Biology subjects

Mosley, A. L.

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

26 records · Page 2Linked to original sources

bioRxiv2022

Asparagine starvation suppresses histone demethylation through iron depletion

Nutrient availability can impact epigenome to modify gene expression and dictate cell fate decision (Etchegaray and Mostoslavsky, 2016; Kinnaird et al., 2016). -ketoglutarate is an indispensable substrate for the Jumonji family of histone demethylases (JHDMs) mediating most of the cellular demethylation reactions on histone subunits (Schvartzman et al., 2018). Since -ketoglutarate is an intermediate of the tricarboxylic acid (TCA) cycle and a product of transamination, its intracellular levels are regulated by the metabolism of several amino acids (Baksh et al., 2020; Carey et al., 2015; Raffel et al., 2017; Vardhana et al., 2019). Here we show that asparagine starvation suppresses global histone demethylation. This process is neither due to the change of expression of histone modifying enzymes, nor due to the change of intracellular level of -ketoglutarate. Rather, asparagine starvation reduces intracellular pool of labile iron (Fe2+), which is a key cofactor for the JHDMs to function. Mechanistically, asparagine starvation post-transcriptionally suppresses the expression of iron responsive element binding protein 2 (IREB2), an iron sensing protein, which then reduces the mRNA expression of the transferrin receptor (TFRC), a major carrier for iron uptake (Hentze et al., 2010). Furthermore, iron supplementation to the culture medium restores histone demethylation and alters global gene expression to accelerate cell death under conditions of asparagine starvation. Collectively, our results uncover that suppression of iron-dependent histone demethylation is part of the cellular adaptive response to asparagine starvation.

cell biology↗

bioRxiv2022

Spinophilin limits metabotropic glutamate receptor 5 scaffolding to the postsynaptic density and cell type-specifically mediates excessive grooming

BackgroundConstitutive knockout of the obsessive-compulsive disorder-associated protein, disks large associated protein 3 (SAPAP3), results in repetitive motor dysfunction, such as excessive grooming, caused by increased metabotropic glutamate receptor 5 (mGluR5) activity in striatal direct- and indirect pathway medium spiny neurons (dMSNs and iMSNs, respectively). However, MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Here, we investigate the MSN subtype-specific roles of the striatal signaling hub protein, spinophilin, in mediating repetitive motor dysfunction associated with mGluR5 function. MethodsQuantitative proteomics and immunoblotting were utilized to identify how spinophilin impacts mGluR5 phosphorylation and protein interaction changes. Plasticity and repetitive motor dysfunction associated with mGluR5 action was measured using our novel conditional spinophilin mouse model that had spinophilin knocked out from striatal dMSNs or/and iMSNs. ResultsLoss of spinophilin only in iMSNs decreased performance of a novel motor repertoire, but loss of spinophilin in either MSN subtype abrogated striatal plasticity associated with mGluR5 function and prevented excessive grooming caused by SAPAP3 knockout mice and treatment with the mGluR5-specific positive allosteric modulator (VU0360172) without impacting locomotion-relevant behavior. Biochemically, we determined spinophilins protein interaction correlates with grooming behavior and loss of spinophilin shifts mGluR5 interactions from lipid-raft associated proteins toward postsynaptic density (PSD) proteins implicated in psychiatric disorders. ConclusionsThese results identify spinophilin as a novel striatal signaling hub molecule in MSNs that cell subtype-specifically mediates behavioral, functional, and molecular adaptations associated with repetitive motor dysfunction in psychiatric disorders.

neuroscience↗

bioRxiv2021

Quantitative proteomic and metabolomic profiling reveals altered mitochondrial metabolism and folate biosynthesis pathways in the aging Drosophila eye

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease, that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. Here, we profiled the aging proteome and metabolome of the Drosophila eye, and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Notably, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. Our integration of the proteomic, metabolomic and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. We propose that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases.

biochemistry↗

bioRxiv2020

Boosting detection of low abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to TMT multiplexes

The study of low abundance proteins is a challenge to discovery-based proteomics. Mass-spectrometry (MS) applications, such as thermal proteome profiling (TPP) face specific challenges in detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low abundance subunits of the Cleavage and Polyadenylation Factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40x for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (Tm) calculations for other unrelated proteins in the samples, with a high positive correlation between Tm estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of affinity purified protein complex as an isobaric trigger channel within a TMT multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.

biochemistry↗

bioRxiv2020

Inflect: Optimizing Computational Workflows for Thermal Proteome Profiling Data Analysis

The use of CETSA and Thermal Proteome Profiling (TPP) analytical methods are invaluable for the study of protein-ligand interactions and protein stability in a cellular context. These tools have increasingly been leveraged in work ranging from understanding signaling paradigms to drug discovery. Consequently, there is an important need to optimize the data analysis pipeline that is used to calculate protein melt temperatures (Tm) and relative melt shifts from proteomics abundance data. Here we report a user-friendly analysis of the melt shift calculation workflow where we describe the impact of each individual calculation step on the final output list of stabilized and destabilized proteins. This report also includes a description of how key steps in the analysis workflow quantitatively impacts the list of stabilized/destabilized proteins from an experiment. We applied our findings to develop a more optimized analysis workflow that illustrates the dramatic sensitivity of chosen calculation steps on the final list of reported proteins of interest in a study and will make the R based program Inflect available for research community use. Overall, this work provides an essential resource for scientists as they analyze data from TPP and CETSA experiments and implement their own analysis pipelines geared towards specific applications.

systems biology↗

bioRxiv2020

The genetic and physical interactomes of the Saccharomyces cerevisiae Hrq1 helicase

The human genome encodes five RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) that participate in various processes underpinning genomic stability. Of these enzymes, the disease-associated RECQL4 is comparatively understudied due to a variety of technical challenges. However, Saccharomyces cerevisiae encodes a functional homolog of RECQL4 called Hrq1, which is more amenable to experimentation and has recently been shown to be involved in DNA inter-strand crosslink (ICL) repair and telomere maintenance. To expand our understanding of Hrq1 and the RecQ4 subfamily of helicases in general, we took a multi-omics approach to define the Hrq1 interactome in yeast. Using synthetic genetic array analysis, we found that mutations of genes involved in processes such as DNA repair, chromosome segregation, and transcription synthetically interact with deletion of HRQ1 and the catalytically inactive hrq1-K318A allele. Pull-down of tagged Hrq1 and mass spectrometry identification of interacting partners similarly underscored links to these processes and others. Focusing on transcription, we found that hrq1 mutant cells are sensitive to caffeine and that mutation of HRQ1 alters the expression levels of hundreds of genes. In the case of hrq1-K318A, several of the most highly upregulated genes encode proteins of unknown function whose expression levels are also increased by DNA ICL damage. Together, our results suggest a heretofore unrecognized role for Hrq1 in transcription, as well as novel members of the Hrq1 ICL repair pathway. These data expand our understanding of RecQ4 subfamily helicase biology and help to explain why mutations in human RECQL4 cause diseases of genomic instability.

genetics↗

bioRxiv2019

Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impact of missense mutations on the proteome

Temperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

systems biology↗

bioRxiv2019

RNA Polymerase II CTD phosphatase Rtr1 prevents premature transcription termination

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). Using disruption-compensation (DisCo) protein-protein interaction network analysis, interaction changes were observed within the termination machinery as a consequence of deletion of the serine 5 RNAPII CTD phosphatase Rtr1. Interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1{Delta}, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. These changes could be the result of altered interactions between the termination machinery and/or increased levels of premature termination of RNAPII. Transcriptome analysis in rtr1{Delta} cells found decreased pervasive transcription and a shift in balance of expression of sense and antisense transcripts. Globally, rtr1{Delta} leads to decreases in noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS)-dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes and increased efficiency of snRNA termination. Additionally, premature termination increases globally at protein-coding genes where NNS is recruited during early elongation. The effects of rtr1{Delta} on RNA expression levels were erased following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade terminated noncoding RNAs. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature RNAPII termination of mRNAs and ncRNAs. Additionally, Rtr1 phosphatase activity facilitates low-level elongation of noncoding transcripts that impact the transcriptome through RNAPII interference.\n\nAUTHOR SUMMARYMany cellular RNAs including those that encode for proteins are produced by the enzyme RNA Polymerase II. In this work, we have defined a new role for the phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of transcription to the 3 end of the gene where the nascent RNA from protein-coding genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 leads to changes in the interactions between RNA polymerase II and the termination machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its target gene types including protein coding genes and noncoding RNAs. Evidence suggests that the premature termination observed in RTR1 knockout cells occurs through the termination factor and RNA binding protein Nrd1 and its binding partner Nab3. Additionally, many of the prematurely terminated noncoding RNA transcripts are degraded by the Rrp6-containing nuclear exosome, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through preventation of Nrd1-directed termination.

systems biology↗