Biology subjects

Ren, H.

Publications and source records attributed to Ren, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

P450-Catalyzed Biaryl Macrocyclization of Leaderless Ribosomal Peptides

Macrocyclic peptides containing biaryl motifs, predominantly derived from natural products, are valuable scaffolds due to their structural rigidity and potent bioactivity. However, current synthetic methods remain constrained by the absence of broadly applicable strategies across chemocatalytic and biocatalytic platforms. Here, we discover a versatile P450 enzyme, GpeC, capable of facilitating oxidative C-C/O/N cross-coupling for peptide biaryl macrocyclization (PBC) with leader-independent activity. Crystal structure analysis of GpeC rationalizes leader-independent property and reveals adaptive recognition that enables access to diverse biaryl-linked macrocycles. GpeC exhibits exceptional substrate promiscuity, accommodating 19 of 20 canonical amino acids and diverse noncanonical analogs within a minimal tetrapeptide scaffold. The efficient semi-synthesis of the natural product Rubrin further demonstrated the versatility of GpeC. We further introduced lytic to tetraregion (LTT), a one-step, single enzyme synthesis for modular synthesis of biaryl-cyclized tetrapeptide. Overall, GpeCs robustness and programmability position it as a broadly applicable biocatalyst for the synthesis of biaryl macrocyclic peptides.

biochemistry↗

bioRxiv2024

δ-α cell-to-cell interactions modulate pancreatic islet Ca2+ oscillation modes

Glucose-induced pancreatic islet hormone release is tightly coupled with oscillations in cytoplasmic free Ca2+ concentration of islet cells, which is regulated by a complex interplay between intercellular and intracellular signaling. {delta} cells, which entangle with cells located at the islet periphery, are known to be important paracrine regulators. However, the role of {delta} cells in regulating Ca2+ oscillation pattern remains unclear. Here we show that {delta}- cell-to-cell interactions are the source of variability in glucose-induced Ca2+ oscillation pattern. Somatostatin secreted from {delta} cells prolonged the islets oscillation period in an cell mass-dependent manner. Pharmacological and optogenetic perturbations of {delta}- interactions led islets to switch between fast and slow Ca2+ oscillations. Continuous adjustment of {delta}- coupling strength caused the fast oscillating islets to transition to mixed and slow oscillations. We developed a mathematical model, demonstrating that the fast-mixed-slow oscillation transition is a Hopf bifurcation. Our findings provide a comprehensive understanding of how {delta} cells modulate islet Ca2+ dynamics and reveal the intrinsic heterogeneity of islets due to the structural composition of different cell types. HighlightsO_LISomatostatin slows down islet Ca2+ oscillations in an cell mass-dependent manner. C_LIO_LIPharmacological and optogenetic perturbations of {delta}- interaction cause islet Ca2+ oscillation mode switching. C_LIO_LIContinuous tuning of {delta}- interaction strength induces fast-mixed-slow oscillation transition successively. C_LIO_LIMathematical modeling shows the fast-mixed-slow oscillation transition as a Hopf bifurcation. C_LI

systems biology↗

bioRxiv2024

Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

Human ANP32A/B (huANP32A/B) poorly support the polymerase activity of avian influenza viruses (AIVs), thereby limiting interspecies transmission of AIVs from birds to humans. The SUMO-interacting motif (SIM) within NS2 promotes the adaptation of AIV polymerase to huANP32A/B via a yet undisclosed mechanism. Here we show that huANP32A/B are SUMOylated by the E3 SUMO ligase PIAS2, and deSUMOylated by SENP1. SUMO modification of huANP32A/B results in the recruitment of NS2, thereby facilitating huANP32A/B-supported AIV polymerase activity. Such a SUMO-dependent recruitment of NS2 is mediated by its association with huANP32A/B via the SIM-SUMO interaction module, where K68/K153-SUMO in huANP32A or K68/K116-SUMO in huANP32B interacts with the NS2-SIM. The SIM-SUMO interactions between huANP32A/B and NS2 function to promote AIV polymerase activity by positively regulating AIV vRNP-huANP32A/B interactions and AIV vRNP assembly. Our study offers insights into the mechanism of NS2 SIM in facilitating AIVs adaptation to mammals.

microbiology↗

bioRxiv2024

Locally released somatostatin triggers cAMP and Ca2+ signaling in primary cilia to modulate pancreatic β-cell function

