well as primarily cultured VSMCs from WT and Tymp-/- mice, we elucidated the essential role of TYMP in regulating MMP2 expression and activation. TYMP deficiency or inhibition by tipiracil, a selective TYMP inhibitor, led to reduced MMP2 production, release, and activation in VSMCs. Additionally, TYMP was found to promote pro-inflammatory cytokine expression systemically, and its absence attenuates TNF--stimulated activation of MMP2 and AKT. By co-culturing VSMCs and platelets, we observed that TYMP-deficient platelets had a reduced inhibitory effect on VSMC proliferation compared to WT platelets. Moreover, TYMP appeared to enhance the expression of activated TGF{beta}1 in cultured VSMCs in vitro and in human AAA vessel walls in vivo. TYMP also boosted the activation of thrombospondin-1 type 1 repeat domain-enhanced TGF{beta}1 signaling, resulting in increased connective tissue growth factor production. ConclusionOur findings collectively demonstrated that TYMP serves as a novel regulatory force in vascular biology, exerting influence over VSMC functionality and inflammatory responses that promote the development of AAA. Translational PerspectiveThymidine phosphorylase (TYMP) is increased in the vessel walls of patients with abdominal aortic aneurysm (AAA), and TYMP deficiency in mice reduces the incidence of AAA, suggesting that TYMP plays a crucial role in AAA development. This could be attributed to TYMPs role in enhancing systemic inflammation and thrombosis, inhibiting vascular smooth muscle cell function, increasing the activation of matrix metalloproteinase and AKT, as well as enhancing the expression of TGF{beta}1 and connective tissue growth factor. Tipiracil is an FDA-approved drug known to inhibit TYMP-enhanced thrombosis. Targeting TYMP with tipiracil could represent a promising new therapeutic strategy for AAA development.

cell biology↗

bioRxiv2024

Discovery of Alkenyl Oxindole as a Novel PROTAC Moiety for Targeted Protein Degradation via CRL4DCAF11 Recruitment

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of hetero-bifunctional compounds by conjugating different alkenyl oxindoles with the BRD4 inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent hetero-bifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in vivo. Our research provides new employable PROTAC moieties for targeted protein degradation, providing new possibilities for drug discovery.

biochemistry↗

bioRxiv2024

LncRNA-CIR6 Mediates Repair of Infarcted Hearts by Inducing MSCs Differentiation into Cardiogenic Cells through CDK1

PurposeThis study aims to investigate the induction effect of LncRNA-CIR6 on MSCs differentiation into Cardiogenic Cells in vitro and in vivo. MethodsIn addition to pretreatment with the Ro-3306 (CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was transfected into C57BL/6 mice heart by 100 L of AAV9-cTnT-LncRNA-CIR6-ZsGreen i.v. After 3 weeks of transfection followed by AMI surgery, hUCMSCs (5x105/100 L) were injected by i.v 1 week later. Cardiac function was evaluated using VEVO 2100 and electric mapping 9 days after cell injection. IF, Evans blue-TTC, Masson staining, FACS, and WB were used to determine relevant indicators. ResultsLncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00{+/-}0.58)% and hUCMSCs (95.43{+/-}2.13)% into cardiogenic cells, as determined by the expression of cTnT. Compared with MI group, cardiac contraction and conduction function in MI heart treated by LncRNA-CIR6 or combined with MSCs injection groups were significantly increased as well as the area of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was in Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0-50/190-255 in the sequence. CDK1 is a protein found to be related to the proliferation and differentiation of cardiomyocytes is which located in the functional region of LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed high expression of CDK1. LncRNA-CIR6 mediates repair of infarcted hearts by inducing MSCs differentiation into cardiogenic cells through CDK1. ConclusionsLncRNA-CIR6 mediates repair of infarcted hearts by inducing MSCs differentiation into cardiogenic cells through CDK1.

molecular biology↗

bioRxiv2023

A 3-component module maintains sepal flatness in Arabidopsis

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding, through the interplay between biochemical and biomechanical cues. In contrast, certain organs maintain their flat posture over several days. Here we identified a pathway, which is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular and mechanical approaches, our results demonstrate that global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin signaling to down-regulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Our work unravels a 3-component module, which relates hormonal patterns to organ curvature, and actively maintains sepal flatness during its growth.

