Browsing by Author "Ray, W. Keith"
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- Characterization of the adult Aedes aegypti early midgut peritrophic matrix proteome using LC-MSWhiten, Shavonn R.; Ray, W. Keith; Helm, Richard F.; Adelman, Zach N. (PLOS, 2018-03-23)The Aedes aegypti mosquito is the principal vector of arboviruses such as dengue, chikungunya, yellow fever, and Zika virus. These arboviruses are transmitted during adult female mosquito bloodfeeding. While these viruses must transverse the midgut to replicate, the blood meal must also reach the midgut to be digested, absorbed, or excreted, as aggregation of blood meal metabolites can be toxic to the female mosquito midgut. The midgut peritrophic matrix (PM), a semipermeable extracellular layer comprised of chitin fibrils, glycoproteins, and proteoglycans, is one such mechanism of protection for the mosquito midgut. However, this structure has not been characterized for adult female Ae. aegypti. We conducted a mass spectrometry based proteomic analysis to identify proteins that comprise or are associated with the adult female Ae. aegypti early midgut PM. Altogether, 474 unique proteins were identified, with 115 predicted as secreted. GO-term enrichment analysis revealed an abundance of serine-type proteases and several known and novel intestinal mucins. In addition, approximately 10% of the peptides identified corresponded to known salivary proteins, indicating Ae. aegypti mosquitoes extensively swallow their own salivary secretions. However, the physiological relevance of this remains unclear, and further studies are needed to determine PM proteins integral for midgut protection from blood meal derived toxicity and pathogen protection. Finally, we describe substantial discordance between previously described transcriptionally changes observed in the midgut in response to a bloodmeal and the presence of the corresponding protein in the PM. Data are available via ProteomeXchange with identifier PXD007627.
- Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with CoccidiosisGilbert, Elizabeth R.; Cox, Chasity M.; Williams, Patricia M.; McElroy, Audrey P.; Dalloul, Rami A.; Ray, W. Keith; Barri, Adriana; Emmerson, Derek A.; Wong, Eric A.; Webb, Kenneth E. Jr. (PLOS, 2011-01-31)Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001. Conclusions/Significance Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.
- Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent MannerSlade, William O.; Ray, W. Keith; Hildreth, Sherry B.; Winkel, Brenda S. J.; Helm, Richard F. (MDPI, 2017-07-10)Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.
- Fasting differentially alters the hypothalamic proteome of chickens from lines with the propensity to be anorexic or obeseLiu, Lingbin; Yi, Jiaqing; Ray, W. Keith; Vu, Lucas T.; Helm, Richard F.; Siegel, Paul B.; Cline, Mark A.; Gilbert, Elizabeth R. (Springer Nature, 2019)Background: The hypothalamus is the ultimate modulator of appetite and energy balance and therefore sensitive to changes in nutritional state. Chicks from lines selected for low (LWS) and high (HWS) body weight are hypophagic and compulsive eaters, respectively, and differ in their propensity to become obese and in their hypothalamic mRNA response to fasting. Methods: As fasting-induced changes in hypothalamic proteins are unknown, we investigated the hypothalamic proteomes of 5-day old LWS and HWS chicks in the fed and fasted states using a label-free liquid chromatographytandem mass spectrometry (LC-MS/MS) approach. Results: A total of 744 proteins were identified in the chicken hypothalamus, and 268 differentially abundant proteins were identified among four pairwise comparisons. Ninety-five proteins were associated with the response to fasting in HWS chicks, and 23 proteins were associated with the response to fasting in LWS chicks. Fasting-responsive proteins in HWS chicks were significantly enriched in ATP metabolic processes, glyoxylate/dicarboxylate metabolism, and ribosome function. There was no enrichment for any pathways in LWS chicks in response to fasting. In the fasted and fed states, 159 and 119 proteins differed between HWS and LWS, respectively. Oxidative phosphorylation, citric acid cycle, and carbon metabolism were the main pathways associated with differences between the two lines of chicks. Enzymes associated with metabolic pathways differed between HWS and LWS in both nutritional states, including fumarase, aspartate aminotransferase, mitochondrial GOT2, 3-hydroxyisobutyrate dehydrogenase, chondrogenesis associated lipocalin, sialic acid synthase, arylamine N-acetyltransferase, pineal gland isozyme NAT-3, and succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial. Conclusions: These results provide insights into the hypothalamic metabolic pathways that are affected by nutritional status and the regulation of appetite and eating behavior.