Somatostatin is produced and released from {delta}-cells within pancreatic islets of Langerhans and constitutes one of the most important negative regulators of islet hormone secretion. Somatostatin receptors (SSTR) are abundantly expressed in the islet cells and coupled to Gi and lowering of intracellular cAMP. We find that both SSTR3 and SSTR5 localize to primary cilia of {beta}-cells, and that activation of these receptors lowers the ciliary cAMP concentration. cAMP produced in the cytosol can enter the cilium through diffusion, but activation of ciliary SSTR3 specifically counteracts the rise of cAMP in the cilium. Moreover, we find that islet {delta}-cells are positioned near primary cilia of the other islet cell types and that somatostatin secretion is directed towards primary cilia. While acute exposure to somatostatin caused a rapid lowering of ciliary cAMP, sustained exposure promoted nuclear entry of the cilia-dependent transcription factor GLI2 through a mechanism that operated in parallel with the canonical Hedgehog pathway and depended on ciliary Ca2+ signaling. We also find that primary cilia length is reduced in islets from human donors with type-2 diabetes, which is associated with a reduction in cilia-{delta}-cell interactions. Our findings show that islet cell primary cilia constitute an important target of somatostatin action that endows it with the ability to regulate islet cell function beyond the acute suppression of hormone release.

cell biology↗

bioRxiv2024

Hepatic Macrophage Migration Inhibitory Factor Promotes Pancreatic Cancer Liver Metastasis in NAFLD

How pathological livers shape tumors, thereby driving pancreatic ductal adenocarcinoma (PDAC) metastasis to the liver, is poorly understood. In the present study, we focus on examining key molecules implicated in this process and assessing their translational significance. We demonstrated that patients with combined non-alcoholic fatty liver disease (NAFLD) have approximately a ninefold increased risk of developing liver metastasis compared to those without NAFLD. In mice model, NAFLD fosters an immunosuppressive microenvironment with increased tumor cell pluripotency and focal adhesion. Mechanistically, NAFLD-induced MIF mediated the progression of PDAC liver metastasis by attracting CD44 positive pancreatic cells. Hepatic MIF knockdown significantly reduced metastases burden with decreased stem-like cancer cells, tumor associated macrophages (TAMs) infiltration and focal adhesion. Targeting the MIF-CD44 axis by either a MIF tautomerase inhibitor, IPG1576, or by CD44 knockdown in tumor cells significantly attenuate liver metastasis of PDAC within the NAFLD context. Patients with PDAC liver metastasis and NAFLD had elevated hepatic MIF expression and increased number of stem-cell like cancer cells. Collectively, our study highlights a pivotal role for MIF-CD44 axis in cancer stemness and offer novel avenues for tailoring therapeutic strategies to individual patients with NAFLD as an underlying condition.

cancer biology↗

bioRxiv2024

CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment

The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explored the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibited growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single- cell transcriptome levels. This mutation induced premature stem-like cell exhaustion, accelerated the maturation of GABAergic neurons, and disrupted neurodevelopmental and synaptic pathways. Additionally, it specifically hindered CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrored the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.

molecular biology↗

bioRxiv2024

MixtureFinder: Estimating DNA mixture models for phylogenetic analyses

In phylogenetic studies, both partitioned models and mixture models are used to account for heterogeneity in molecular evolution among the sites of DNA sequence alignments. Partitioned models require the user to specify the grouping of sites into subsets, and then assume that each subset of sites can be modelled by a single common process. Mixture models do not require users to pre-specify subsets of sites, and instead calculate the likelihood of every site under every model, while co-estimating the model weights and parameters. While much research has gone into the optimisation of partitioned models by merging user-specified subsets, there has been less attention paid to the optimisation of mixture models for DNA sequence alignments. In this study, we first ask whether a key assumption of partitioned models - that each user-specified subset can be modelled by a single common process - is supported by the data. Having shown that this is not the case, we then design, implement, test, and apply an algorithm, MixtureFinder, to select the optimum number of classes for a mixture model of Q matrices for the standard models of DNA sequence evolution. We show this algorithm performs well on simulated and empirical datasets and suggest that it may be useful for future empirical studies. MixtureFinder is available in IQ-TREE2, and a tutorial for using MixtureFinder can be found here: http://www.iqtree.org/doc/Complex-Models#mixture-models.