plant biology↗

bioRxiv2023

Semantical and Topological Protein Encoding Toward Enhanced Bioactivity and Thermostability

Protein engineering is a pivotal aspect of synthetic biology, involving the modification of amino acids within existing protein sequences to achieve novel or enhanced functionalities and physical properties. Accurate prediction of protein variant effects requires a thorough understanding of protein sequence, structure, and function. Deep learning methods have demonstrated remarkable performance in guiding protein modification for improved functionality. However, existing approaches predominantly rely on protein sequences, which face challenges in efficiently encoding the geometric aspects of amino acids local environment and often fall short in capturing crucial details related to protein folding stability, internal molecular interactions, and bio-functions. Furthermore, there lacks a fundamental evaluation for developed methods in predicting protein thermostability, although it is a key physical property that is frequently investigated in practice. To address these challenges, this paper introduces a novel pre-training framework that integrates sequential and geometric encoders for protein primary and tertiary structures. This framework guides mutation directions toward desired traits by simulating natural selection on wild-type proteins and evaluates variant effects based on their fitness to perform specific functions. We assess the proposed approach using three benchmarks comprising over 300 deep mutational scanning assays. The prediction results showcase exceptional performance across extensive experiments when compared to other zero-shot learning methods, all while maintaining a minimal cost in terms of trainable parameters. This study not only proposes an effective framework for more accurate and comprehensive predictions to facilitate efficient protein engineering, but also enhances the in silico assessment system for future deep learning models to better align with empirical requirements. The PyTorch implementation are available at https://github.com/tyang816/ProtSSN.

bioengineering↗

bioRxiv2023

Protein Engineering with Lightweight Graph Denoising Neural Networks

Protein engineering faces challenges in finding optimal mutants from the massive pool of candidate mutants. In this study, we introduce a deep learning-based data-efficient fitness prediction tool to steer protein engineering. Our methodology establishes a lightweight graph neural network scheme for protein structures, which efficiently analyzes the microenvironment of amino acids in wild-type proteins and reconstructs the distribution of the amino acid sequences that are more likely to pass natural selection. This distribution serves as a general guidance for scoring proteins toward arbitrary properties on any order of mutations. Our proposed solution undergoes extensive wet-lab experimental validation spanning diverse physicochemical properties of various proteins, including fluorescence intensity, antigen-antibody affinity, thermostability, and DNA cleavage activity. More than 40% of PO_SCPLOWROTC_SCPLOWLGN-designed single-site mutants outperform their wild-type counterparts across all studied proteins and targeted properties. More importantly, our model can bypass the negative epistatic effect to combine single mutation sites and form deep mutants with up to 7 mutation sites in a single round, whose physicochemical properties are significantly improved. This observation provides compelling evidence of the structure-based models potential to guide deep mutations in protein engineering. Overall, our approach emerges as a versatile tool for protein engineering, benefiting both the computational and bioengineering communities.

bioengineering↗

bioRxiv2023

Growth couples temporal and spatial fluctuations of tissue properties during morphogenesis

Living tissues display fluctuations - random spatial and temporal variations of tissue properties around their reference values - at multiple scales. It is believed that such fluctuations may enable tissues to sense their state or their size. Recent theoretical studies developed specific models of fluctuations in growing tissues and predicted that fluctuations of growth show long-range correlations. Here we elaborated upon these predictions and we tested them using experimental data. We first introduced a minimal model for the fluctuations of any quantity that has some level of temporal persistence or memory, such as concentration of a molecule, local growth rate, or mechanical property. We found that long-range correlations are generic, applying to any such quantity, and that growth couples temporal and spatial fluctuations, through a mechanism that we call fluctuation stretching -- growth enlarges the lengthscale of variation of this quantity. We then analysed growth data from sepals of the model plant Arabidopsis and we quantified spatial and temporal fluctuations of cell growth using the previously developed Cellular Fourier Transform. Growth appears to have long-range correlations. We compared different genotypes and growth conditions: mutants with lower or higher response to mechanical stress have lower temporal correlations and longer-range spatial correlations than wild-type plants. Finally, we used theoretical predictions to merge experimental data from all conditions and developmental stages into an unifying curve, validating the notion that temporal and spatial fluctuations are coupled by growth. Altogether, our work reveals kinematic constraints on spatiotemporal fluctuations that have an impact on the robustness of morphogenesis. Significance StatementHow do organs and organisms grow and achieve robust shapes in the face of subcellular and cellular variability? In order to address this outstanding mystery, we investigated the variability of growth at multiple scales and we analysed experimental data from growing plant tissues. Our results support the prediction that tissue expansion couples temporal memory of growth with spatial variability of growth. Our work reveals a constraint on the spatial and temporal variability of growth that may impact the robustness of morphogenesis.

