Biology subjects

Doud, E. H.

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

7 recordsLinked to original sources

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↗

bioRxiv2023

In situ cryoET reveals inhibitor-induced perturbations in Pf80S GTPase interactions

Malaria parasites rely heavily on rapid, high fidelity protein synthesis to infect and replicate in human erythrocytes, making translation an attractive target for new antimalarials. Here, we have determined in situ structures of Pf80S ribosomes in thirteen conformational and compositional states from cryoFIB-milled Plasmodium falciparum-infected human erythrocytes across the stages of asexual intraerythrocytic parasite replication. We observe eight active translation intermediates, enabling us to define the native malarial translation elongation cycle, which surprisingly features a bifurcation at the decoding stage of the cycle that has not previously been described. Examination of perturbations in the distribution of ribosomes among these states in the presence of a malaria-specific translation inhibitor suggests that the inhibitor impedes PfeEF2 and PfeEF1 interactions with the ribosome. We integrated our in situ cryoET data with proteomic and ultrastructural data to arrive at a deeper understanding of malarial translation, which will inform development of new therapies.

biophysics↗

bioRxiv2023

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of the nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the rigidity and arrangement of the cortical microtubules. Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 phenocopies lack of PPKL, strongly suggesting a functional relationship between these two signaling proteins. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated proteins SPM1 in PPKL-depleted parasites, suggesting PPKL regulates the cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway. ImportanceToxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase, PPKL, appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

cell 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

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

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

Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo

The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2 phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2 axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision.

physiology↗