Biology subjects

Jiang, K.

Publications and source records attributed to Jiang, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Metabolic dysfunction promoted by mitochondrial DNA mutation burden drives retinal degeneration

Retinal degenerative diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and glaucoma, have been linked to mitochondrial dysfunction. However, the impact of mitochondrial DNA (mtDNA) mutation accumulation in the context of these retinopathies has yet to be thoroughly explored. Our previous studies focused on the retinal phenotype observed in the PolgD257A mutator mice (D257A), revealing the effects of aging and mtDNA mutation accumulation in the retina. We have reported that this model exhibited significant morphological and functional deficits in the retina by 6 months of age, with notable alterations in the retinal pigment epithelium (RPE) occurring as early as 3 months, including changes in the cristae density and reduction in length of mitochondria. This study investigated how mtDNA mutations affect the metabolic interaction between the retina and RPE in young (3 months) and old (12 months) wild-type (WT) and D257A mice. We assessed cellular energy production using freshly dissected retina samples from both groups through Seahorse analysis, immunofluorescence, and Western blot experiments. The analysis of aged D257A retina punches revealed significantly reduced basal and maximal mitochondrial respiration, along with increased mitochondrial reserve capacity compared to WT. However, glycolytic flux, measured as a function of extracellular acidification rate (ECAR), did not differ between WT and D257A mice. Both D257A retina and RPE exhibited decreased expression of essential electron transport proteins involved in oxidative phosphorylation. Additionally, we observed a reduction in the expression of glucose transporter 1 (GLUT-1) and lactate transporter (MCT1) at the apical surface of the RPE. Enzymes associated with glycolysis, including hexokinase II and lactate dehydrogenase A, were significantly lower in the aged D257A retina, while hexokinase I and pyruvate kinase 2 were upregulated in the RPE. These findings indicate that the accumulation of mtDNA mutations leads to impaired metabolism in both the retina and RPE. Furthermore, it suggests that glucose from the choroidal blood supply is being utilized by the RPE rather than being transported to the neural retina. Mitochondrial dysfunction in RPE promotes a glycolytic state in these cells, leading to reduced availability of metabolites and, consequently, diminished overall retinal function. These results are essential for advancing our understanding of the mechanisms underlying retinal degeneration and provide a new perspective on the role of mtDNA mutations in these diseases.

cell biology↗

bioRxiv2025

DJ-1 deficiency and aging: dual drivers of retinal mitochondrial dysfunction.

We have previously extensively characterized the role of DJ-1 in oxidative stress regulation in the retina and RPE during aging. However, the DJ-1 protein also plays a role in regulating mitochondrias response to oxidative stress by translocating to the mitochondria where it helps clear generated reactive oxygen species (ROS). To study the effects of aging and oxidative stress in the retina, the DJ-1 KO mouse was analyzed. Freshly dissected ex vivo retinal punches were analyzed for real-time live cell metabolism. Total DNA and protein were isolated from RPE, and retina of 3- and 15-month-old DJ-1 WT and DJ-1 KO mice. The mitochondrial DNA (mtDNA) genome was divided into four discrete regions (RI-RIV), and lesions/10kb were quantified using long-extension PCR. mtDNA content was analyzed using RT-qPCR. Protein levels of OXPHOS complexes, POLG, OGG1, SOD2, and PGC1 were measured by western blotting. Seahorse analysis detected significantly decreased basal and maximal OCR in 3- and 15-month-old DJ-1 KO compared to age-matched DJ-1 WT. In the RPE, a significant decrease in the protein levels of the NDUFB8 subunit of CI, the SDHB subunit of CII, and MTCO1 of CIV in 15-month-old DJ-1 KO mice compared to 15-month-old DJ-1 WT, while the ATP5A subunit of CV was significantly decreased in 3-month-old DJ-1 KO mice compared to 3-month-old DJ-1 WT. In the retina, significantly decreased levels of NDUFB8 subunit of CI and MTCO1 of CIV were detected in the in 3-month-old DJ-1 KO mice compared to 3-month-old DJ-1 WT. We observed a significant increase in mtDNA gene content in 15-month-old DJ-1 KO RPE and retina compared to age-matched DJ-1 WT. The PGC1 levels significantly decreased in 3- and 15-month-old RPE lysates compared to their age group DJ-1 WT. However, in the retina, there was only a decrease in DJ-1 WT with aging. The POLG levels increased when 15-month-old DJ-1 KO lysates were compared to 15-month-old DJ-1 WT. The mtDNA lesions in the RPE detected a trend of increase in 15-month-old DJ-1 WT and DJ-1 KO RPE in all regions compared to 3-month-old mice. In the retina, a significant increase in mtDNA lesions/10kb accumulation in the 15-month-old DJ-1 WT RIV was detected compared to 3-month-old DJ-1 WT. The OGG1 levels significantly increased in 3-month-old retinal lysates compared to the 3-month-old DJ-1 WT. Our findings suggest that DJ-1 is critical for mitochondrial regulation and function in RPE and retina. The observed changes reflect mitochondrial dysfunction related to absence of DJ-1.