biophysics↗

bioRxiv2023

Decoupling of the Onset of Anharmonicity between a Protein and Its Surface Water around 200 K

The protein dynamical transition at [~] 200 K, where the biomolecule transforms from a harmonic, non-functional form to an anharmonic, functional state, has been thought to be slaved to the thermal activation of dynamics in its surface hydration water. Here, by selectively probing the dynamics of protein and hydration water using elastic neutron scattering and isotopic labelling, we found that the onset of anharmonicity in the two components around 200 K are decoupled. The one in protein is an intrinsic transition, whose characteristic temperature is independent of the instrumental resolution time, but varies with the biomolecular structure and the amount of hydration, while the one of water is merely a resolution effect.

biophysics↗

bioRxiv2023

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma

MicroRNA (miR)-200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c-based gene therapy to inhibit OSCC growth and metastasis has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA encoding miR-200c delivered via non-viral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were utilized to transfect pDNA encoding miR-200c into OSCC cells and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line-derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and metastasis and improve clinical outcomes in OSCC treatment. (227 words)

bioengineering↗

bioRxiv2023

Conditional Protein Denoising Diffusion Generates Programmable Endonucleases

Deep learning-based methods for generating functional proteins address the growing need for novel biocatalysts, allowing for precise tailoring of functionalities to meet specific requirements. This emergence leads to the creation of highly efficient and specialized proteins with wide-ranging applications in scientific, technological, and biomedical domains. This study establishes a pipeline for protein sequence generation with a conditional protein diffusion model, namely CPDiffusion, to deliver diverse sequences of proteins with enhanced functions. CPDiffusion accommodates protein-specific conditions, such as secondary structure and highly conserved amino acids (AAs). Without relying on extensive training data, CPDiffusion effectively captures highly conserved residues and sequence features for a specific protein family. We applied CPDiffusion to generate artificial sequences of Argonaute (Ago) proteins based on the backbone structures of wild-type (WT) Kurthia massiliensis Ago (KmAgo) and Pyrococcus furiosus Ago (PfAgo), which are complex multi-domain programmable endonucleases. The generated sequences deviate by up to nearly 400 AAs from their WT templates. Experimental tests demonstrated that the majority of generated proteins show unambiguous activity in DNA cleavage for both KmAgo and PfAgo, with many of them exhibiting superior activity as compared to the WT. These findings underscore CPDiffusions remarkable success rate to generate novel sequences for proteins of complex structures and functions in a single step with enhanced activity. This approach facilitates the design of enzymes with multi-domain molecular structures and intricate functions through in silico generation and screening, all accomplished without any supervision from labeled data.

bioengineering↗

bioRxiv2023

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and {gamma}-IR. CP-8 and CP-23 effectively inhibited {gamma}-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23s efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/552990v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dcec56org.highwire.dtl.DTLVardef@1202a09org.highwire.dtl.DTLVardef@a7e7b6org.highwire.dtl.DTLVardef@15d8925_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Hypoxia-sensing by the Histone Demethylase UTX (KDM6A) Controls Colitogenic CD4+ T cell Fate and Mucosal Inflammation

Hypoxia is a feature of inflammatory conditions [e.g., inflammatory bowel disease (IBD)] and can exacerbate tissue damage in these diseases. To counteract hypoxias deleterious effects, adaptive responses have evolved which protect against hypoxia-associated tissue injury. To date, much attention has focused on hypoxia-activated HIF (hypoxia-inducible factor) transcription factors in these responses. However, recent work has identified epigenetic regulators that are also oxygen-sensitive, but their role in adaptation to hypoxic inflammation is currently unclear. Here, we show that the oxygen-sensing epigenetic regulator UTX is a critical modulator of colitis severity. Unlike HIF transcription factors that act on gut epithelial cells, UTX functions in colitis through its effects on immune cells. Hypoxia results in decreased CD4+ T cell IFN-{gamma} production and increased CD4+ regulatory T cells, and these findings are recapitulated by T cell-specific UTX deficiency. Hypoxia impairs the histone demethylase activity of UTX, and loss of UTX function leads to accumulation of repressive H3K27me3 epigenetic marks at IL12/STAT4 pathway genes (Il12rb2, Tbx21, and Ifng). In a colitis mouse model, T cell-specific UTX deletion ameliorates colonic inflammation, protects against weight loss, and increases survival. Together these findings implicate UTXs oxygen-sensitive histone demethylase activity in mediating protective, hypoxia-induced pathways in colitis.

