Biology subjects

Mosley, A. L.

Publications and source records attributed to Mosley, A. L..

At least 19 recordsLinked to original sources

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

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

Quantifying the Structural and Energetic Consequences of EXOSC3 S1 Domain Variants from a Comparative Assessment of {lambda}-Dynamics with Two Charge-Changing Perturbation Strategies

Charge-changing perturbations are notoriously dificult to investigate with alchemical free energy calculations. The routine use of periodic boundary conditions and electrostatic approximations, such as particle-mesh Ewald (PME), may produce finite-size efect errors that become non-negligible as a perturbation changes a simulation cells net charge away from zero. Two prevalent strategies exist to correct for these errors: the analytic correction (AC) and co-alchemical ion (CI) methods. Both correction schemes have been found to produce comparable relative free energy results for small molecule perturbations, but these methods have not been compared using {lambda}-dynamics ({lambda}D) free energy calculations or for protein side chain mutations. Recently, we investigated relative folding and binding free energies ({Delta}{Delta}Gs) of a series of EXOSC3 variants involved in a rare neurodegenerative disorder, including D132A, G135R, and G191D charge-change perturbations, with a simplified AC scheme in {lambda}D. In this study, these perturbations are reevaluated with the CI scheme for comparison with AC to identify the best correction strategy for {lambda}D. The collected AC- and CI-corrected {Delta}{Delta}Gs show excellent agreement with a mean unsigned error of 0.4 kcal/mol. However, reduced sampling proficiency and increased dificulties of evaluating multisite perturbations with the CI method suggest that a simplified AC approach may be more generalizable for future {lambda}D calculations. Previously, the use of the CI approach with {lambda}D has been limited due to a lack of infrastructure available to users to simplify its more involved setup procedure. This study introduces an automated workflow for implementation of the CI approach with {lambda}D, laying the foundation for future comparisons between charge-change correction schemes. These studies facilitated analysis of the {lambda}D trajectories to identify structural changes within EXOSC3 and the RNA exosome complex that clearly rationalize the calculated {Delta}{Delta}Gs for the D132A, G135R, G191C, and G191D EXOSC3 variants, providing insight into potential disease-causing mechanisms of EXOSC3 modifications.

biophysics↗

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

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

IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer that is unresponsive to hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. Here we use a derivative of DNQ, isobutyl-deoxynyboquinone (IB-DNQ) that is more potent and specific in killing TNBC cells than NQO1-activator {beta}-lapachone while displaying strong NQO1-dependence. We evaluated the cellular signaling changes that occur following 4-hour treatment of TNBC cells with either single agent or combination IB-DNQ and / or PARP inhibitor (Rucaparib). Short treatments (4 hours) with IB-DNQ alone or combined with the PARP inhibitor Rucaparib revealed few changes in protein abundance but significant rapid alterations in protein phosphorylation and thermal stability, with clear synergy in the combination treatment. Key phosphorylated targets linked to RNA Polymerase II inhibition and DNA damage response were altered during our short time treatment. Thermal proteome profiling (TPP) identified novel, combination-specific changes in protein biophysical state suggesting new therapeutic vulnerabilities in TNBC cells. Our findings highlight how even brief treatments can uncover distinct biophysical protein changes via TPP, offering a resource for mechanistic studies of IB-DNQ mechanism of action and the development of NQO1-activated therapeutics for TNBC treatment.

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

Gemcitabine plus nab-paclitaxel preserves skeletal and cardiac mass and function in a murine model of pancreatic cancer cachexia

More than 85% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, a debilitating syndrome characterized by the loss of muscle and fat and remains an unmet medical need. While chemotherapy remains an effective treatment option, it can also induce weight and muscle loss in patients with cancer. Gemcitabine combined with nab paclitaxel (GnP) is a first line treatment option for patients with PDAC but GnPs effect on cachexia has not been comprehensively investigated. We interrogated the effects of GnP in a murine model of pancreatic cancer cachexia. Mice were orthotopically implanted with the cachexia inducing pancreatic cell line (KPC) and were administered GnP or vehicle. The controls underwent sham surgery. We defined GnP effects on cachexia and tumor burden by evaluating muscle and cardiac mass and function, fat mass, bone morphometry, and hematology measurements. We completed RNA sequencing and deep proteome profiling in skeletal and cardiac muscle. KPC+GnP reduced tumor burden over 50% and increased survival compared to KPC. KPC vehicle group had more than 15% muscle mass loss and decreased left ventricular mass, this was not present in KPC+GnP when compared to controls. RNA Seq and deep proteomics analyses suggested that muscle and cardiac dysfunction pathways activated in KPC group were either reversed or decreased in KPC+GnP. In all, our data suggests that GnP protects against muscle and cardiac wasting in an experimental model of PDAC cachexia. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/536434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d394f7org.highwire.dtl.DTLVardef@1d8fdaforg.highwire.dtl.DTLVardef@f709d4org.highwire.dtl.DTLVardef@a668e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2023

Mechanisms of spinophilin-dependent pancreas dysregulation underlying diabesity

ObjectiveSpinophilin is an F-actin binding and protein phosphatase 1 (PP1) targeting protein that acts as a scaffold of PP1 to its substrates. Spinophilin knockout (Spino-/-) mice have decreased fat mass, increased lean mass, and improved glucose tolerance, with no difference in feeding behaviors. While spinophilin is enriched in neurons, its roles in non-neuronal tissues, such as beta cells of the pancreatic islets, are unclear. Methods & ResultsWe have corroborated and expanded upon previous studies to determine that Spino-/- mice have decreased weight gain and improved glucose tolerance in two different models of obesity. Using proteomics and immunoblotting-based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas and observed increased interactions of spinophilin with exocrine, ribosomal, and cytoskeletal protein classes that mediate peptide hormone production, processing, and/or release in Leprdb/db and/or high fat-fed (HFF) models of obesity. Moreover, loss of spinophilin specifically in pancreatic beta cells improved glucose tolerance without impacting body weight. ConclusionOur data further support a role for spinophilin in mediating pathophysiological changes in body weight and whole-body metabolism associated with obesity and provide the first evidence that spinophilin mediates obesity-dependent pancreatic dysfunction that leads to deficits in glucose homeostasis or diabesity.

cell biology↗

bioRxiv2022

Obtaining Increased Functional Proteomics Insights from Thermal Proteome Profiling through Optimized Melt Shift Calculation and Statistical Analysis

Thermal Proteome Profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein- protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with advancements and improvements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is even more apparent as TPP findings have reported more subtle melt shift changes related to protein post-translational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. To benchmark InflectSSP, we have reanalyzed two publicly available datasets to demonstrate the performance of this publicly available R based program for TPP data analysis. Additionally, we report new findings following temporal treatment of human cells with the small molecule Thapsigargin which induces the unfolded protein response (UPR). InflectSSP analysis of our UPR study revealed highly reproducible target engagement over time while simultaneously providing new insights into the dynamics of UPR induction.

biophysics↗

bioRxiv2022

Prenatal methadone exposure selectively alters protein expression in primary motor cortex: implications for synaptic function

As problematic opioid use has reached epidemic levels over the past two decades, the annual prevalence of opioid use disorder (OUD) in pregnant women has also increased 333%. Yet, how opioids affect the developing brain of offspring from mothers experiencing OUD remains understudied and not fully understood. Animal models of prenatal opioid exposure have discovered many deficits in the offspring of prenatal opioid exposed mothers, such as delays in the development of sensorimotor function and long-term locomotive hyperactivity. In attempt to further understand these deficits and link them with protein changes driven by prenatal opioid exposure, we used a mouse model of prenatal methadone exposure (PME) and preformed an unbiased multi-omic analysis across many sensoriomotor brain regions known to interact with opioid exposure. The effects of PME exposure on the primary motor cortex (M1), primary somatosensory cortex (S1), the dorsomedial striatum (DMS), and dorsolateral striatum (DLS) were assessed using quantitative proteomics and phosphoproteomics. PME drove many changes in protein and phosphopeptide abundance across all brain regions sampled. Gene and gene ontology enrichments were used to assess how protein and phosphopeptide changes in each brain region were altered. Our findings showed that M1 was uniquely affected by PME in comparison to other brain regions. PME uniquely drove changes in M1 glutamatergic synapses and synaptic function. Immunohistochemical analysis also identified anatomical differences in M1 for upregulating the density of glutamatergic and downregulating the density of GABAergic synapses due to PME. Lastly, comparisons between M1 and non-M1 multi-omics revealed conserved brain wide changes in phosphopeptides associated with synaptic activity and assembly, but only specific protein changes in synapse activity and assembly were represented in M1. Together, our studies show that lasting changes in synaptic function driven by PME are largely represented by protein and anatomical changes in M1, which may serve as a starting point for future experimental and translational interventions that aim to reverse the adverse effects of PME on offspring.

neuroscience↗

bioRxiv2022

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro crosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations in S. cerevisiae RAD6 that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression. Significance StatementTranscription by RNAPII is tightly coordinated with mechanisms that control chromatin structure. Disruption of this interplay leads to deleterious effects on gene expression and genome architecture. Proteins that associate with RNAPII during transcription elongation play an important role in coupling histone modifications to active transcription. Paf1C, a conserved member of the RNAPII active elongation complex, is required for the ubiquitylation of histone H2B, a modification with effects on nucleosome stability and the methylation and acetylation state of chromatin. Here, we provide new insights into how a conserved domain in Paf1C, which we previously showed to be necessary and sufficient for Paf1C-mediated stimulation of H2B ubiquitylation, interacts with the ubiquitin conjugase for H2B thereby guiding its specificity.

molecular biology↗