neuroscience↗

bioRxiv2025

Aerobic glycolysis and lactate regulate histone H3K18Lactylation occupancy to fine-tune gene expression in developing and mature retina

High aerobic glycolysis in retinal photoreceptors, as in cancer cells, is implicated in mitigating energy and metabolic demands. Lactate, a product of glycolysis, plays a key role in epigenetic regulation through histone lactylation in cancer. Here, we demonstrate that increased ATP production during retinal development is achieved primarily through augmented glycolysis. Histone lactylation, especially H3K18La, parallels enhanced glycolysis and lactate in developing retina and in retinal explants. Multi-omics analyses, combined with confocal imaging, reveal the localization of H3K18La near H3K27Ac in euchromatin at promoters of active retinal genes. H3K18La and gene expression are also concordant with glucose metabolism in retinal explants. Evaluation of accessible chromatin at H3K18La marked promoters uncovers an enrichment of GC-rich motifs for transcription factors of SP, KMT and KLF families, among others, indicating specificity of H3K18La-mediated gene regulation. Our results highlight glycolysis/lactate/H3K18La as a regulatory axis in fine-tuning gene expression in developing and mature retina.

molecular biology↗

bioRxiv2025

Stress Granule Formation Drives ZBP1-dependent Necroptosis in Non-obstructive Azoospermia and Aging testes

Male infertility and age-related reproductive decline remain major unmet medical challenges, with limited understanding of the underlying mechanisms. Here, we identify a stress granule-driven necroptosis pathway involving ZBP1 and RIPK3 as a central driver of non-obstructive azoospermia (NOA), a severe form of male infertility marked by loss of spermatogenesis. We show that heat stress or environmental insults activate eIF2 kinases, inducing stress granules that recruit ZBP1 and RIPK3 to form a signaling complex. This leads to RIPK3 activation, MLKL phosphorylation, and necroptotic death of spermatogonia and Sertoli cells. Genetic ablation of Zbp1 or Ripk3 protects mice from heat-induced testicular atrophy, highlighting their essential role in testicular cell death. Notably, this same necroptosis pathway is also activated in aged human testes, suggesting a shared mechanism driving both male infertility and age-related testicular degeneration.

molecular biology↗

bioRxiv2025

Saturation mapping of MUTYH variant effects using DNA repair reporters

Variants of uncertain significance (VUS) limit the actionability of genetic testing. A prominent example is MUTYH, a base excision repair factor associated with polyposis and colorectal cancer, which has a pathogenic variant carrier rate approaching 1 in 50 individuals in some populations. To systematically interrogate variant function in MUTYH, we coupled deep mutational scanning with a DNA repair reporter containing its lesion substrate, 8OG:A. Our variant-to-function map covers >97% of all possible MUTYH point variants (n=10,941) and achieves 100% accuracy classifying the pathogenicity of known clinical variants (n=247). Leveraging a large clinical registry, we observe significant associations with colorectal polyps and cancer, with more severely impaired missense variants conferring greater risk. We recapitulate known functional differences between pathogenic founder alleles, and highlight sites of complete missense intolerance, including residues that intercalate DNA and coordinate essential Zn2+ or Fe-S clusters. This map provides a resource to resolve the 1,032 existing missense VUS and 90 variants with conflicting interpretations in MUTYH, and demonstrates a scalable strategy to interrogate other clinically relevant DNA repair factors.