evolutionary biology↗

bioRxiv2024

PFKP regulates AXL-MET oncogenic and metabolic pathways in lung cancer

ObjectivePFKP (Phosphofructokinase, Platelet Type isoform), as an essential metabolic enzyme, contributes to the high glycolysis rates seen in cancers, while its role in oncogenic pathways, especially from a non-metabolic aspect, is not fully understood. MethodsHere we performed a comprehensive analysis of published RNA-seq, microarray data, and immunohistochemistry of tissue microarray to evaluate the significance of PFKP expression in non-small cell lung cancer (NSCLC). Functionally, we tested the cell proliferation, colony formation, invasion, and migration upon PFKP knockdown in lung cancer cells. Mechanistically, we performed RNA-seq, DIA-mass spectrum, western blot, and qPCR to probe the change of cell signaling pathways upon PFKP silencing. Co-immunoprecipitation and mass spectrum were used to uncover potential PFKP interacting proteins. ResultsWe found that PFKP was highly expressed in NSCLC and was related to poor patient survival. Knockdown of PFKP significantly inhibited cell proliferation, colony formation, invasion, and migration of NSCLC cells. Mechanistically, we found that PFKP can directly bind with AXL and promote its phosphorylation at Y779, thus activating the AXL signaling pathway and promoting MET phosphorylation. In addition, several glycolysis, glutaminolysis, and TCA cycle proteins were downregulated following PFKP silencing. ConclusionsThese data demonstrate that PFKP, beyond its known role in glycolysis, also has a distinct non-metabolic function in affecting lung cancer progression by directly interacting with the AXL-MET axis, thus indicating a potential therapeutic target for lung cancer.

cancer biology↗

bioRxiv2024

Cell circuits underlying nanomaterial specific respiratory toxicology

Nanomaterials emerged as boundless resource of innovation, but their shape and biopersistence related to respiratory toxicology raise longstanding concerns. The development of predictive safety tests for inhaled nanomaterials, however, is hampered by limited understanding of cell type-specific responses. To advance this knowledge, we used single-cell RNA-sequencing to longitudinally analyze cellular perturbations in mice, caused by three carbonaceous nanomaterials of different shape and toxicity upon pulmonary delivery. Focusing on nanomaterial-specific dynamics of lung inflammation, we found persistent depletion of alveolar macrophages by fiber-shaped nanotubes. While only little involvement was observed for alveolar macrophages during the initiation phase, they emerged, together with infiltrating monocyte-derived macrophages, as decisive factors in shifting inflammation towards resolution for spherical nanomaterials, or chronic inflammation for fibers. Fibroblasts, central for fibrosis, sensed macrophage and epithelial signals and emerged as orchestrators of nanomaterial-induced inflammation. Thus, the mode of actions identified in this study will significantly inspire the precision of future in vitro testing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/579746v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d6f061org.highwire.dtl.DTLVardef@3fabb1org.highwire.dtl.DTLVardef@21040eorg.highwire.dtl.DTLVardef@1915c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge, while incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to 7 genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expressions of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/L, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased FAD biosynthesis, enhanced flux entering the TCA cycle and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories. Impact StatementIn this study, yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and successfully incorporating modules for large-scale disulfide bond formation. KmYACs were stably propagated in K. marxianus without compromising the normal growth of the host, irrespective of the selection of telomeres (either Tetrahymena or K. marxianus) and configuration (either linear or circular). KmYACs notably enhanced the expressions of various heterologous proteins, with further yield improvement by supplementing lysine or arginine in the medium. Our findings affirm KmYAC as a robust and versatile platform for transferring large-scale function modules, demonstrating immense potential for both industrial applications and fundamental research.

synthetic biology↗

bioRxiv2023

Attenuating amyloid-beta pathology in mice with in situ programmed astrocytes

Astrocytes are abundant cells in the central nervous system that provide trophic support for neurons and clear detrimental factors, such as A{beta} oligomers (A{beta}Os). However, in the brains of Alzheimers disease (AD) patients, astrocytes lose these physiological functions. Here, we genetically engineered astrocytes with an anti-A{beta}O chimeric antigen receptor (CAR), constructed by replacing the antigen-binding domain of MerTK with an A{beta}O-specific single-chain variable fragment, to direct their phagocytic activity against A{beta}Os. CAR-engineered astrocytes (CAR-As) showed significantly enhanced phagocytosis of A{beta}Os due to effective activation of Rac1, Cdc42 and RhoA and markedly decreased release of pro-inflammatory cytokines due to inhibition of the NF-{kappa}B and cytokine receptor signalling pathways. Consistently, in situ CAR-As markedly ameliorated the cognitive deficits of APP/PS1 transgenic mice possibly by clearing A{beta}Os and creating a non-inflammatory microenvironment for neuronal survival and the restoration of microglia to a healthy phenotype. Our present study is the first to introduce a CAR-A-based therapy, validate its feasibility and effectiveness, and highlight its potential application for the treatment of AD and other brain disorders.

