Protein & Cell

Chromatin organization plays a critical role in regulating gene expression. Chromatin compaction represses gene expression by physically restricting the access of the transcriptional machinery to DNA, while spatial proximity between enhancers and promoters, often mediated by chromatin loops, is essential for gene activation. To investigate the regulatory mechanisms underlying loop formation and chromatin compaction, as well as their effects on gene expression, we developed CRISPR-FOIL (utilizing CRISPR to FOld and ILluminate chromosomal DNA), a novel programmable platform for engineering chromatin loops and inducing chromatin compaction in live cells. CRISPR-FOIL anchors pairs of genomic loci in proximity by engineered single-guide RNAs (sgRNAs), resulting in an artificial chromatin loop. The fused two CRISPR-Sirius gRNAs enable genomic loci to be visualized through fluorescent RNA coat proteins in various colors. In addition, multiple CRISPR-FOIL complexes can act cooperatively to drive chromatin compaction. These results establish CRISPR-FOIL as a powerful tool for engineering chromatin organization in live cells and highlight its potential as a therapeutic platform for gene regulation and disease control.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

Compact CRISPR nucleases are attractive for therapeutic genome editing because their small coding sequences facilitate delivery by adeno-associated virus. Type II-D Cas9 (Cas9d) enzymes constitute the most compact Cas9 subtype, yet only a few orthologs have demonstrated mammalian genome-editing activity, leaving it unclear whether this activity is general or exceptional. Here, we mined the IMG/M metagenomic database and identified five previously uncharacterized MG102-like Cas9d orthologs ([~]950 amino acids) that share the hallmark genomic, sequence, and structural features of type II-D Cas9. Two of them, Cas9d-1 and Cas9d-4, recognized a 5-NRC-3 protospacer-adjacent motif and edited endogenous human loci with efficiencies up to 20.1%, exceeding Streptococcus pyogenes Cas9 at one site, while producing deletion-biased outcomes and no detectable off-target activity. Notably, both orthologs edited more efficiently than the sole previously validated member of this lineage, MG102-2, when assayed side by side under identical conditions. These findings establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV- compatible scaffolds for in vivo therapeutic genome editing.

Compact type II-C Cas9 nucleases are attractive for therapeutic genome editing because their small size enables packaging into adeno-associated viral (AAV) vectors, and their extended protospacer-adjacent motifs (PAMs) reduce off-target cleavage while expanding targeting scope. Yet characterized type II-C orthologs have edited mammalian cells far less efficiently than the canonical SpCas9. Here, we used embedding-based metagenomic mining of >4.7 x 10 proteins, combined with AlphaFold3 structure prediction and locus-context analysis, to identify three previously uncharacterized compact type II-C Cas9 orthologs, NsuCas9 (1,092 aa), PsuCas9 (1,084 aa), and GfoCas9 (1,074 aa), and benchmarked them in vitro and in human HEK293T cells. All three are robust RNA-guided nucleases with distinct PAM specificities (N CC, N NYAA, and N RHAA, respectively), divergent thermal profiles, and asymmetric sgRNA cross-compatibility. In human cells, PsuCas9 with an N ATAA PAM reaches 78.4% indels and matches or exceeds SpCas9 at multiple loci, representing the first natural compact type II-C ortholog reported to do so, while GfoCas9 and NsuCas9 add complementary coverage. All three show a strong deletion-biased repair signature and no detectable editing across 33 predicted off-target sites. These compact, high-fidelity nucleases expand the CRISPR targeting space for AAV-deliverable therapeutic editing.

Programmable endonucleases such as CRISPR/Cas9 provide powerful tools to edit mammalian genomes by engaging cellular mechanisms of DNA double-strand break (DSB) repair. CRISPR-catalysed homology-directed repair (CRISPR-HDR), though generally less efficient than other modes of DNA repair, holds particular promise to enable precise sequence replacement by targeted insertion of a homologous DNA template1,2. While recent studies have reported appreciable levels of HDR in cardiomyocytes in vivo3, skeletal muscle myofibres have historically been considered refractory to HDR-mediated genome editing4. Furthermore, how repair outcomes differ across tissues after systemic delivery of CRISPR/Cas9 editors, whether precise HDR editing can be achieved in regenerative tissue stem cells, and how developmental timing influences accessibility to CRISPR-induced repair remain unclear. Here, we use an adeno-associated virus (AAV)-delivered in vivo GFP-to-BFP colour-switching reporter system (AAV-GFP-to-BFP) to examine in vivo CRISPR-HDR with cellular- and tissue-level resolution. We find that postnatal cardiac muscle, skeletal muscle, and muscle stem cells undergo templated HDR at different rates across discrete developmental stages in mice. While HDR-edited muscle stem cells and myofibres were readily detectable after in vivo editing in juvenile mice, editing in neonatal mice yielded more efficient HDR in cardiac tissue. Based on these results, we adapted the CRISPR-HDR approach to rescue the therapeutically relevant Dmd mutation in mdx mice, demonstrating recoding to the wild-type protein sequence in both skeletal and cardiac muscles. These results provide a framework for advancing donor-templated DNA repair in living postnatal animals, and reveal unexpected cellular, developmental, and disease-related constraints on precise, therapeutic in vivo gene correction.

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

Human Dicer (hDicer) is a key enzyme in the RNA interference (RNAi) pathway that generates [~]21-22 nt micro-RNA (miRNAs) and small interfering RNAs (siRNAs). We have previously shown that hDicer also generates tRNA-derived small RNAs (tsRNAs), which mediate nuclear gene silencing and regulate hundreds of disease-associated genes. As powerful and evolutionarily conserved cellular regulators, tsRNAs emerged as an important class of small RNAs. Therefore, it is essential to understand their biogenesis. However, the molecular and structural basis of tRNA cleavage by hDicer, as well as the role of chemical modifications such as 5-methylcytosine (m5C), in this process, remain unknown. Here, we present the first structural insights into hDicer in complex with tRNA, obtained by cryo-electron microscopy (cryo-EM), selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and molecular dynamics (MD) simulations. Our results reveal that tRNAs adopt alternative conformations that are recognized and processed by hDicer. Furthermore, we show that tRNA cleavage by hDicer is facilitated by the m5C modification deposited by Nop2/SUN RNA methyltransferase 2 (NSUN2). Collectively, our findings redefine tRNAs as bona fide hDicer substrates and uncover a modification-dependent biogenetic pathway that reshapes the current understanding of the origins and regulation of human small RNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/744379v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@ad78aborg.highwire.dtl.DTLVardef@cd3a7dorg.highwire.dtl.DTLVardef@1bb2594org.highwire.dtl.DTLVardef@1a0427e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Precise installation of DNA methylation at selected loci offers a powerful strategy for regulating gene expression without altering DNA sequence, but existing plant epigenome editors are constrained by limited efficiency, locus dependence, and genome-wide off-target methylation. Here, we developed SunTag-MQ1v variants incorporating TRBIP1, which promotes removal of the antagonistic H3K4me3 mark, and CHLAMY, an oligomerizing alpha crystalline domain protein from Chlamydomonas reinhardtii. TRBIP1 enhanced methylation and silencing at the Arabidopsis FWA promoter but caused widespread off-target methylation and severe developmental defects. Adding CHLAMY produced SunTag-CHLAMY-TRBIP1-MQ1v (designated as SunTag-NOVA), which successfully overcame the lethality and widespread off-target effects associated with direct TRBIP1-MQ1v fusions. We demonstrate that CHLAMY drives higher-order oligomerization of the editing complex, which enhances target specificity and mitigates off-target accumulation. SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences. These results show that combining local chromatin modification with controlled effector assembly can improve targeted DNA methylation, and establish SunTag-NOVA as a specific epigenome-editing platform for plants.