genomics↗

bioRxiv2025

Precision in Spinal Cord Injury Research: A Novel Electromagnetic Impactor for a Consistent Porcine Model

PurposeReplicating spinal cord injury (SCI) in large animals is necessary for evaluating therapeutics for potential human translation, yet there is currently no commercial, standardized device for inducing SCI. We present the fabrication and testing of a custom impactor device for producing repeatable contusion SCI in porcine models. MethodsWe first designed and built the device. Mechanical modeling was subsequently utilized to calibrate our benchtop testing setup. Benchtop verification was performed to measure impact force post calibration. We then used the device to generate a contusion SCI model in 2 pigs and the results were compared to an uninjured pig. Intraoperative ultrasound was used to visualize a hematoma in the injured spinal cord. Hematoxylin-eosin (H&E) and Massons trichrome staining were used to confirm injury presence on ex vivo spinal cord samples. ResultsMechanical modeling forces matched benchtop impact forces within 1.4 N, indicating successful calibration of the testing setup. Our device demonstrated repeatability and the potential for modulating injury severity on the benchtop. Impactor forces were demonstrated across a range from 12.8 to 67.6 N, with variability remaining within 0.2 to 0.7 N standard deviation. The device induced two contusion injuries of different severity in vivo, confirmed by intraoperative ultrasound imaging and post-excision histology of the spinal cord. ConclusionOur impactor device is a major advancement towards producing repeatable and titratable contusions in large animal SCI models.

bioengineering↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2024

Restoration of PKM1 improves functional maturation of human stem-cell derived-β cell by regulating PEP metabolism

Human stem cell-derived {beta} (SC-{beta}) cells still exhibit limited glucose response required for insulin secretion due to glycolytic bottlenecks, yet how these metabolic abnormalities impact glucose response and functional maturation of SC-{beta} cells remains unclear. In this study, we identified a metabolic checkpoint located at PEP accumulation that impeded the functional maturation, which was rescued by restoration of pyruvate kinase 1 (PKM1). Glucose-tracing metabolomics in human stem cell-derived islets revealed abnormal glycolytic PEP accumulation at resting condition, resulting in impaired calcium response and insulin secretion upon high glucose or glycolytic metabolite stimulation. Mechanistically, elevated PEP significantly raised intracellular basal calcium levels, leading to downregulated expression of genes involved in TCA cycle elucidated by single cell transcriptomics. Furthermore, the activity of pyruvate kinase, which metabolizes PEP, was notably reduced due to low PKM1 expression. By overexpressing PKM1, the impairment of TCA-related genes caused by PEP accumulation was reversed via modulating PEP metabolism, resulting in enhanced calcium responses and insulin secretion upon high glucose stimulation. Together, we discovered a novel role of PKM1-regulated PEP metabolism in mediating the functional maturation of human SC-{beta} cells. This study highlights the importance of metabolic reprogramming in human SC-{beta} cell maturation, advancing cell therapy approaches for diabetes treatment.

cell biology↗

bioRxiv2024

TIA1 Mediates Divergent Inflammatory Responses to Tauopathy in Microglia and Macrophages

The RNA binding protein TIA1 is known to regulate stress responses. Here we show that TIA1 plays a much broader role in inflammatory cells, being required for the microglial sensome. We crossed TIA1 cKO mice (using a CX3CR1 driven cre element) to PS19 MAPT P301S tauopathy mice. The peripheral macrophages of TIA1 cKO mice exhibited a hyper-inflammatory phenotype with increased cytokine signaling, as expected. Surprisingly, the brains of these mice showed striking reductions in inflammation, including decreases in microglial inflammatory cytokines (TNF and IL-1{beta}) and sensome markers (CLEC7A, TREM2, ITGAX); these reductions were accompanied by corresponding decreases in tau pathology. Analysis of the brain TIA1 protein interactome identified brain selective TIA1 protein mediated pathways, including strong interactions with the microglial protein C1q, which directs pruning of dystrophic neurons. These results uncover a previously unknown regulatory role for TIA1 in microglial activation in the context of neurodegenerative disease and highlights the divergent regulation of two mononuclear phagocytic lineages: microglia and macrophages.