immunology↗

bioRxiv2023

Growth directions and stiffness across cell layers determine whether tissues stay smooth or buckle

From smooth shapes to buckles, nature exhibits organs of various shapes and forms. How cells grow to produce smooth shaped leaves and sepals remain unclear. Here, we show that growth along the longitudinal axis during early developmental stages and comparable stiffness across both epidermal layers of Arabidopsis sepals are essential for smoothness, as seen in the wild type. We identified a mutant (as2-7D) with ectopic expression of ASYMMETRIC LEAVES 2 (AS2) on the outer epidermis. Our analysis reveals that ectopic AS2 expression causes the outer epidermis of as2-7D sepals to buckle during early stages of sepal development. We show that buckling of the outer epidermis occurs due to conflicting cell growth directions and unequal tissue stiffness across the epidermal layers. Overexpression of cyclin-dependent kinase (CDK) inhibitor Kip-related protein 1 (KRP1) in as2-7D restores sepal smoothness by aligning the growth directions of the outer epidermal cells along the longitudinal axis, while also increasing the overall stiffness of the outer epidermis. Furthermore, buckling is associated with the convergence of auxin efflux transporter protein PIN-FORMED 1 (PIN1) to generate outgrowth in the sepals at later stages, suggesting that buckling can initiate outgrowths. Our findings suggest that in addition to molecular cues influencing tissue mechanics, tissue mechanics can also modulate molecular signals, giving rise to well-defined shapes. One-Sentence SummaryThe asymmetric leaves 2-7D mutant sepals buckle due to discoordination of growth between the two epidermal layers.

developmental biology↗

bioRxiv2023

A Medium Chain Fatty Acid, 6-hydroxyhexanoic acid (6-HHA), Protects Against Obesity and Insulin Resistance

Obesity, a worldwide health problem, increases the risk for developing metabolic diseases such as insulin resistance and diabetes. It is well recognized that obesity-associated chronic inflammation plays a key role in the pathogenesis of systemic metabolic dysfunction. Previously, we revealed an anti-inflammatory role for spent culture supernatants isolated from the oral commensal bacterial species Streptococcus gordonii (Sg-SCS). Here, we identified that 6-hydroxyhexanoic acid (6-HHA), a medium chain fatty acid (MCFA), is the one of the key components of Sg-SCS. We found that treatment of 6-HHA in mice fed a high-fat diet (HFD) significantly reduced HFD-mediated weight gain which was largely attributed to a decrease in fat mass. Systemically, 6-HHA improves obesity-associated glucose intolerance and insulin resistance. Furthermore, administration of 6-HHA suppressed obesity-associated systemic inflammation and dyslipidemia. At the cellular level, treatment of 6-HHA ameliorated aberrant inflammatory and metabolic transcriptomic signatures in white adipose tissue of mice with diet-induced obesity (HFD). Mechanistically, we found that 6-HHA suppressed adipocyte-proinflammatory cytokine production and lipolysis, the latter through Gi-mediated signaling. This work provides direct evidence for the anti-obesity effects of a novel MCFA, which could be a new therapeutic treatment for combating obesity. KEY POINTSO_LIHydroxyhexanoic medium chain fatty acids (MCFAs) are dietary and bacterial-derived energy sources, however, the outcomes of using MCFAs in treating metabolic disorders are diverse and complex. C_LIO_LIThe MCFA 6-hydroxyhexanoic acid (6-HHA) is a metabolite secreted by the oral bacterial commensal species Streptococcus gordonii; here we investigated its role in modulating high-fat diet (HFD)-induced metabolic dysfunction. C_LIO_LIIn a murine model of obesity, we found 6-HHA-mediated improvement of diet-mediated adiposity, insulin resistance and inflammation were in part due to actions on white adipose tissue (WAT). C_LIO_LI6-HHA suppressed proinflammatory cytokine production and lipolysis through Gi-mediated signaling in differentiated white adipocytes. C_LI

physiology↗

bioRxiv2023

Mn2+-Induced Structural Flexibility Enhances the Entire Catalytic Cycle and the Cleavage of Mismatches in Prokaryotic Argonaute Proteins

