Biology subjects

Doud, E. H.

Publications and source records attributed to Doud, E. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular mechanisms underlying amyloid lowering by aducanumab: differential and comparative effects of sex and IgG reveal the post-treatment disease brain

INTRODUCTIONImproving the predictive validity of preclinical studies for Alzheimers disease (AD) requires rigorous evaluation of therapeutic efficacy, safety, and sex-specific responses in translationally relevant models. As amyloid-targeting monoclonal antibodies continue to advance clinically, there is an urgent need to define the molecular milieu that persists after amyloid is reduced and disease progression continues. Leveraging the NIA-funded MODEL-AD Preclinical Testing Core, we investigated the biochemical, functional, and multi-omic signatures associated with chronic administration of murine chimeric aducanumab (chAdu) in 5XFAD mice, including the contribution of IgG-mediated effects. METHODSMale and female 5XFAD mice were treated weekly with chAdu beginning at 8 months of age and compared to age-and sex-matched murine IgG2a{kappa} isotype (IgG) and saline controls. Plasma and brain pharmacokinetics, amyloid-beta (A{beta}), behavioral assessments, and treatment-emergent anti-drug antibodies (ADAs) were quantified. Post-treatment transcriptomic and proteomic analyses were performed to assess molecular pathways associated with chAdu and IgG exposure following 17-week treatment. RESULTSchAdu produced sex-dependent changes in A{beta}, including increased plasma A{beta}42:40 and reductions in brain A{beta} which were associated with mild behavioral impairments in the absence of improvements in cognitive function. IgG control treatment produced similar reductions, indicating biologically active IgG-mediated processes independent of A{beta}-targeted specificity. Treatment-emergent ADAs occurred in 10% of chAdu-treated mice and were associated with reduced drug exposure and efficacy. Multi-omics analyses confirmed sex-dependent and IgG-mediated effects at both the transcriptomic and proteome level revealing disease-associated genes and proteins not altered despite reductions in amyloid with treatment. DISCUSSIONThese findings demonstrate sex-dependent PK and pharmacodynamic responses to chAdu, identify biologically meaningful IgG-driven effects, and reveal molecular signatures that persist after amyloid reduction. This work provides biological insights into pathways that may remain insufficiently addressed following amyloid lowering; revealing novel targets for future drug discovery to prevent and treat disease.

neuroscience↗

bioRxiv2026

A CAMKK2-UBR4-19S Proteasome Axis Regulates Chondrocyte Proteostasis and SOX9 Stability

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

cell biology↗

bioRxiv2026

EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery

Pathogenic variants in EXOSC3, a noncatalytic subunit of the RNA exosome, cause pontocerebellar hypoplasia type 1B (PCH1B), yet substantial variability in disease severity is observed among individuals carrying different EXOSC3 alleles. The molecular mechanisms of RNA exosome dysfunction in individuals carrying EXOSC3 p.G191 variants remains unresolved. To address this, we generated CRISPR/Cas9-engineered human cell models harboring EXOSC3 p.G191 variants and performed integrated transcriptomic, proteomic, and computational structural analyses. EXOSC3 p.G191 variants produced extensive, allele- and dosage-dependent alterations in gene expression and splicing, with heterozygous variants causing broad but attenuated disruption relative to homozygous EXOSC3 G191D/G191D cells. All EXOSC3 G191 variants promoted increased skipping of exon 3 in EXOSC3 transcripts, generating a short isoform predicted to encode an unstable proteoform. Molecular dynamics and {lambda}-dynamics simulations predicted substantial thermodynamic destabilization of all EXOSC3 G191 variant proteoforms, consistent with reduced protein abundance and thermal stability measured by global proteomics and PISA. At the protein complex level, EXOSC3 p.G191 variants were associated with coordinated decreases in all RNA exosome core subunits and the exonuclease EXOSC10, consistent with destabilization of RNA exosome assembly and orphan protein decay. In contrast, the catalytic exonuclease DIS3 and multiple independent RNA processing pathways were upregulated, indicating compensatory recalibration of RNA metabolism. Together, these findings link variant-induced alternative splicing, RNA exosome complex destabilization, and adaptive network responses to phenotypic variability in EXOSC3 p.G191-associated disease.

systems biology↗

bioRxiv2026

Characterization of Lysine Methylation During Neuronal Differentiation of LUHMES cells.