neuroscience↗

bioRxiv2024

Inhibition on neutrophil extracellular traps by oligomeric procyanidins alleviate chemotherapy-induced chronic kidney injury via gut-kidney axis

Cisplatin is one of the most widely used chemotherapeutic agents for various solid tumors in the clinic, but its use is limited by adverse effects in normal tissues. In particular, cisplatin administration often damages the kidneys. However, little is known about how to alleviate cisplatin-induced chronic kidney disease (CKD) specifically. Here, we found that repeated low-dose cisplatin (RLDC) recruited susceptible neutrophils to the proximal tubule, thereby promoting the progression of CKD in the mouse model. Mechanically, cisplatin destroyed the intestinal epithelium, which induced dysregulation of gut flora and intestinal leakage. Under the dual stimulation of intestinal bacteria and cisplatin, neutrophils become extremely susceptible to form Neutrophil extracellular traps (NETs), accumulating in the proximal tubule and promoted chronic inflammation, fibrosis, and chronic hypoxia, leading to poor regeneration that promoted CKD progression. NETs provided a scaffold for tissue factors (TF) adhesion and metalloid-matrix protease 9 (MMP-9) activation, which triggered local ischemia and hypoxia. In addition, NETs promoted inflammasome construction through NOD-like receptor thermal protein domain associated protein 3 (NLRP3) shear and secretion of mature interleukin-18 (IL18), which subsequently released interferon- {gamma} (IFN-{gamma}), contributing to renal interstitial fibrosis. We proposed that oligomeric procyanidins (OPCs) ameliorated RLDC-induced CKD through multi-targeting damage induced by NETs. OPCs ameliorated microcirculatory disorders and inhibited inflammation by protecting the intestinal mucosa barrier and subsequent bacterial endotoxin translocation. Furthermore, we found that OPCs altered the susceptibility of neutrophils to form NETs. In summary, OPC alleviates CKD by inhibiting NETs production via TF/MMP-9 and IL-18-NLRP3 pathways. OPCs protect the kidney through anti-inflammatory and antioxidant activities, and restore the balance of the intestinal flora.

immunology↗

bioRxiv2024

Using Functional Genomic Data in Monocytes/Macrophages and Genotyping to Nominate Disease-Driving Single Nucleotide Polymorphisms and Target Genes in Juvenile Idiopathic Arthritis

IntroductionGWAS have identified multiple regions that confer risk for juvenile idiopathic arthritis (JIA). However, identifying the single nucleotide polymorphisms (SNPs) that drive disease risk is impeded by the SNPs that identify risk loci being in linkage disequilibrium (LD) with hundreds of other SNPs. Since the causal SNPs remain unknown, it is difficult to identify target genes and use genetic information to inform patient care. We used genotyping and functional data in primary human monocytes/macrophages to nominate disease-driving SNPs on JIA risk haplotypes and identify their likely target genes. MethodsWe identified JIA risk haplotypes using Immunochip data from Hinks et al (Nature Gen 2013) and the meta-analysis from McIntosh et al (Arthritis Rheum 2017). We used genotyping data from 3,939 children with JIA and 14,412 healthy controls to identify SNPs that: (1) were situated within open chromatin in multiple immune cell types and (2) were more common in children with JIA than the controls (p< 0.05). We intersected the chosen SNPs (n=846) with regions of bi-directional transcription initiation characteristic of non-coding regulatory regions detected using dREG to analyze GRO-seq data. Finally, we used MicroC data to identify gene promoters interacting with the regulatory regions harboring the candidate causal SNPs. ResultsWe identified 190 SNPs that overlap with dREG peaks in monocytes and126 SNPs that overlap with dREG peaks in macrophages. Of these SNPs, 101 were situated within dREG peaks in both monocytes and macrophages, suggesting that these SNPs exert their effects independent of the cellular activation state. MicroC data in monocytes identified 20 genes/transcripts whose promoters interact with the enhancers harboring the SNPs of interest. ConclusionSNPs in JIA risk regions that are candidate causal variants can be further screened using functional data such as GRO-seq. This process identifies a finite number of candidate causal SNPs, the majority of which are likely to exert their biological effects independent of cellular activation state in monocytes. Three-dimensional chromatin data generated with MicroC identifies genes likely to be influenced by these SNPs. These studies demonstrate the importance of investigations into the role of innate immunity in JIA.