neuroscience↗

bioRxiv2023

Non-modular Fatty Acid Synthases Yield Unique Acylation in Ribosomal Peptides

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries1, 2. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machineries from other natural product families3-8. Here, we report lipoavitides, a class of RiPP/fatty acid hybrid lipopeptides that display a unique, membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N-terminus. The HMP is formed via condensation of isobutyryl-CoA and methylmalonyl-CoA catalyzed by a 3-ketoacyl-ACP synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

biochemistry↗

bioRxiv2023

Autoantibodies against eukaryotic translation elongation factor 1 delta (EEF1D) in two patients with autoimmune cerebellar ataxia

To report a novel autoantibody against eukaryotic translation elongation factor 1 delta (EEF1D) in two patients with autoimmune cerebellar ataxia (ACA). The patients on one center with cerebellar ataxia of unknown cause, who were tested positive with tissue-based indirect immunofluorescence assay (TBA) on rat cerebellum sections and negative for comprehensive anti-neural autoantibodies panel, were investigated for novel autoantibody identification. Tissue-immunoprecipitation (TIP) combined with mass spectrometric (MS) analysis was used to identify the target antigen. The EEF1D protein was identified from complex precipitated by serum and cerebrospinal fluid (CSF) of one patient, while the specific binding of autoantibodies and EEF1D were confirmed by subsequent neutralization experiment, recombinant cell-based indirect immunofluorescence assay (CBA) and western blot. In consequently test using CBA, another anti-EEF1D positive ACA patient was identified and confirmed by western blot. All enrolled 30 health doners and 15 connective tissue diseases patients without neurological disorders were anti-EEF1D negative. The two anti-EEF1D positive patients presented similar clinical manifestations, their outcomes supported the effectiveness of immunotherapy, but the cerebellar atrophy occurred before treatment may be irreversible. Symptoms of one patient worsened again after the weaning of pulse corticosteroid therapy. The identification of anti-EEF1D autoantibody provides a new potential biomarker for early diagnosis and recurrence prevention of ACA.

immunology↗

bioRxiv2023

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimized a bacterial CRISPR-hybrid system coupled with FACS, and identified novel high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allowed effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

synthetic biology↗

bioRxiv2023

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.

biophysics↗

bioRxiv2023

Haplotype-resolve genome assembly and resequencing provide insights into the origin and domestication of modern rose

Modern rose (Rosa hybrida) is a recently formed interspecific hybrid and has become one of the most important and widely cultivated ornamentals. Here, we report the haplotype-resolved chromosome-scale genome assembly of the tetraploid R. hybrida Samantha and a genome variation map of 233 Rosa accessions involving various wild species, and old and modern cultivars. The two subgenomes of Samantha show no significant global bias in gene loss but substantial expression bias, and homoeologous exchanges are frequently observed between subgenomes. Population genomic and genomic composition analyses reveal the contributions of wild Rosa species to modern roses, and highlight that R. odorata and its derived cultivars contribute more to modern roses than R. chinensis Old Blush. Furthermore, selective sweeps during modern rose breeding associated with major agronomic traits, including continuous flowering, floral organ identity, flower senescence, and disease resistance, are identified. This study provides insights into the genetic basis of modern rose origin and breeding history, and offers unprecedented genomic resources for rose improvement.

genomics↗

bioRxiv2023

Leading Basic Modes of Spontaneous Activity Drive Individual Functional Connectivity Organization in the Resting Human Brain

Spontaneous activity of the human brain provides a window to explore intrinsic principles of functional organization. However, most studies have focused on interregional functional connectivity. The principles underlying rich repertoires of instantaneous activity remain largely unknown. We apply a novel eigen-microstate analysis to three resting-state functional MRI datasets to identify basic modes that represent fundamental activity patterns that coexist over time. We identify a few (i.e., five) leading basic modes that dominate activity fluctuations. Each of these modes exhibits a distinct functional system-dependent coactivation pattern and corresponds to specific cognitive profiles. In particular, the spatial pattern of the first leading basis mode shows the separation of activity between the default-mode and primary and attention regions. We further reconstruct individual functional connectivity as the weighted contribution of these leading basic modes based on theoretical modelling. Moreover, these leading basic modes capture sleep deprivation-induced changes in brain activity and interregional connectivity, primarily involving the default-mode and task-positive regions. Our findings reveal a dominant set of basic modes of spontaneous activity that reflect multiplexed interregional coordination and drive conventional functional connectivity, furthering the understanding of the functional significance of spontaneous brain activity.

neuroscience↗

bioRxiv2023

Genome mining unveils a class of ribosomal peptides with two amino termini

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

biochemistry↗