Over one-third of human lysine methyltransferases (KMTs) and lysine demethylases (KDMs)-the enzymes responsible for adding or removing methylation on lysine residues within proteins-are linked to neurodevelopmental disorders (NDDs). Consequently, several studies have explored the roles of specific KMTs or KDMs in neuronal differentiation, and alterations in histone methylation patterns have been identified. It is now widely recognized that KMTs and KDMs also target non-histone proteins, yet knowledge of how non-histone lysine methylation changes during neuronal differentiation remains limited. Here, we address this gap using quantitative mass spectrometry-based proteomics to identify and measure changes in non-histone lysine methylation at three different stages in the Lund human mesencephalic (LUHMES) neuronal differentiation model. We identify 74 lysine methylation sites with significant differences in abundance across differentiation. Our analysis reveals lysine methylation on many non-histone proteins involved in neuronal differentiation and neurodevelopment, including signaling molecules, cytoskeletal proteins, RNA splicing factors, and transcription factors. Overall, this work broadens the understanding of non-histone lysine methylation in a neuronal differentiation model and offers a valuable resource of lysine methylation sites on proteins of biological and clinical significance for future research.

neuroscience↗

bioRxiv2025

Striatal spinophilin enhances D2R interaction with cytosolic proteins to mediate persistent D2R agonist-induced locomotor suppression.

Loss of dopamine neurons in Parkinson disease (PD) leads to motor deficits. Dopamine D2 receptor (D2R) agonists treat PD-associated motor deficits by acting on postsynaptic receptors located within the striatum that have been upregulated due dopamine loss. However, mechanisms that contribute to increased D2R activity in PD to enhance D2R function are poorly described. Spinophilin is a protein phosphatase 1 targeting protein that is expressed in postsynaptic dendritic spines and interacts with postsynaptic D2Rs. However, how spinophilin regulates D2R function is unknown. In the current study, we found that genetic knockout of spinophilin limited the suppression of locomotion caused by the D2R agonist, quinpirole. Mechanistically, we found that spinophilin is required for quinpirole-induced increases in the interaction of the D2R with intracellular proteins, suggesting spinophilin mediates agonist-induced D2R internalization. Therefore, our data support future studies targeting the spinophilin/D2R interaction to enhance the efficacy of current PD therapeutics.

neuroscience↗

bioRxiv2025

Activation of PP2A-B56α leads to aberrant EGFR signaling and proliferative phenotypes in PDAC

Pancreatic ductal adenocarcinoma (PDAC) stands to become the second most deadly cancer by 2030. The small GTPase, KRAS, is mutated in over 90% of PDAC patients and considered the primary driver mutation. Despite being an almost ubiquitous event, KRAS mutations have been difficult to target therapeutically, particularly KRASG12D, the most common mutation in PDAC. In addition to these pharmacological challenges, KRAS mutations have been shown to drive signaling plasticity and therapeutic resistance through phosphorylation cascades in most cancers. Protein phosphatases are master regulators of kinase signaling, however the contribution of phosphatase deregulation to mutant KRAS cancer phenotypes is poorly understood. Protein phosphatase 2A (PP2A) inhibits effectors downstream of KRAS, placing this family of enzymes as key regulators of PDAC oncogenic signaling. However, our previous studies utilizing small molecule activating compounds of PP2A show a heterogeneous response in PDAC, with some cell lines displaying increased oncogenic signaling despite induction of phosphatase activity. Similarly, specific PP2A subunits exhibit both tumor suppressive and oncogenic functions depending on the cellular context. Therefore, understanding the role of PP2A in regulating cancer phenotypes is critical for the future development of therapeutic strategies that leverage this phosphatase. Here, we determined the impact of the specific PP2A subunit, B56, on PDAC phenotypes using both genetic and pharmacological activation strategies in human PDAC cell lines and genetic mouse models. We demonstrate that while PP2A-B56 suppresses specific oncogenic pathways, B56 activation exacerbates PDAC proliferative phenotypes and decreases overall survival in vivo, potentially through increased epidermal growth factor receptor (EGFR) signaling. EGFR is a critical signaling node in PDAC as inhibition or loss of EGFR prevents KRAS-driven tumorigenesis and increased EGFR activity is associated with poor patient outcome. The activation of EGFR by PP2A-B56 is in part mediated through increased expression and processing of EGFR ligands, specifically amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), and epiregulin. Furthermore, pharmacological PP2A activation in combination with EGFR inhibitors mitigates this signaling and increases cell death. Together, these studies implicate a previously undescribed non-canonical role for PP2A-B56 in EGFR signaling that contributes to PDAC progression.

cancer biology↗

bioRxiv2025

PP2A activation drives aberrant macropinocytosis and cell death in pancreatic ductal adenocarcinoma