genetics↗

bioRxiv2024

Rapid protein evolution by few-shot learning with a protein language model

Directed evolution of proteins is critical for applications in basic biological research, therapeutics, diagnostics, and sustainability. However, directed evolution methods are labor intensive, cannot efficiently optimize over multiple protein properties, and are often trapped by local maxima. In silico-directed evolution methods incorporating protein language models (PLMs) have the potential to accelerate this engineering process, but current approaches fail to generalize across diverse protein families. We introduce EVOLVEpro, a few-shot active learning framework to rapidly improve protein activity using a combination of PLMs and protein activity predictors, achieving improved activity with as few as four rounds of evolution. EVOLVEpro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with up to 100-fold improvement of desired properties. We showcase EVOLVEpro for five proteins across three applications: T7 RNA polymerase for RNA production, a miniature CRISPR nuclease, a prime editor, and an integrase for genome editing, and a monoclonal antibody for epitope binding. These results demonstrate the advantages of few-shot active learning with small amounts of experimental data over zero-shot predictions. EVOLVEpro paves the way for broader applications of AI-guided protein engineering in biology and medicine.

bioengineering↗

bioRxiv2024

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock

Hundreds of bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have recently been identified. Biological functions have been ascribed to an increasing number of microproteins, some of which play roles in bacterial stress responses. In this work, we provide evidence that GndA, a frameshifted 36-amino acid microprotein encoded in an internal open reading frame overlapping the 6-phosphogluconate dehydrogenase (6PGD) coding sequence (CDS), functions in the response to heat shock. We demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA is hydrophobic and associates with the pyruvate dehydrogenase complex and respiratory complex I (RCI). Consistent with a role in cellular homeostasis, GndA mutations alter the transcriptional responses to heat shock. These results demonstrate that GndA promotes bacterial cell growth at high temperature via a mechanism distinct from that of the 6PGD protein that it overlaps. GndA thus expands the paradigm of bacterial microproteins that function in cellular stress responses.

biochemistry↗

bioRxiv2023

Electroadhesive hydrogel interface for prolonged mucosal theranostics

Establishing a robust and intimate mucosal interface that allows medical devices to remain within lumen-confined organs for extended periods has valuable applications, particularly for gastrointestinal (GI) theranostics. Here, we report the development of e-GLUE, an electroadhesive hydrogel interface for robust and prolonged mucosal retention following electrical activation. Notably, this novel mucosal adhesion mechanism can increase the adhesion energy of hydrogels on the mucosa by up to 30-fold and enable in vivo GI retention of e-GLUE devices for up to 30 days. Strong mucosal adhesion occurs within one minute of electrical activation, despite the presence of luminal fluid, mucus exposure, and organ motility, thereby ensuring compatibility with complex in vivo environments. In swine studies, we demonstrate the utility of e-GLUE for mucosal hemostasis, sustained local delivery of therapeutics, and intimate biosensing in the GI tract. This system can enable improved treatments for various health conditions, including gastrointestinal bleeding, inflammatory bowel disease, and diagnostic applications in the GI tract and beyond.

bioengineering↗

bioRxiv2023

Comparison of the three-dimensional chromatin structures of adolescent and adult peripheral blood B cells: implications for the study of pediatric autoimmune diseases