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn2+ bound in the pockets, as verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn2+ in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn2+ in solution enhances the flexibility of diverse pAgo proteins. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn2+, leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn2+-induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicated that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, Mn2+-induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn2+ in solution, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.

biophysics↗

bioRxiv2023

De Novo Generation and Prioritization of Target-Binding Peptide Motifs from Sequence Alone

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

bioengineering↗

bioRxiv2023

Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming the hollow polar tube that transports the sporoplasm components including nucleus into the host cell to achieve the invasion. Due to the complicated topological changes occurring in this process, the formation of sporoplasm is unclear. Here, electron microscopy observation and DiI staining confirmed that during spore germination, a large number of vesicles derived from the polaroplast, nucleus and other cytoplasm were transported out via the polar tube. Meanwhile, the posterior vacuole and plasma membrane remained in the empty spore coat. In addition, there was no DiI-labeled membrane around the nucleus in mature spores, whereas a DiI-labeled limit membrane wrapping nucleus was found at the tip of the extruded polar tube, suggesting that the membrane of sporoplasm was formed outside the mature spore. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were located at the polaroplast in mature spores, in the extruded polar tube and on the sporoplasm membrane, which indicated that the polaroplast transported via the polar tube finally became the limiting membrane of the sporoplasm. Golgi-tracker green and Golgi marker protein syntaxin 6 were also found the same model, which was consistent with the transported polaroplast derived from Golgi transformed into the novel sporoplasm membrane during spore germination. ImportanceMicrosporidia, obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection of microsporidia is its unique invasion apparatus which includes the polar filament, polaroplast and posterior vacuole. When the spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that will transport the sporoplasm components including nucleus into a host cell to achieve the invasion. Knowledge of structure difference between polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting elongated nucleus and other sporoplasm components. And we confirm that the transported polaroplast finally transforms into the novel sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of polar tube and origin of the sporoplasm membrane, which serve as foundations for clarifying the microsporidian infection mechanism.

pathology↗

bioRxiv2023

TRAF4 is crucial for the propagation of ST2+ memory Th2 cells in IL-33-mediated type 2 airway inflammation

Tumor necrosis factor receptor (TNF)-associated factor 4 (TRAF4) is an important regulator of type 2 responses in the airway; however, the underlying cellular and molecular mechanisms remain elusive. Herein, we generated T cell-specific TRAF4-deficient (CD4cre-Traf4fl/fl) mice and investigated the role of TRAF4 in interleukin (IL)-33 receptor (ST2, suppression of tumorigenicity 2)-expressing memory Th2 cells (ST2+ mTh2) in IL-33-mediated type 2 airway inflammation. We found that in vitro polarized TRAF4-deficient (CD4cre-Traf4fl/fl) ST2+ mTh2 cells exhibited decreased IL-33-induced proliferation as compared with TRAF4-sufficient (Traf4fl/fl) cells. Moreover, CD4cre-Traf4fl/fl mice showed less ST2+ mTh2 cell proliferation and eosinophilic infiltration in the lungs than Traf4fl/fl mice in the preclinical models of IL-33-mediated type 2 airway inflammation. Mechanistically, we discovered that TRAF4 was required for the activation of AKT/mTOR and ERK1/2 signaling pathways as well as the expression of transcription factor Myc and nutrient transporters (Slc2a1, Slc7a1, and Slc7a5), signature genes involved in T cell growth and proliferation, in ST2+ mTh2 cells stimulated by IL-33. Taken together, the current study reveals a previously unappreciated role of TRAF4 in ST2+ mTh2 cells in IL-33-mediated type 2 pulmonary inflammation, opening up avenues for the development of new therapeutic strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/528093v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18a8f48org.highwire.dtl.DTLVardef@1c4e29corg.highwire.dtl.DTLVardef@f98f72org.highwire.dtl.DTLVardef@38aacb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