- Females, but not males, require protein degradation in the hippocampus for contextual fear memory formationMartin, Kiley; Musaus, Madeline; Navabpour, Shaghayegh; Gustin, Aspen; Ray, W. Keith; Helm, Richard F.; Jarome, Timothy J. (2021-08)Strong evidence supports a role for protein degradation in fear memory formation. However, these data have been largely done in only male animals. Here, we found that following contextual fear conditioning, females, but not males, had increased levels of proteasome activity and K48 polyubiquitin protein targeting in the dorsal hippocampus, the latter of which occurred at chaperones or RNA processing proteins. In vivo CRISPR-dCas9-mediated repression of protein degradation in the dorsal hippocampus impaired contextual fear memory in females, but not males. These results suggest a sex-specific role for protein degradation in the hippocampus during the consolidation of a contextual fear memory.
- Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparumElahi, Rubayet; Ray, W. Keith; Dapper, Christie; Dalal, Seema; Helm, Richard F.; Klemba, Michael (2019-11-26)Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from ei/0 hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.
- The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironmentVu, Lucas T.; Orbach, Sophia M.; Ray, W. Keith; Cassin, Margaret E.; Rajagopalan, Padmavathy; Helm, Richard F. (Biomed Central, 2017-06-20)Background: Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. Methods: To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. Results: All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based β-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. Conclusions: Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.
- Levels of Germination Proteins in Bacillus subtilis Dormant, Superdormant, and Germinating SporesChen, Yan; Ray, W. Keith; Helm, Richard F.; Melville, Stephen B.; Popham, David L. (PLOS, 2014-04-21)Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiplereaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.
- Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory FormationFarrell, Kayla; Musaus, Madeline; Navabpour, Shaghayegh; Martin, Kiley; Ray, W. Keith; Helm, Richard F.; Jarome, Timothy J. (Frontiers, 2021-09-29)Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.
- Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturationJimenez-Vicente, Emilio; Yang, Zhi-Yong; Ray, W. Keith; Echavarri-Erasun, Carlos; Cash, Valerie L.; Rubio, Luis M.; Seefeldt, Lance C.; Dean, Dennis R. (2018-06-22)Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as assembly factors. Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
- Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in ratsFarrell, Kayla; Auerbach, Aubrey; Liu, Catherine; Martin, Kiley; Pareno, Myasia; Ray, W. Keith; Helm, Richard F.; Biase, Fernando; Jarome, Timothy J. (2023-11-10)Background Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. Methods We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. Results We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. Conclusions These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).
- Sex-Specific Linear Polyubiquitination Is a Critical Regulator of Contextual Fear Memory FormationMusaus, Madeline; Farrell, Kayla; Navabpour, Shaghayegh; Ray, W. Keith; Helm, Richard F.; Jarome, Timothy J. (Frontiers, 2021-07-09)Strong evidence supports that protein ubiquitination is a critical regulator of fear memory formation. However, as this work has focused on protein degradation, it is currently unknown whether polyubiquitin modifications that are independent of the proteasome are involved in learning-dependent synaptic plasticity. Here, we present the first evidence that atypical linear (M1) polyubiquitination, the only ubiquitin chain that does not occur at a lysine site and is largely independent of the proteasome, is critically involved in contextual fear memory formation in the amygdala in a sex-specific manner. Using immunoblot and unbiased proteomic analyses, we found that male (49) and female (14) rats both had increased levels of linear polyubiquitinated substrates following fear conditioning, though none of these protein targets overlapped between sexes. In males, target protein functions involved cell junction and axonal guidance signaling, while in females the primary target was Adiponectin A, a critical regulator of neuroinflammation, synaptic plasticity, and memory, suggesting sex-dependent functional roles for linear polyubiquitination during fear memory formation. Consistent with these increases, in vivo siRNA-mediated knockdown of Rnf31, an essential component of the linear polyubiquitin E3 complex LUBAC, in the amygdala impaired contextual fear memory in both sexes without affecting memory retrieval. Collectively, these results provide the first evidence that proteasome-independent linear polyubiquitination is a critical regulator of fear memory formation, expanding the potential roles of ubiquitin-signaling in learning-dependent synaptic plasticity. Importantly, our data identify a novel sex difference in the functional role of, but not a requirement for, linear polyubiquitination in fear memory formation.
- Sinorhizobium meliloti chemotaxis to quaternary ammonium compounds is mediated by the chemoreceptor McpXWebb, Benjamin A.; Compton, K. Karl; Saldaña, Rafael Castañeda; Arapov, Timofey D.; Ray, W. Keith; Helm, Richard F.; Scharf, Birgit E. (Wiley-Blackwell, 2017-01-01)The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC–MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpXPR and McpX34-306) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpXPR with dissociation constants (Kd) in the nanomolar range for choline and glycine betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plantderived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding.