The ankyrin repeat is one of the most abundant protein-protein interaction motifs in eukaryotes yet occurs in only a small number of ion channels. These channels are all members of the transient receptor potential (TRP) superfamily and contain prominent ankyrin repeat domains (ARDs) in their cytoplasmic N termini. In TRP vanilloid 4 (TRPV4), the importance of this domain has been highlighted by the finding that gain-of-function neuromuscular disease-causing missense mutations cluster on the ARD surface. Little is known currently about the extent of the TRPV4-ARD interactome, nor how it may be altered by disease-causing mutations. Here, we utilized a human proteome microarray to profile the ARD interactomes of WT and mutant TRPV4. Probing of the microarray with TRPV4-ARD revealed 78 interactors, including proteins related to ubiquitination and small GTPase signaling, such as the ubiquitin ligase NEDD4L and the RhoGEF ARHGEF10. In parallel experiments, we also identified the deubiquitinase OTUB2 as an interactor of the proximal N terminus. Comparison of the ARD interactomes of WT and mutant TRPV4 revealed 21 interactions affected by disease-causing mutations. Strikingly, one of these interactors, ARHGEF10, is also mutated in neuromuscular disease. Cell-based studies confirmed that ARHGEF10 exhibits a reduced capacity to co-immunoprecipitate with mutant TRPV4. Furthermore, calcium imaging studies demonstrated that ARHGEF10 overexpression suppressed TRPV4 channel activity, but that this inhibition is abrogated by disease-causing mutations. Together, these findings provide insights into the functional roles of an ion channel ARD, as well as their disruption in disease, and offer a resource for future cell-based studies.

Leukemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) known for its specificity towards Ras homolog family member A (RhoA). LARG plays a crucial regulatory role in various cellular processes such as migration, proliferation, invasion, and metastasis by facilitating the exchange of GDP to GTP on RhoA. Phosphorylation of LARG at S1288 by ribosomal S6 kinase 2 (RSK2) promotes RhoA activation. However, the precise mechanism remains unclear. Here, we demonstrate the essential role and mechanism by which S1288 phosphorylation facilitates LARG-mediated invasiveness in response to Epidermal Growth Factor (EGF). Upon EGF stimulation, RSK2 phosphorylates LARG at S1288, thereby promoting the membrane translocation of LARG. Furthermore, phosphorylation of LARG at S1288 markedly enhances the assembly of the LARG-RhoA complex and subsequent activation of RhoA through GTP loading. Analysis of patient-derived Glioblastoma Multiforme (GBM) cell lines revealed a correlation between RSK activation and LARG S1288 phosphorylation. Moreover, GBM tissue samples showed LARG S1288 phosphorylation and RhoA-GTP-bound RhoA. Elucidating the regulatory mechanisms governing this process is crucial for the development of LARG-targeted therapeutic interventions.

Gastric cancer (GC) is one of major global cancers that is highly heterogeneous and has a poor prognosis, especially in cases associated with Helicobacter pylori (H. pylori) infection. H. pylori promote metastasis via mechanisms like endoplasmic reticulum stress (ERS). Telomere-protecting protein 1 (TPP1), a telomere-protecting protein, is overexpressed in GC and linked to poor outcomes. This study investigated TPP1's role in H. pylori-induced ERS and its implications for GC metastasis.We analyzed TPP1 expression using both Starbase data and clinical samples. Functional assays (e.g., migration, invasion, wound healing) were performed in GC cell lines with TPP1 knockdown or overexpression. The interaction between TPP1 and ERS-related proteins was assessed by immunoprecipitation and immunofluorescence. The role of TPP1 in GC metastasis was further validated in a xenograft mouse model. TPP1 was upregulated in GC tissues and cell lines, correlating with poor prognosis. TPP1 knockdown inhibited GC cell metastasis but not proliferation, while TPP1 overexpression enhanced metastasis. H. pylori enhanced TPP1 expression by stabilizing Enhancer of Zeste Homolog 1 (EZH1), which in turn binds to the TPP1 promoter. TPP1 interacted with 78 kDa Glucose-regulated Protein (GRP78), disrupting its binding to PKR-like ER kinase (PERK) and activating ERS. Blocking ERS reversed the pro-metastatic effects of TPP1 overexpression. In vivo, TPP1 knockdown significantly reduced GC metastasis in nude mice xenograft model. TPP1, induced by H. pylori, promoted GC metastasis by enhancing ERS via interaction with GRP78. Targeting the TPP1/ERS axis may offer a novel therapeutic strategy for GC.