Echovirus 18 (E18) is a predominant pathogen causing aseptic meningitis in children, and post-E18 infection frequently triggers myasthenia gravis-like autoimmune neurological damage. This pathological process relies on neonatal Fc receptor (FcRn)-mediated IgG transcytosis across mucosal barriers, and FcRn also acts as an essential functional receptor required for E18 attachment and uncoating during host cell invasion. At present, no E18-specific prophylactic vaccine has been clinically approved, and anti-FcRn monoclonal antibodies are the available therapeutics to alleviate autoantibody-mediated tissue injury. We constructed an integrated automated phylogenetic pipeline named evolution_conservation, which enables rapid tracing of the evolutionary position and genetic relatedness of clinical isolates to identify closely related strains from previous outbreaks. Serving as an in silico alternative to animal experiments, this pipeline supports reference-guided vaccine design and longitudinal comparative assessment of vaccine safety and efficacy, facilitates identification of patient populations presenting rare post-viral sequelae, and accelerates clinical trial progression. In this study, we inserted the pre-screened linear epitope E18-3 into a truncated hepatitis B core (HBc) scaffold to generate chimeric virus-like particles (VLPs). A non-invasive floss-based gingival mucosal immunization mouse model was established, with subcutaneous Freunds adjuvant immunization set as the control group. ELISA results confirmed that gingival mucosal delivery of particulate HBc-E18-3 VLPs alone could induce sustained high levels of antigen-specific intestinal IgG in vivo. Drawing on research paradigms of therapeutic neoantigen vaccines for tumor recurrence prevention, the evolution_conservation bioinformatic pipeline and mucosal VLP platform described herein establish an innovative framework for developing antigen-competitive prophylactic and therapeutic vaccines targeting FcRn for myasthenia gravis and autoimmune encephalitis.

A major goal for hepatitis C virus (HCV) vaccine development is to elicit broadly neutralizing antibodies (bNAbs) against the E1E2 glycoprotein complex located on the viral surface. Inducing HCV bNAbs requires engagement of their germline B cell precursors. HCV glycoproteins usually do not bind and activate inferred germline precursors of bNAbs (igl-bNAbs), possibly because most circulating strains contain non-conserved isolate-specific residues, even in bNAb epitopes. Here, we generated stabilized native-like soluble E1E2 (sE1E2) antigens based on a consensus sequence of HCV (HepCon) to limit the exposure of antigenically rare residues. The antigenicity and glycosylation profiles show that HepCon sE1E2 resembles a native-like E1E2 heterodimer. HepCon sE1E2 induced cross-reactive neutralizing antibody responses as a soluble protein immunogen and as membrane-anchored mRNA-delivered immunogen in animals. Importantly, HepCon sE1E2 engages multiple igl-bNAbs against two major epitopes: antigenic region 3 (AR3), which is targeted by igl-bNAbs derived from the widely expressed human VH1-69 B cell gene, and antigenic region 4 (AR4), which is only present on native-like E1E2. Nanoparticles with HepCon sE1E2 efficiently activated B cell lines expressing AR3 and AR4 igl-bNAb B cell receptors in vitro. Finally, using HepCon sE1E2 we elucidated the atomic contacts of an AR3 igl-bNAb by cryo-electron microscopy. Thus, HepCon sE1E2 is a promising candidate for germline-targeting vaccination strategies.

De novo mutations underlying early-onset systemic autoinflammatory diseases have identified key regulators of innate immunity, including pathways that drive IL-1-mmediated inflammation. Here we describe two unrelated girls presenting in infancy with systemic inflammation and sterile lung abscesses, who harbor the same de novo gain-of-function mutation in dysferlin (DYSF; p.P1449L) Myeloid expression of DYSF P1449L enhances COP-I binding, promotes dysferlin retention in the ER-Golgi, and disrupts vesicle trafficking and membrane homeostasis. Dysferlin-mutant monocytes and M2-like macrophages exhibit ectopic perinuclear NLRP3 inflammasome activation, increased IL-1{beta} production, and inflammatory cell death. Mutant M2-like macrophages further display defects in membrane expansion, exocytosis, efferocytosis, and debris clearance, promoting neutrophil recruitment and DAMP-signal amplification that culminate in sterile abscess formation. These findings identify dysferlin as a regulator of membrane homeostasis in myeloid cells, establish defective membrane-stress adaptation as trigger of NLRP3 inflammasome activation, and define a novel IL-1 mediated autoinflammatory disease caused by gain-of-function DYSF mutations.