Pancreatic cancer is highly aggressive with a five-year survival rate of just 13%. Metabolic rewiring in response to oncogenic signals plays a critical role in pancreatic ductal adenocarcinoma (PDAC) survival, tumor growth, and metastasis. These alterations make PDAC tumors dependent on anabolic metabolism for survival, highlighting a unique vulnerability that can be therapeutically exploited. However, during nutrient deprivation, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids in a process called, macropinocytosis. This process can be induced downstream of oncogenic KRAS expression, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, emphasizing the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process remain poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that regulates a wide variety of cell signaling pathways, including KRAS, and is commonly dysregulated in human PDAC tumors. Here, we show that acute PP2A activation prevents macropinosome processing leading to cell death. Furthermore, we demonstrate that PP2A posttranslationally regulates the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determine that PP2A activating compounds can function synergistically with metabolic inhibitors, supporting a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging.

cancer biology↗

bioRxiv2025

Suppression of PP2A-B56α Drives EMT in EGFR Mutant Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in the United States and [~]50% of these patients present with metastatic disease at diagnosis. Epithelial-to-Mesenchymal Transition (EMT) is an important initiating step in the metastatic cascade that allows cells to acquire the migratory and invasive phenotypes necessary for dissemination. The transcriptional reprogramming that takes place during EMT has been well described in multiple cancer types; however, the posttranslational regulatory mechanisms that govern EMT are poorly understood. Protein Phosphatase 2A (PP2A) is serine/threonine (ser/thr) phosphatase that accounts for 50% of cellular ser/thr phosphatase activity and is critically important in regulating signaling homeostasis. PP2A dysregulation has been implicated in cell state regulation, EMT, and metastasis, but the roles of individual PP2A complexes are poorly understood. Our data indicate that suppression of the specific PP2A complex, PP2A-B56, results in decreased expression of epithelial markers and increased expression of mesenchymal markers consistent with EMT. These molecular changes are associated with migratory and invasive phenotypes both in vitro and in vivo. Furthermore, these migratory phenotypes can be rescued with B56 overexpression. Together, these findings implicate B56 as a key regulator of cellular plasticity and highlight the dynamic nature by which PP2A-B56 posttranslationally regulates NSCLC EMT.

cancer biology↗

bioRxiv2025

EXOSC3 S1-domain variants implicated in PCH1b alter RNA exosome cap subunit abundance and thermal stability disrupting rRNA processing and targeting of AU-rich mRNA

Missense variants in EXOSC3, an RNA exosome subunit, have been identified in patients with PCH1b. We investigated three missense variants in the S1 domain of EXOSC3, including one variant of uncertain significance (VUS) and two pathogenic variants (hence S1 variants). EXOSC3 S1 variant cell lines were generated using CRISPR-Cas9 resulting in widespread proteome changes including decreases in some RNA exosome subunits paired with increases in the catalytic subunit DIS3. Thermal stability, analyzed by PISA, revealed extensive destabilization of RNA exosome cap subunits and the cap-associated exonuclease EXOSC10. Functionally, S1 variants altered rRNA processing with corresponding protein compensation observed in rRNA processing proteins outside the RNA exosome. Exogenous overexpression of EXOSC3 rescues many molecular defects caused by S1 variants suggesting that protein destabilization and turnover strongly contribute to molecular defects. Overall, our findings define the mechanisms through which cells respond to EXOSC3 S1 variant disruption of RNA processing homeostasis.

systems biology↗

bioRxiv2025

Application of Whole Proteome Thermal Shift Assays to Define PERK-dependent Changes in Protein Homeostasis during the Unfolded Protein Response

The Unfolded Protein Response (UPR) is a cellular pathway activated by sensory proteins, including the protein kinase PERK (EIF2AK3), that monitors perturbations in the endoplasmic reticulum (ER). Using tunicamycin, which induces ER stress by thwarting N-glycosylation, we monitored system-wide changes in the proteome using PISA (Proteome Integral Solubility Alteration) and abundance analysis. Global proteomics revealed precise changes in membrane- and ER-associated proteins through widespread induction of ER-associated degradation (ERAD) while normalized PISA (nPISA) analyses selectively identified pathway changes associated with drug mechanism of action. nPISA analysis following tunicamycin treatment in cells, in combination with genetic disruption of PERK, facilitated identification of novel proteins involved in PERK-dependent and -independent processes and how those changes intersect with PERK function specifically during the ER stress response. Overall, protein-centered multiomics analyses defined the precise proteome alterations in tunicamycin-induced ER stress, highlighting the consequences of PERK disruption on ER-mitochondrial homeostasis.

systems biology↗

bioRxiv2025

An Essential Adaptor for Apicoplast Fission and Inheritance in Malaria Parasites

Blood-stage Plasmodium falciparum parasites rely on a non-photosynthetic plastid, the apicoplast, for survival, making it an attractive target for antimalarial intervention. Like the mitochondrion, the apicoplast cannot be generated de novo and must be inherited by daughter parasites during cell division. This inheritance relies on coordinated apicoplast positioning and fission, but the molecular mechanisms controlling these processes remain poorly understood. Here, we identify a previously uncharacterized P. falciparum protein (Pf3D7_0613600), which we name PfAnchor, as a key regulator of apicoplast fission. Using Ultrastructure Expansion Microscopy (U-ExM), we show that PfAnchor localizes to the apicoplast throughout the asexual blood-stage. Conditional depletion disrupts apicoplast fission, leading to incomplete cytokinesis and parasite death. Notably, loss of the apicoplasts elongated branched structure via azithromycin treatment rescues these defects, underscoring Anchors specific role in apicoplast fission. Immunoprecipitation identified an interaction with the dynamin-like GTPase PfDyn2, a key mediator of both apicoplast and mitochondrial fission, establishing PfAnchor as the first apicoplast-specific dynamin adaptor protein. Our findings define PfAnchor as an essential factor for apicoplast fission and inheritance in P. falciparum blood-stage parasites, highlighting parasite-specific organelle division as a potential vulnerability for therapeutic intervention.

cell biology↗

bioRxiv2025

Loss of Cathepsin K impairs collagen biogenesis and enhances actin polymerization in trabecular meshwork

Trabecular meshwork (TM) dysfunction and extracellular matrix (ECM) dysregulation contribute to increased intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Earlier, we provide a proof-of-concept study identifying the regulation and the role of Cathepsin K (CTSK), a potent collagenase, in ECM homeostasis, actin bundling, and IOP regulation. Better understanding of the loss of CTSK function in TM remains unclear. Using siRNA-mediated knockdown of CTSK (siCTSK) in human TM cells, this study investigated the role of CTSK in actin and ECM homeostasis using an unbiased proteomics approach. Loss of CTSK significantly disrupted collagen biogenesis and ECM homeostasis. CTSK depletion also increased intracellular calcium levels, with proteomics data suggesting possible involvement of calcium-regulatory proteins. Additionally, PRKD1 activation enhanced actin polymerization through the LIMK1/SSH1/cofilin pathway, promoting focal adhesion maturation. Despite increased apoptotic markers (CASP3, CASP7, TRADD, PPM1F), caspase 3/7 activation was not induced, suggesting apoptosis-independent cellular remodeling. Notably, RhoQ and myosin motor proteins were significantly downregulated, indicating altered mechanotransduction in TM cells. These findings highlight the role of CTSK in maintaining ECM homeostasis, calcium signaling, and cytoskeletal regulation in TM. Its depletion induces actin polymerization, which may influence aqueous humor outflow. Targeting CTSK-related pathways may provide novel therapeutic strategies for regulating IOP and preventing glaucoma progression.

cell biology↗

bioRxiv2025

Disulfide bonds are required for cell division, cell envelope biogenesis and antibiotic resistance proteins in mycobacteria

Mycobacteria, including Mycobacterium tuberculosis--the etiological agent of tuberculosis--have a unique cell envelope critical for their survival and resistance. The cell envelopes assembly and maintenance influence permeability, making it a key target against multidrug-resistant strains. Disulfide bond (DSB) formation is crucial for the folding of cell envelope proteins. The DSB pathway in mycobacteria includes two enzymes, DsbA and VKOR, required for survival. Using bioinformatics and cysteine profiling proteomics, we identified cell envelope proteins dependent on DSBs. We validated via in vivo alkylation that key proteins like LamA (MmpS3), PstP, LpqW, and EmbB rely on DSBs for stability. Furthermore, chemical inhibition of VKOR results in phenotypes similar to those of {Delta}vkor. Thus, targeting DsbA-VKOR systems could compromise both cell division and mycomembrane integrity. These findings emphasize the potential of DSB inhibition as a novel strategy to combat mycobacterial infections.

microbiology↗

bioRxiv2025

Proximity labeling and orthogonal nanobody pulldown (ID-oPD) approaches to map the spinophilin interactome uncover a putative role for spinophilin in protein homeostasis.

Spinophilin is a dendritic spine enriched scaffolding and protein phosphatase 1 targeting protein. To detail spinophilin interacting proteins, we created an Ultra-ID and ALFA-tagged spinophilin encoding construct that permits proximity labeling and orthogonal nanobody pulldown (ID-oPD) of spinophilin-associated protein complexes in heterologous cells. We identified 614 specific, and 312 specific and selective, spinophilin interacting proteins in HEK293 cells and validated a subset of these using orthogonal approaches. Many of these proteins are involved in mRNA processing and translation. In the brain, we determined that spinophilin mRNA is highly neuropil localized and that spinophilin may normally function to limit its own expression but promote the expression of other PSD-associated proteins. Overall, our use of an ID-oPD approach uncovers a novel putative role for spinophilin in mRNA translation and synaptic protein expression specifically within dendritic spines.

neuroscience↗

bioRxiv2024

Target deconvolution of an insulin hypersecretion-inducer acting through VDAC1 with a distinct transcriptomic signature in beta-cells

Obesity, insulin resistance, and a host of environmental and genetic factors can drive hyperglycemia, causing {beta}-cells to compensate by increasing insulin production and secretion. In type 2 diabetes (T2D), {beta}-cells under these conditions eventually fail. Rare {beta}-cell diseases like congenital hyperinsulinism (HI) also cause inappropriate insulin secretion, and some HI patients develop diabetes. However, the mechanisms of insulin hypersecretion and how it causes {beta}-cell dysfunction are not fully understood. We previously discovered small molecules (e.g. SW016789) that cause insulin hypersecretion and lead to a loss in {beta}-cell function without cell death. Here, we uncover the protein target of SW016789 and provide the first time-course transcriptomic analysis of hypersecretory responses versus thapsigargin-mediated ER stress in {beta}-cells. In mouse MIN6 and human EndoC-{beta}H1 {beta}-cells, we identified and validated VDAC1 as a SW016789 target using photoaffinity proteomics, cellular thermal shift assays, siRNA, and small molecule inhibitors. SW016789 raises membrane potential to enhance Ca2+ influx, potentially through VDAC1. Chronically elevated intracellular Ca2+ appears to underpin the negative impacts of hypersecretion, as nifedipine protected against each small molecule hypersecretion inducer we tested. Using time- course RNAseq, we discovered that hypersecretion induced a distinct transcriptional pattern compared to ER stress. Clustering analyses led us to focus on ER-associated degradation (ERAD) as a potential mediator of the adaptive response. SW016789 reduced the abundance of ERAD substrate OS-9 and pharmacological inhibition of ERAD worsened {beta}-cell survival in response to hypersecretory stress. Changes in other ERAD components in MIN6 and EndoC-{beta}H1 at the protein level were minor with either SW016789 or thapsigargin. However, immunostaining for core ERAD components SEL1L, HRD1, and DERL3 in non-diabetic and T2D human pancreas revealed altered distributions of SEL1L/HRD1 and SEL1L/DERL3 rations in {beta}-cells of T2D islets, in alignment with altered ERAD in stressed {beta}-cells. We conclude that hypersecretory stimuli, including SW016789- mediated VDAC1 activation, cause enhanced Ca2+ influx and insulin release. Subsequent differential gene expression represents a {beta}-cell hypersecretory response signature that is reflected at the protein level for some, but not all genes. A better understanding of how {beta}-cells induce hypersecretion and the mechanisms of negative feedback on secretory rate may lead to the discovery of novel therapeutic targets for T2D and HI.

cell biology↗

bioRxiv2024

Quantitative analysis of non-histone lysine methylation sites and lysine demethylases in breast cancer cell lines

Growing evidence shows that lysine methylation is a widespread protein post-translational modification that regulates protein function on histone and non-histone proteins. Numerous studies have demonstrated that dysregulation of lysine methylation mediators contributes to cancer growth and chemotherapeutic resistance. While changes in histone methylation are well documented with extensive analytical techniques available, there is a lack of high-throughput methods to reproducibly quantify changes in the abundances of the mediators of lysine methylation and non-histone lysine methylation (Kme) simultaneously across multiple samples. Recent studies by our group and others have demonstrated that antibody enrichment is not required to detect lysine methylation, prompting us to investigate the use of Tandem Mass Tag (TMT) labeling for global Kme quantification sans antibody enrichment in four different breast cancer cell lines (MCF-7, MDA-MB-231, HCC1806, and MCF10A). To improve the quantification of KDMs, we incorporated a lysine demethylase (KDM) isobaric trigger channel, which enabled 96% of all KDMs to be quantified while simultaneously quantifying 326 Kme sites. Overall, 142 differentially abundant Kme sites and eight differentially abundant KDMs were identified between the four cell lines, revealing cell line-specific patterning.

cancer biology↗

bioRxiv2024

Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex.

The modified E. coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37{degrees}C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a comparison of these variations of biotin ligases has not been reported in Saccharomyces cerevisiae. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive activity from biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically-induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.

molecular biology↗

bioRxiv2023

Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

molecular biology↗