Ovarian cancer (OC) remains a leading cause of gynecological cancer-related mortality, largely due to metabolic reprogramming and aggressive progression. Zinc finger protein 280A (ZNF280A), a poorly characterized transcriptional regulator, has recently been implicated in tumorigenesis, but its mechanistic role in OC remains undefined. Here, we identify ZNF280A as an oncogenic driver that promotes OC progression through transcriptional regulation of acrosomal vesicle protein 1 (ACRV1) and activation of the PI3K/AKT signaling pathway. ZNF280A expression was markedly elevated in OC tissues and cell lines and correlated with advanced clinicopathological features and poor patient survival. Functional assays revealed that ZNF280A knockdown inhibited OC cell proliferation, migration, and tumorigenesis while inducing apoptosis both in vitro and in vivo. Mechanistically, ZNF280A enhanced ACRV1 transcription by interacting with the transcription factor CUX2, thereby facilitating its recruitment to the ACRV1 promoter. Elevated ZNF280A or ACRV1 expression activated PI3K/AKT signaling and increased glycolytic enzyme expression (PKM2, LDHA), glucose uptake, lactate production, ATP generation, and extracellular acidification rate, whereas pharmacological inhibition of AKT or glycolysis abrogated these effects. Collectively, our findings establish ZNF280A as a key regulator of metabolic reprogramming in OC through the CUX2-ACRV1-PI3K/AKT axis, highlighting this pathway as a potential therapeutic target in ovarian cancer.

ROR1 is a member of the receptor tyrosine kinase (RTK) family that plays a crucial role during organogenesis of bone and neural systems by regulating non-canonical Wnt signaling. Misregulation of ROR1 is additionally a causative factor for carcinogenesis in solid and liquid tumors. However, we have a poor understanding of how ROR1 activity is regulated. We employed a recently developed single-molecule method termed SiMPull-POP to study the oligomeric state of ROR1. RTK function is typically triggered by ligand binding, which promotes self-assembly of RTKs to form dimers and in some cases oligomers. However, our data indicate that ROR1 does not follow this paradigm. Instead, ROR1 forms dimers and oligomers in a process that is not affected by the presence of the ROR1 ligand Wnt5a. Additional experiments indicate that the transmembrane domain of ROR1 has a strong tendency to self-assemble, suggesting that this domain modulates ROR1 dimerization. Investigation into a regulatory mechanism for ROR1 self-assembly led to evaluation of the role of the lipid cholesterol, which plays pleiotropic roles in Wnt signaling. Cholesterol was found to promote the assembly of ROR1, and our results point to the transmembrane domain as the region where cholesterol exerts this regulatory effect. Taken together, our results indicate that ROR1 self-assembles in human cells; however, unlike other RTKs, this process is not stabilized by ligand binding but is instead facilitated by membrane cholesterol.

Initially discovered in Drosophila, the Hippo pathway is pivotal for tissue growth and organ homeostasis. It is regulated by both extrinsic and intrinsic signals and exerts its effect via a core kinase cascade, in which large tumor suppressor 1 and 2 (LATS1/2) plays a key role. LATS1 has also been shown to regulate mitotic progression by phosphorylating myosin phosphatase targeting subunit 1 (MYPT1) to counteract the activity of polo-like kinase 1 (PLK1), a mitotic master kinase. Herein we demonstrate that the hexosamine biosynthetic pathway regulates the Hippo pathway via LATS1. We show that LATS1 interacts with the O-GlcNAc transferase (OGT) and is O-GlcNAcylated. Via electron transfer dissociation mass spectrometry, we mapped the O-GlcNAcylation sites to be S479/S482/T484/T485. O-GlcNAcylation attenuates LATS1 protein stability, and downregulates the phosphorylation level of its downstream substrates, such as MYPT1. Subsequently, decreased MYPT1-pS473 levels enhance PLK1-pT210 levels and drive mitotic progression. Importantly, we demonstrate that in Drosophila O-GlcNAcylation of LATS1 promotes the wing size. Thus, this study suggests that O-GlcNAcylation links extrinsic glucose levels to LATS1 in the Hippo pathway and cell proliferation.

The transferrin receptor 1 (TfR1)-transferrin (TF) axis is central to iron homeostasis and represents a validated route for delivering biologics across the blood-brain barrier (BBB). We developed human-specific anti-TfR1 nanobodies (NewroBus) that exploit this pathway, but their lack of cross-reactivity with rodent TfR1 limits conventional preclinical testing. To overcome this, we generated knock-in (KI) rats in which the coding sequences of the endogenous Tfrc and Tf genes were replaced with their human counterparts, producing animals that express human TfR1 and/or human TF under physiological control. Rats homozygous for both humanized alleles were viable and fertile, indicating functional replacement of their rodent orthologs, but exhibited erythropoietic abnormalities and altered iron distribution-reduced splenic and increased hepatic iron-suggesting incomplete compensation. In contrast, heterozygous rats displayed only mild, subclinical microcytosis and hypochromia while maintaining normal BBB integrity and near-physiological iron homeostasis. Using these heterozygous humanized Tfrc rats, we demonstrated that a biologic engineered to engage human TfR1, NewroBus, fused to a therapeutic payload such as TNFα-neutralizing nanobodies, achieved significant BBB penetration and CNS exposure. These results validate the translational relevance of this model for studying TfR1-mediated drug delivery. Overall, the humanized TfR1-TF axis is compatible with life and systemic iron regulation, albeit with gene-dosage-dependent effects on compensation. These KI rats provide a unique, translationally relevant platform for evaluating the pharmacokinetics, CNS penetration, efficacy, and safety of human-specific biologics engineered to access the brain via human TfR1-mediated transcytosis.

The MYC oncogene encodes a transcription factor that regulates cell growth, metabolism, and proliferation. Its dysregulation is a hallmark of many cancers, including prostate cancer. Elevated c-MYC expression promotes tumor progression and therapy resistance, yet c-MYC remains a challenging therapeutic target due to its intrinsically disordered structure and lack of enzymatic activity. Identifying upstream regulators of MYC activity may reveal new therapeutic strategies. α/β-Hydrolase domain-containing protein 5 (ABHD5) is best known as a coactivator of the triglyceride lipase PNPLA2, facilitating intracellular lipolysis. However, recent studies have suggested a tumor-suppressive role for ABHD5 in various cancers, including prostate cancer, though the molecular mechanisms remain unclear. Here, we identify ABHD5 as a suppressor of c-MYC-driven transcriptional programs in prostate cancer cells. Transcriptomic profiling in 22Rv1 cells revealed that ABHD5 overexpression downregulates MYC target genes and reduces c-MYC protein levels. In contrast, ABHD5 knockout increased c-MYC protein expression, enhanced cell proliferation, and markedly elevated colony-forming capacity. ABHD5 deficiency also conferred resistance to the pharmacologic c-MYC inhibitor 10058-F4. Notably, PNPLA2 knockout failed to phenocopy these effects, indicating that ABHD5's tumor-suppressive function is independent of its canonical lipolytic role. Furthermore, ABHD5 overexpression continued to suppress c-MYC in PNPLA2-deficient cells, confirming a lipase-independent mechanism. These findings define a previously unrecognized role for ABHD5 as a negative regulator of c-MYC and highlight a novel, noncanonical pathway linking lipid metabolism regulators to oncogene control in prostate cancer.

G⍺, the alpha subunit of the most abundant heterotrimeric G protein in the brain, mediates signaling by opioids and by many neuromodulators to inhibit neural function. An open question is whether activated G⍺-GTP directly binds to and regulates effector molecules, like all other animal G⍺ proteins, or if it signals solely by releasing Gβγ subunits. Using mouse brain lysates as native source of G⍺ and its potential effectors, we analyzed immunopurified G⍺ protein complexes by mass spectrometry. Pre-activating G⍺ in the lysates with GTPγS resulted in a ∼6 fold increase in the amount of the small G protein GTPase activators RASA3 and RASA2 in the purified complexes, the largest increase among all G⍺-associated proteins, making RASA2/3 candidate G⍺ effectors. Using purified recombinant proteins, we found that RASA3 binds directly to G⍺-GTPγS more strongly than it does to G⍺-GDP. We also found that the addition of Ca, a second messenger produced by the G⍺ pathway that opposes G⍺ signaling, strengthened binding of RASA3 to G⍺-GDP . A C-terminal fragment of RASA3 containing a predicted Ca site was sufficient to bind G⍺, albeit more weakly and without a preference for the activated state of G⍺. We present a model in which RASA3 could mediate G⍺ signaling using two distinct G⍺-binding sites: one on full-length RASA3 that preferentially binds active G⍺-GTP, and a second on the RASA3 C-terminus that binds inactive G⍺ in the presence of Ca.