Post-COVID-19 syndrome (PCS) is characterized by fatigue, neurological impairment and systemic symptoms. This heterogeneity of symptoms hinders biomarker development. Here, we profiled extracellular-vesicle (EV) surface markers in plasma and CSF from 61 participants with PCS (COVIDpost), 80 recovered controls (COVIDreco), and 10 participants with non-SARS-CoV-2 post-viral syndromes. EVs were analysed by bead-based multiplex flow cytometry using tetraspanin-directed (TSPN) and phosphatidylserine-directed lactadherin (PS) detection. Amongst 37 targets covering tetraspanins and vasculature-, immunity- and stemness-associated markers, none met a 1% false-discovery-rate threshold. However, L1-regularized logistic regression under fully nested 5x5 cross-validation identified a distributed plasma EV profile, with mean out-of-fold areas under the receiver operating characteristic curve (AUCs) of 0.788 (95% CI 0.715 - 0.852) for TSPN and 0.716 (95% CI 0.636 - 0.792) for PS detection. Across the pooled COVIDpost and COVIDreco population, EV classification scores covaried with clinical group differences, but did not track clinical severity within either cohort. These PCS-EV classification scores decreased at one-year follow-up in COVIDpost participants. Our findings identify an internally cross-validated multivariable EV surface profile associated with COVIDpost versus COVIDreco status and support independent validation and exploration of EV-based biomarkers in post-viral fatigue syndromes.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with epidemic potential. Despite the threat NiV poses, no therapeutics are licensed to treat infection. Studies have shown that monoclonal antibodies (mAb) can protect animals against NiV and the related Hendra virus (HeV). The best studied mAb, m102.4, has been used to treat infected patients on a compassionate basis, and has entered clinical trials. However, there is a need to define additional mAbs with therapeutic potential, which could be combined with m102.4 to improve neutralising potency and breadth. Here, we isolated five high affinity mAbs from an mRNA immunised pig, which bound the G glycoprotein derived from NiV Malaysia strain (NiV-M), and one of which (mAb A2) also bound HeV G. Aligned with this, all mAbs neutralised NiV-M pseudovirus but only mAb A2 neutralised pseudovirus representing the NiV Bangladesh (NiV-B) strain. mAb A2 and the most potent NiV-M neutralising mAb, C1, showed minimal competition with each other and m102.4, suggesting recognition of non-overlapping epitopes. Single-particle cryogenic electron microscopy of the NiV-M G receptor binding domain complexed to A1 and C2 Fab fragments revealed distinct epitopes that did not overlap with the receptor-binding site, targeted by m102.4, suggesting action through steric impedance of receptor binding or interference downstream of receptor engagement. Inoculation of mAb A2 to hamsters did not provide complete protection against NiV-B challenge (60% survival), however, a split dose of mAb A2 and m102.4 provided the same protection as m102.4 alone (100% survival). Collectively, these data demonstrate the potential of the porcine model for isolation of therapeutic candidate mAbs, which contribute both to our understanding of the NiV G antigenic landscape, and the development of mAb combinations, that exert complementary mechanisms of neutralisation, for therapeutic intervention.

Influenza A virus antigenic drift forces annual vaccine reformulation, motivating the search for conserved epitope candidates that could support broadly protective vaccines. We systematically screened influenza A virus sequences (H1N1, H3N2, H5N1; nine viral proteins) to define 98 conserved candidate regions, 38 of which were identical across the H1N1, H3N2, and H5N1 consensus sequences, all in the polymerase complex and nucleoprotein (PB2, PB1, PA, NP), whereas the ten surface-glycoprotein (HA/NA) candidates were subtype-specific. We then benchmarked two protein-language-model (ESM-2) features against alignment conservation. Group-masked log-probability correlated moderately with MSA conservation (Spearman rho = 0.25 to 0.39 for HA) but provided no incremental value for T-cell epitope discrimination (change in AUROC +0.004, p = 0.46); attention-derived contact-density was not a valid solvent-accessibility proxy. A curated antibody-epitope benchmark (22 clusters, 5 neutralization-supported) was underpowered for a high-confidence B-cell test. We document data-quality and reproducibility pitfalls (length heterogeneity, coordinate mapping, and pseudoreplication) and release the auditable benchmark. These results provide an auditable candidate resource and show that, in the evaluated benchmarks, ESM-2 sequence scores did not improve epitope prioritization beyond alignment-derived conservation.

Lung cancer remains the leading cause of cancer-related mortality worldwide, with cigarette smoking (CS) representing its primary risk factor. In addition to promoting tumorigenesis, chronic CS exposure contributes to chemotherapy resistance, although the underlying mechanisms remain poorly understood. Here, we established a long-term CS exposure model by repeatedly treating Lewis lung carcinoma (LLC) cells with cigarette smoke extract (CSE). After 4 months of exposure, CSE-treated cells exhibited enhanced proliferation, migration, and resistance to chemotherapy-induced cell death. Mechanistically, chronic CSE exposure suppressed receptor-interacting protein kinase 3 (RIPK3) expression by upregulating the epigenetic regulator enhancer of zeste homolog 2 (EZH2), which promoted repressive histone methylation at the RIPK3 promoter. Loss of RIPK3 impaired chemotherapy-induced cell death primarily by inhibiting ferroptosis rather than necroptosis. Importantly, genetic depletion or pharmacological inhibition of EZH2 restored RIPK3 expression and sensitized lung cancer cells to gemcitabine treatment both in vitro and in vivo. Furthermore, analysis of human lung cancer datasets revealed an inverse correlation between EZH2 and RIPK3 expression, with RIPK3 levels progressively decreasing with smoking history. Collectively, these findings identify the EZH2/RIPK3 axis as a critical mediator of smoking-associated chemoresistance and uncover a previously unrecognized role for RIPK3 in ferroptosis regulation. Targeting EZH2-mediated RIPK3 suppression may represent a promising therapeutic strategy to overcome chemoresistance in lung cancer patients with a history of smoking.

A missense variant in TM6SF2 (transmembrane 6 superfamily member 2, TM6SF2E167K) is a major risk factor for steatotic liver disease1, while protecting against coronary artery disease2. TM6SF2 is a polytopic resident protein of the smooth endoplasmic reticulum (ER) and ER-Golgi intermediate compartment that promotes lipidation of hepatic ApoB-containing lipoproteins before secretion into the circulation. Here, we used cryo-electron microscopy (cryo-EM) to determine the structures of TM6SF2 and TM6SF2E167K at 3.64 [A] and 3.58 [A] resolution, respectively. TM6SF2 comprises 10 transmembrane helices that bind a single cholesterol molecule within a transmembrane cavity. The protein assembles into homodimers and homotetramers that interact with ApoB. Structural and biochemical analyses show that the E167K substitution reduces cholesterol binding and ApoB interaction without disrupting overall protein structure. Expression of wild-type, but not mutant, TM6SF2 restores hepatic triglyceride secretion in TM6SF2-deficient hepatocytes. Together, these findings establish the first structural framework for the bulk lipidation step in hepatic lipoprotein biogenesis, the principal pathway for hepatic triglyceride and cholesterol export into the circulation.

Cells in multicellular eukaryotic systems are diverse biological units, with characteristics and functions determined by their molecular profiles. CRISPR-Cas9 genome editing has been widely used across biology to modulate gene expression and study gene function. However, there is currently no versatile and scalable method for editing a cells genome in response to endogenous cellular signals. Here, we report the engineering of a CRISPR guide RNA that efficiently confers genome editing in response to the catalytic activity of a target microRNA (miRNA) within a cell. miRNAs are short non-coding RNAs that are widely conserved across eukaryotes and can cleave their target RNA through almost perfect base pairing. In mammals, miRNAs are largely involved in development and homeostasis as well as disease progression and developmental disorders. To leverage these properties for genome editing, we developed a cuffed guide RNA (cgRNA) which is composed of a permutated order of sequence domains from the commonly used single guide RNA (sgRNA). These permutated domains were then concatenated with a miRNA target sequence, yielding a warped guide RNA that is inactive until cleaved by a complementary miRNA. We demonstrated that cgRNA enabled efficient miRNA activity-dependent genome editing in human and mouse cell lines. Biochemical and structural analyses revealed three stages of inhibition of the CRISPR genome-editing pathway for unprocessed cgRNA. Utilizing a lentiviral library of cgRNAs containing miRNA targets covering mouse genome-wide miRNAs, we identified miRNA cleavage activities and their sequence specificities in mouse embryonic stem cells and during smooth muscle cell differentiation. Furthermore, we showed that endogenous mRNA expression could be irreversibly recorded into a DNA sequence using a cgRNA targeted by a synthetic miRNA repeat. cgRNA is a simple, robust, miRNA activity-gated genome editing system that could facilitate the development of cell state-specific genome editing, the mapping of miRNA activity and gene expression landscapes, and the recording of molecularly determined cell states during the long-term progression of multicellular systems.