Background/PurposeKnowledge of the 3D genome is essential to elucidate genetic mechanisms driving autoimmune diseases. The 3D genome is distinct for each cell type, and it is uncertain whether cell lines faithfully recapitulate the 3D architecture of primary human cells or whether developmental aspects of the pediatric immune system require use of pediatric samples. We undertook a systematic analysis of B cells and B cell lines to compare 3D genomic features encompassing risk loci for juvenile idiopathic arthritis (JIA), systemic lupus (SLE), and type 1 diabetes (T1D). MethodsWe isolated B cells from healthy individuals, ages 9-17. HiChIP was performed using CTCF antibody, and CTCF peaks were identified. CTCF loops within the pediatric were compared to three datasets: 1) self-called CTCF consensus peaks called within the pediatric samples, 2) ENCODEs publicly available GM12878 CTCF ChIP-seq peaks, and 3) ENCODEs primary B cell CTCF ChIP-seq peaks from two adult females. Differential looping was assessed within the pediatric samples and each of the three peak datasets. ResultsThe number of consensus peaks called in the pediatric samples was similar to that identified in ENCODEs GM12878 and primary B cell datasets. We observed <1% of loops that demonstrated significantly differential looping between peaks called within the pediatric samples themselves and when called using ENCODE GM12878 peaks. Significant looping differences were even less when comparing loops of the pediatric called peaks to those of the ENCODE primary B cell peaks. When querying loops found in juvenile idiopathic arthritis, type 1 diabetes, or systemic lupus erythematosus risk haplotypes, we observed significant differences in only 2.2%, 1.0%, and 1.3% loops, respectively, when comparing peaks called within the pediatric samples and ENCODE GM12878 dataset. The differences were even less apparent when comparing loops called with the pediatric vs ENCODE adult primary B cell peak datasets.The 3D chromatin architecture in B cells is similar across pediatric, adult, and EBVtransformed cell lines. This conservation of 3D structure includes regions encompassing autoimmune risk haplotypes. ConclusionThus, even for pediatric autoimmune diseases, publicly available adult B cell and cell line datasets may be sufficient for assessing effects exerted in the 3D genomic space.

genomics↗

bioRxiv2023

Engineering synthetic phosphorylation signaling networks in human cells

Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Here, we describe the engineering of artificial phosphorylation networks in which "push-pull" motifs--reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities--are assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells. We demonstrate that the composability of our design scheme enables model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression. We leverage these capabilities to engineer cell-based cytokine controllers that dynamically sense and suppress activated T cells. Our work introduces a generalizable approach for designing and building phosphorylation signaling circuits that enable user-defined sense-and- respond function for diverse biosensing and therapeutic applications.

synthetic biology↗

bioRxiv2023

Structure-based virtual screening identifies small molecule inhibitors of O-fucosyltransferase SPINDLY

Protein O-glycosylation is a nutrient-signaling mechanism that plays essential roles in maintaining cellular homeostasis across different species. In plants, SPINDLY (SPY) and SECRET AGENT (SEC) catalyze posttranslational modifications of hundreds of intracellular proteins by O-fucose and O-linked N-acetylglucosamine, respectively. SPY and SEC play overlapping roles in cellular regulation and loss of both SPY and SEC causes embryo lethality in Arabidopsis. Using structure-based virtual screening of chemical libraries followed by in vitro and in planta assays, we identified a SPY O-fucosyltransferase inhibitor (SOFTI). Computational analyses predicted that SOFTI binds to the GDP-fucose-binding pocket of SPY and competitively inhibits GDP-fucose binding. In vitro assays confirmed that SOFTI interacts with SPY and inhibits its O-fucosyltransferase activity. Docking analysis identified additional SOFTI analogs that showed stronger inhibitory activities. SOFTI treatment of Arabidopsis seedlings decreased protein O-fucosylation and caused phenotypes similar to the spy mutants, including early seed germination, increased root hair density, and defect in sugar-dependent growth. By contrast, SOFTI had no visible effect on the spy mutant. Similarly, SOFTI inhibited sugar-dependent growth of tomato seedlings. These results demonstrate that SOFTI is a specific SPY O-fucosyltransferase inhibitor and a useful chemical tool for functional studies of O-fucosylation and potentially for agricultural management.

plant biology↗

bioRxiv2023

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

molecular biology↗