Test systems enabling preclinical assessment of drug effects in relevant models are essential for optimizing the selection of candidate therapeutics before their further clinical translation. Xenograft tissue slice tandem co-culture (XTCC) models were developed as ex vivo systems for visualizing glioblastoma (GBM) tumor growth and invasion into the complex host tissue structures of the brain. Work here tested the XTCC model for delineating specific drug effects, in particular inhibition of invasion as major issue in GBM. The established chemotherapeutic Temozolomide (TMZ) and three promising candidates - two histone deacetylase (HDAC) inhibitors, Vorinostat and Entinostat, and the neuropeptide Apamin - were tested. XTCCs were generated by placing G55T2 or U87-MG cell-derived tumor xenograft tissue slices onto murine cortical brain slices. Upon drug treatment, effects on growth, invasion, proliferation, and apoptosis were analyzed by immunohistochemistry. Differences in invasion capacity were seen between the two cell lines. Profound invasion-inhibitory effects of 100 μM TMZ were accurately monitored and substantially higher than inhibition of the bulk tumor mass. Likewise, the extent of single-cell invasion into the normal brain tissue was massively inhibited by Vorinostat and especially by Entinostat, indicating that HDAC inhibitor treatment is particularly efficient in inhibiting GBM cell invasion. Despite the absence of inhibitory effects of Apamin in 2D cell culture, G55T2 XTCCs revealed ∼70% reduced GBM invasion, associated with a substantial inhibition of proliferation as indicated by loss of Ki-67 positivity. Taken together, we show the suitability of the XTCC models for monitoring tumor growth and, in particular anti-invasive effects of drugs.

The oncogenic role of VAV1, a GTPase guanine nucleotide exchange factor (GEF) with cytoplasmic and nuclear localizations, has been previously reported in multiple malignancies. Most of the mechanisms underlying this pro-tumoral activity have been linked to the cytoplasmic expression of this GEF. To date, the contribution of nuclear VAV1 to cancer development remains poorly understood. Here, using models of pancreatic ductal adenocarcinomas (PDAC), the most common subtype of pancreatic cancer, we provide evidence of a novel mechanism driving oncogenic gene expression in PDAC cells that is regulated by nuclear VAV1. We show that VAV1 wild-type, unlike its mutant lacking the nuclear localization signal (NLS), localizes to the nucleus of PDAC cells where it increases GLI transcriptional activity without affecting the expression of GLI factors (GLI1, GLI2 and GLI3). Interestingly, this VAV1 NLS-deficient mutant loses interaction with Importin β1 but maintains ability to activate RAC1. Further analysis showed that VAV1 and GLI1 endogenously interact in PDAC cells, and knockdown of VAV1 reduces the expression of a set of GLI target genes including BCL2. We found VAV1 bound to the GLI binding motif present within the BCL2 promoter region and demonstrate the requirement of VAV1 to maintain BCL2 expression and promoter activity. Finally, we showed that VAV1 is necessary for the binding of GLI1 and its coactivator the histone acetyltransferase PCAF to this regulatory element. Taken together, our data supports a role for VAV1 in GLI1 transcriptional regulation, elucidating a new mechanism of function for nuclear VAV1 in PDAC cells.

Megalencephalic leukoencephalopathy with subcortical cysts is a rare leukodystrophy primarily caused by mutations in two genes: MLC1, encoding a membrane protein of unknown function, and GlialCAM, a cell adhesion molecule. Although MLC1 has been implicated in downregulating signaling pathways, its molecular mechanisms remain elusive. Recently, the orphan G protein-coupled receptor GPRC5B was identified as a novel interactor of both GlialCAM and MLC1, with dominant heterozygous mutations found in MLC patients, suggesting that GlialCAM and MLC1 may regulate cell signaling via GPRC5B. Here, we show that GPRC5B exhibits constitutive activity, which is inhibited by MLC1, likely through interference with GPRC5B oligomerization. Conversely, GlialCAM enhances β-arrestin 2 recruitment, leading to its own mislocalization from cell-cell junctions. MLC-associated GPRC5B mutants show enhanced maturation and increased stability at the plasma membrane, retain normal constitutive activity and responsiveness to MLC1 and GlialCAM but display increased affinity for GlialCAM and localize to cell-cell junctions in its presence. Notably, co-expression of GlialCAM with these mutants does not induce GlialCAM mislocalization. We propose a model in which finely tuned interactions among GPRC5B, GlialCAM, and MLC1 regulate receptor signaling. These findings provide the first biochemical evidence of GlialCAM and MLC1 modulating GPRC5B activity, suggesting a biochemical explanation for the gain-of-function phenotype observed in GPRC5B MLC mutants. Importantly, our work supports the potential of targeting GPRC5B as a therapeutic strategy in MLC.

Fibroblast growth factor 21 (FGF21) is a hepatokine that regulates systemic metabolism. Here, we delineate a novel regulatory pathway for FGF21 orchestrated by the small GTPase Rab2A. Our previous findings demonstrated that liver-specific deficiency of Rab2A impairs very-low-density lipoproteins lipidation and promotes apolipoprotein B (APOB) accumulation. We now show that accumulated APOB drives the cleavage and activation of cyclic-AMP-responsive-element-binding protein H (CREBH), a key hepatic transcription factor for FGF21 expression. Mechanistically, hepatic Rab2A inhibition protected mice from high-fat diet induced obesity and was associated with markedly elevated circulating FGF21, the phenotype largely rescued by adeno-virus-mediated knockdown of either CREBH or APOB. Collectively, we define a Rab2A-APOB-CREBH axis that is potentially essential for the hepatic regulation of FGF21.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments establish that at room temperature the L99A cavity mutant of T4 lysozyme (L99A T4L) interconverts between two compact folded conformations on the millisecond timescale. These include the native state in which the sidechain of Phe114 is exposed to solvent (E state) and a near native minor state in which the aromatic moiety of Phe114 is buried in the core of the protein (B state, ∼2%). Molecular dynamics simulations that have captured the E to B interconversion in L99A T4L and related mutants suggest that these proteins adopt compact folded conformations other than E and B, yet extensive CPMG studies have not detected such states. In an effort to detect these more elusive conformers experimentally we have recorded Chemical Exchange Saturation Transfer (CEST) experiments, as the widths of minor state dips in the resulting CEST profiles are sensitive to additional, even more sparse, conformers. Analysis of amide N and CH CEST profiles recorded on L99A T4L show that in addition to states E and B, a rare state (I) populated to ∼0.2 % (11.5 C) exchanges rapidly with state B. CEST-based urea m-values establish that all three states are compact, with interconversion between them proceeding via compact transition states. This study highlights the utility of CEST to characterize the free energy surface of a protein by detecting states with a wide range of lifetimes (100 ms to 100 μs) in ways that are not possible using other relaxation-based NMR techniques.

Magnetic resonance imaging (MRI) is a widely used tool for tumor diagnosis, but current contrast agents often lack sufficient sensitivity and specificity. To address these limitations, we developed novel gadolinium-based (Gd) contrast agents conjugated with epidermal growth factor receptor (EGFR)-binding peptides (EBPs) for targeted MRI of EGFR-overexpressing tumors. The synthesized EBP-Gd-DO3A and EBP-(Gd-DO3A) conjugates exhibited high binding affinity and specificity for EGFR-positive cells, as demonstrated through cell binding assays and fluorescence microscopy. Competitive binding assays confirmed EGFR-mediated specificity. In vitro MRI revealed strong contrast enhancement in EGFR-overexpressing tumor cells, while in vivo studies in xenograft models showed increased tumor uptake and prolonged contrast retention. These agents demonstrated superior specificity and sensitivity compared to traditional non-targeted Gd-DO3A, offering a promising approach for improved tumor detection, grading, and prognosis in clinical molecular imaging.

Hundreds of KCNJ2 (Kir2.1) variants of uncertain significance (VUS) have been associated with Andersen-Tawil Syndrome (ATS). Remarkably, most Kir2.1 variants' surface expression and function have been described via deep mutational scans (DMS). These results have provided an unprecedented picture of Kir2.1 structure-function relationships and insights into VUS. However, these studies are limited by the lack of robust validation. We performed a flow cytometry-based Kir2.1 surface expression assay for 70 variants (61 ATS-linked) distributed across the potassium inward rectifier channel, a thermal stability assay of 20 variants with reduced surface expression, a yeast-based functional assay for 20 variants (10 pathogenic or likely pathogenic (P/LP) and 10 VUS), as well as whole-cell patch clamp for 13 variants (4 P/LP and 9 VUS). Kir2.1 cell surface expression results showed that ∼30% of variants have reduced surface expression when co-expressed with WT and that ∼25% disagreed with the DMS datasets. ∼70% of variants with reduced surface tested had reduced thermal stability. Our yeast assay showed all 10 P/LP variants exhibiting LOF and 7 out of 10 VUS were LOF, in contrast to the DMS method. Patch clamp data further validated the yeast assay. Our data underscores the limitations to interpreting the Kir2.1 DMS datasets, demonstrates a proof-of-principle yeast assay as complementary method to better inform ClinVar classifications, and provides several lines of evidence for LOF of 9 Kir2.1 VUS in the process.

Vasoinhibin exerts potent inhibitory effects on angiogenesis and vascular permeability through a minimal three-amino acid sequence, the HGR motif. However, the nature of the vasoinhibin receptor has remained controversial. Here, we identify integrin α5β1 as the endothelial cell-surface binding molecule mediating the actions of the HGR motif. Vasoinhibin binds to α5β1 integrin through this motif, and silencing the integrin α5 subunit abolishes the vasoinhibin-mediated inhibition of endothelial cell proliferation, invasion, permeability, and tube formation in vitro. Likewise, an antibody against integrin α5β1 prevented the antiangiogenic activity of vasoinhibin in the Matrigel plug assay in vivo. Notably, the HGR motif activates integrin α5β1, as reflected by an increase in endothelial cell adhesion to fibronectin, the canonical ligand of integrin α5β1. These findings identify integrin α5β1 as the molecular target of vasoinhibin mediating its antiangiogenic and anti-vasopermeability actions. Furthermore, a novel integrin activation mechanism leading to suppressed angiogenesis is unveiled, thereby challenging the conventional integrin inhibition approach as a therapeutic intervention.

Pathogenic variants of human CDC14A (cell division cycle 14A) are associated either with nonsyndromic deafness DFNB32 or HIIMS, hearing impairment infertile male syndrome. The 623-residue CDC14A protein has two globular domains (residues 17-152 and 217-325) and a 278-residue C-terminal intrinsically disordered region (IDR). To date, 16 recessive variants of human CDC14A are associated with hearing loss. Variants affecting the globular N-terminal domains of human CDC14A are associated with HIIMS while mutations in the IDR cause nonsyndromic deafness DFNB32. Here, we tested the hypothesis that human CDC14A c.1033C>T variant, segregating with nonsyndromic deafness in family PKSN10, introduces a premature translation stop codon (p.R345X) yet the mRNA escapes nonsense-mediated decay (NMD) and produces sufficient active phosphatase to allow for male fertility. Quantitative analyses of CDC14A mRNA in blood leukocytes from PKSN10 family showed CDC14A transcripts are stable including homozygous (p.R345X) transcripts which evade NMD. To further test this hypothesis, we performed biochemical and structural characterizations of truncated CDC14A (ΔC-CDC14A) protein retaining only residues 1 to 345. Kinetic functional studies and X-ray crystallographic findings of purified ΔC-CDC14A protein indicate that it retains structural integrity and phosphatase activity. Molecular genetic reports of DFNB32 and HIIMS, taken together with structural and functional data in this study, indicate that phosphatase activity of ΔC-CDC14A containing the two globular domains is sufficient for male fertility but insufficient for normal hearing. In addition, we show that the C-terminal IDR of CDC14A is required for normal hearing, likely because it is necessary for normal localization of CDC14A in hair cells.

The High Mobility Group Nucleosome-binding (HMGN) proteins are small, abundant nuclear proteins that directly bind nucleosomes and form a major component of chromatin. HMGN proteins localize to enhancers and actively transcribed genes across the genome; however, their roles in regulating chromatin structure and transcription remain poorly understood. Although it is well-established that HMGN proteins bind to the H2A-H2B acidic patch on nucleosomes, other potential nucleosome targeting mechanisms including histone post-translational modifications (PTMs) and histone variants remain unclear. To investigate the nucleosomal binding preferences and function of HMGN proteins, we engineered mouse embryonic stem cells (mESCs) lacking HMGN1 and/or HMGN2 (Hmgn1 mESCs, Hmgn2 mESCs, and Hmgn1Hmgn2 mESCs) and profiled gene expression and localization of architectural proteins. In the absence of these HMGN proteins, ∼1,000 genes were differentially expressed, including cell identity genes, with most genes being downregulated. Nucleosome binding assays revealed preferential binding of HMGN1 and HMGN2 proteins to nucleosomes with acetylated H3 tail residues and nucleosomes containing the histone variant H2A.Z. Additionally, in vitro acetylation assays demonstrated that binding of HMGN1 and HMGN2 to nucleosomes reduces p300-mediated acetylation of H3K18, H3K23, and H3K27 residues. An epiproteomic mass spectrometry analysis of histone tail modifications revealed that Hmgn1Hmgn2 mESCs have increased steady state levels of H3K27me2 and H3K27me3, but not H3 tail acetylation, relative to WT cells. Together, these findings show that HMGN proteins function as both sensors and modulators of the histone PTM landscape inside cells, playing a critical role in the dynamic balance between active and repressive chromatin states.

Biallelic variants in CHKA, which encodes the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, cause an inherited disorder characterized by epilepsy, microcephaly, and intellectual disability. How a deficiency in CHKA activity manifests these neurological symptoms is poorly understood. In this study, we investigated patient-derived fibroblasts with CHKA missense variants to elucidate the molecular and biochemical mechanisms underlying the associated pathologies. CHKA variant fibroblasts exhibited impaired phospholipid and triacylglycerol synthesis, altered mitochondrial morphology and function, elevated reactive oxygen species (ROS) levels, and increased lipid peroxidation, suggesting a mechanism by which defective CHKA activity leads to lipid damage. Treatment with FCCP, a mitochondrial uncoupler, reduced ROS levels and attenuated lipid peroxidation in CHKA patient fibroblasts, suggesting a potential approach to therapeutic intervention.

Long interspersed element 1 (LINE-1, L1) is a eukaryotic retrotransposon which copies itself through an RNA intermediate. The mutagenic insertion mechanism, termed target-primed reverse transcription (TPRT), requires coordinated activities of the encoded ORF2 protein (ORF2p) endonuclease (EN) and reverse transcriptase (RT) domains. TPRT is initiated when ORF2p EN nicks a target site in genomic DNA (gDNA), creating a 3'-OH that primes ORF2p RT for cDNA synthesis using the bound L1 RNA template. L1 insertions occur preferentially at 5'-TTTTT↓AA consensus motifs, a preference that could be driven by either site-specific EN cleavage or by sequence requirements in the subsequent RT priming step, in which the cut gDNA flap must base pair with the poly(A) RNA template. We find that in vitro, EN is promiscuous, cutting linear DNA oligos and plasmids at many non-consensus sites. We discovered a novel cleavage activity on a mismatched substrate that was nicked ∼40-fold faster than duplex DNA containing the consensus site and identify three features enabling rapid cutting. First, L1 EN cuts two nucleotides downstream of mismatches, favoring A-G mismatches or T•G/U•G wobble pairs. Second, both mismatch and consensus sequences are cut >2-fold faster when proximal to a DNA end. Third, end-proximal EN cutting depends on end composition: 5' overhangs cut fastest, followed by 3' overhangs, followed by blunt ends. These results suggest that EN cleavage is based primarily on DNA structure rather than sequence, that many attempted L1 insertions likely fail at the priming step after cleavage, and that DNA mismatches and possibly other alterations to DNA conformation promote EN cleavage, together broadening our understanding of the genomic impact of L1 expression.