Retinol-binding protein 4 (RBP4) exhibits diurnal oscillatory pattern and is elevated under conditions of circadian disruption and in type 2 diabetes mellitus, yet the molecular link between RBP4 and impaired glucose metabolism remains elusive. Here, we overexpressed RBP4 in human hepatoma Huh7 cells and performed integrated RNA sequencing (RNA-seq), Co-immunoprecipitation (Co-IP) coupled with mass spectrometry (MS), and Cleavage Under Targets and Tagmentation (CUT&Tag). We identified BACH1 as a direct RBP4-interacting transcription factor that predominantly binds the TGACTCA motif in promoter regions of genes involved in carbon metabolism pathways. Integrative analysis of RNA-seq and CUT&Tag data uncovered 63 direct target genes co-regulated by RBP4 and BACH1, including known circadian and metabolic regulators SLC7A11, PFKFB3, CTCF, NR1D2 and WEE1 as well as novel candidates SF1 and PIN1. These target genes are significantly enriched in insulin receptor signaling and carbohydrate metabolic pathways. Mechanistically, the RBP4-BACH1 axis reprograms glucose metabolism, linking circadian rhythm disturbances to dysregulated glucose homeostasis. Collectively, our findings establish a functional role for RBP4 in connecting circadian disruption to diabetes and highlight RBP4 as a potential therapeutic target.

O-GlcNAcylation is an important post translational modification that regulates numerous cellular processes, yet tools enabling selective removal of O GlcNAc from individual proteins via endogenous O-GlcNAcase (OGA) in living cells remain limited. Here, we report De-O GlcNAcylation-targeting chimeras (DOGTACs), a chemically induced proximity strategy that selectively reduces O GlcNAc from target proteins by recruiting endogenous OGA. Initial designs incorporating potent competitive OGA inhibitors efficiently engaged OGA but failed to induce de-O-GlcNAcylation, revealing that catalytic competence is essential for productive proximity-driven editing. By attenuating inhibitor potency while retaining sufficient OGA engagement, we developed optimized DOGTACs that promote concentration- and time-dependent, target-specific de-O-GlcNAcylation in living cells without perturbing global O-GlcNAc levels. Furthermore, we successfully applied DOGTAC to additional target proteins across multiple cell lines. Collectively, this work established attenuated competitive inhibitors as effective recruitment modules for catalytic enzyme engagement and a novel framework, DOGTAC, for targeted de-O-GlcNAcylation via endogenous OGA recruitment in living cells.

We report the generation and characterization of a human induced pluripotent stem cell (iPSC) line derived from dermal fibroblasts of a patient with Skogholt disease, a rare maternally inherited neurodegenerative syndrome associated with choroid plexus dysfunction and impaired cerebrospinal fluid (CSF) homeostasis. Patient fibroblasts were reprogrammed using the non-integrating Repro-OSKGM kit. The resulting iPSC line exhibited typical pluripotent morphology, expressed canonical pluripotency markers, maintained a normal karyotype, retained the disease-associated genetic variant, was mycoplasma-free, and demonstrated trilineage differentiation potential. We also made choroid plexus (ChP) like organoids from the generated iPSCs. This patient-specific iPSC line provides a valuable resource for generating choroid plexus organoids and neurons to investigate disease mechanisms and develop therapeutic strategies.