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Item Ablation of arginylation in the mouse N-end rule pathway: Loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations(Public Library of Science, 2009) Brower, Christopher S.; Varshavsky, AlexanderIn the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway’s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ,15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1- deficient males are also infertile, owing to defects in Ate12/2 spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.Item Alleviation of drought stress in Arabidopsis thaliana by 17β-estradiol application(Scientific Research Publishing, 2016-10) Upadhyay, Pallavi; Maier, CameliaAnimal steroidal hormones, including estrogens, are being introduced into the agricultural soil and water supply from increased pharmaceutical and farm waste. Considering the current levels of xenoestrogen contamination of plant environments in view of the climate change induced drought conditions, this study was designed to understand the effect of estradiol (ES) application on Arabidopsis drought stress responses. Estradiol treatment (10 nM, 100 nM) of plants subjected to drought stress conditions by withholding water for 7 days resulted in increased tolerance to drought stress reflected in the significantly higher plant survival rates of 74% and 78%, respectively compared to control plants’ survival rates of 36% (no treatment) and 40% (mock treatment). Estradiol application significantly increased the content of glutathione, proline and H2O2 and significantly enhanced the transcription of the stress responsive genes GSTU3, GER5, HSP101, and HSP70b. A high concentration of ES (10 μM) did not protect plants against drought stress and proved to be toxic. These results provide new insight into the effect of ES on drought-stress responses in Arabidopsis with possible practical agricultural applications regarding the effect of environmental estrogens on crop plants. *This article was published with the assistance of the Texas Woman's University Libraries Open Access Fund. The original article can be found at:http://dx.doi.org/10.4236/ajps.2016.714186Item Analyzing N-terminal arginylation through the use of peptide arrays and degradation assays(Elsevier, 2016) Wadas, Brandon; Piatkov, Konstantin I.; Brower, Christopher S.; Varshavsky, AlexanderNα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (“canonical”) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only α-amino groups of N-terminal residues but also γ-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.Item Androgen and estrogen (α) receptor localization on periaqueductal gray neurons projecting to the rostral ventromedial medulla in the male and female rat(Elsevier, 2008-12) Lloyd, Dayna L.; Murphy, Anne Z.The periaqueductal gray (PAG) is involved in many gonadal steroid-sensitive behaviors, including responsiveness to pain. The PAG projects to the rostral ventromedial medulla (RVM), comprising the primary circuit driving pain inhibition. Morphine administered systemically or directly into the PAG produces greater analgesia in male compared to female rats, while manipulation of gonadal hormones alters morphine potency in both sexes. It is unknown if these alterations are due to steroidal actions on PAG neurons projecting to the RVM. The expression of androgen (AR) and estrogen (ERα) receptors in the PAG of female rats and within this descending inhibitory pathway in both sexes is unknown. The present study used immunohistochemical techniques (1) to map the distribution of AR and ERα across the rostrocaudal axis of the PAG; and (2) to determine whether AR and/or ERα were colocalized on PAG neurons projecting to the RVM in male and female rats. AR and ERα immunoreactive neurons (AR-IR, ERα-IR) were densely distributed within the caudal PAG of male rats, with the majority localized in the lateral/ventrolateral PAG. Females had significantly fewer AR-IR neurons, while the quantity of ERα was comparable between the sexes. In both sexes, approximately 25–50% of AR-IR neurons and 20–50% of ERα-IR neurons were retrogradely labeled. This study provides direct evidence of the expression of steroid receptors in the PAG and the descending pathway driving pain inhibition in both male and female rats and may provide a mechanism whereby gonadal steroids modulate pain and morphine potency.Item Anti-hyperalgesic effects of anti-serotonergic compounds on serotonin- and capsaicin-evoked thermal hyperalgesia in the rat(Elsevier, 2012) Lloyd, Dayna R.; Chen, P.B.; Hargreaves, K.M.The peripheral serotonergic system has been implicated in the modulation of an array of pain states, from migraine to fibromyalgia; however, the mechanism by which serotonin (5HT) induces pain is unclear. Peripherally released 5HT induces thermal hyperalgesia, possibly via modulation of the transient receptor potential V1 (TRPV1) channel, which is gated by various noxious stimuli, including capsaicin. We previously reported in vitro that 5HT increases calcium accumulation in the capsaicin-sensitive population of sensory neurons with a corresponding increase in proinflammatory neuropeptide release, and both are antagonized by pretreatment with 5HT2A and 5HT3 antagonists, as well as the anti-migraine drug sumatriptan. In the current study, we extended these findings in vivo using the rat hind paw thermal assay to test the hypothesis that peripheral 5HT enhances TRPV1-evoked thermal hyperalgesia that can be attenuated with 5HT2A and 5HT3 receptor antagonists, as well as sumatriptan. Thermal hyperalgesia and edema were established by 5HT injection (0.1–10 nmol/100 μl) into the rat hind paw, and the latency to paw withdrawal (PWL) from noxious heat was determined. Rats were then pretreated with either 5HT before capsaicin (3 nmol/10 μl), the 5HT2A receptor antagonist ketanserin or the 5HT3 receptor antagonist granisetron (0.0001–0.1 nmol/100 μl) before 5HT and/or capsaicin, or the 5HT1B/1D receptor agonist sumatriptan (0.01–1 nmol/100 μl) before capsaicin, and PWL was determined. We report that 5HT pretreatment enhances TRPV1-evoked thermal hyperalgesia, which is attenuated with local pretreatment with ketanserin, granisetron, or sumatriptan. We also report that peripheral 5HT induced a similar magnitude of thermal hyperalgesia in male and female rats. Overall, our results provide in vivo evidence supporting an enhancing role of 5HT on TRPV1-evoked thermal hyperalgesia, which can be attenuated by peripheral serotonergic intervention.Item AT1A-mediated activation of kidney JNK1 and SMAD2 in obstructive uropathy: preservation of kidney tissue mass using candesartan(2004-09-01) Wamsley-Davis, Ann; Padda, Ranjit; Truong, Luan D.; Tsao, Chun Chui; Zhang, Ping; Sheikh-Hamad, DavidLiterature suggests the involvement of the renin-angiotensin system and transforming growth factor (TGF)-β in the renal injury that follows chronic ureteric obstruction. SMAD proteins and the JNK1 cascade are essential components of TGF-β signaling machinery, and recent data suggest cooperative interaction between JNK1 and SMAD proteins in TGF-β-mediated gene expression. We used a rat model of chronic unilateral ureteric obstruction to study the effects of candesartan, an AT1A-receptor blocker, on tissue morphology and the activities of JNK1 and SMAD2 protein in the kidney. Ureteric obstruction for 28 days leads to interstitial fibrosis, tubule atrophy, and marked activation of SMAD2 and JNK1, without significant change in p38 kinase or ERK. Candesartan treatment, however, attenuated the chronic tubulointerstitial injury in obstructed kidneys and was associated with significant preservation of kidney tissue mass. Furthermore, treatment with candesartan diminished JNK1 activity and downregulated SMAD2 protein and activity in obstructed kidneys. In conclusion, obstructed kidneys showed chronic tubulointerstitial injury, which was associated with JNK1 and SMAD2 activation. The renoprotective effects afforded by AT1A-receptor blockade in obstructive uropathy are consistent with attenuation of JNK1- and SMAD2-mediated renal injury. obstruction of urine flow complicates many human diseases, such as prostate hypertrophy, abdominopelvic malignancies, and retroperitoneal fibrosis. It is characterized by infiltration of mononuclear inflammatory cells, predominantly macrophages, and is accompanied by activation of cytokines, growth factors, and mediators of apoptosis, the net result of which is the deletion of tubular cells by apoptosis and replacement of renal parenchyma with fibrous tissue. If left untreated, chronic kidney obstruction leads to loss of functional renal parenchyma, and ultimately, the development of kidney failure. Numerous reports implicate the renin-angiotensin system in the pathogenesis of ureteric obstruction, and inhibition of the rennin-angiotensin system, using either angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers, has been shown to attenuate the renal injury that follows ureteric obstruction (19, 23, 37). Activation of the kidney renin-angiotensin system occurs immediately after urteric obstruction, with resultant vasoconstriction and salt and water retention (14). These hemodynamic changes are followed by the activation of a number of cytokines (6, 9, 20, 21, 36, 50), some of which have been directly linked to angiotensin II, such as monocyte chemotactic protein-1 (MCP-1) (20) and transforming growth factor-β (TGF-β) (22), which appears to play a key role in the genesis of the ensuing fibrosis (32). TGF-β signaling is initiated after ligand binding to the TGF-β receptor (15, 33), leading to phosphorylation of SMAD2 or SMAD3 (1, 47, 53). SMAD proteins represent a group of transcription factors involved in gene regulation downstream of the TGF-β receptor. A heteromeric SMAD proteins complex is formed (7), resulting in translocation of the complex to the nucleus (1, 31, 47), where it can interact with transcription factors directly (7), or indirectly (27), to regulate gene expression. It was recently reported that JNK pathway (25, 51), which targets the activation of c-Jun and activated transcription factor-2 (ATF-2) (8, 12, 16, 26), is stimulated rapidly by TGF-β in human fibrosarcoma cells and that JNK activity is essential for TGF-β-induced fibronectin production (17). Furthermore, it was also suggested that SMAD proteins and the JNK1 pathway interact cooperatively in TGF-β signaling (17). These observations assume particular importance, in light of recent data that implicate JNK1 in the development of cell apoptosis after ischemia and UV irradiation in a variety of tissues (2, 3). Apoptosis is a crucial component in the pathogenesis of ureteric obstruction (5, 49). Based on these observations, we hypothesized that JNK1 may be a key contributor to the development of renal injury and fibrosis in obstructive uropathy, and thus renal protection after AT1A-receptor blockade in obstructive uropathy may be related to the downregulation of JNK1 and SMAD proteins. Consistent with this hypothesis, our data suggest activation of SMAD2 and JNK1 signaling in obstructed kidneys in a manner that is AT1A dependent. AT1A- receptor blockade using candesartan downregulates JNK1 and SMAD2, decreases tissue fibrosis, and leads to tissue preservation in obstructed kidneys.Item BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43(Elsevier, 2022) Kasu, Yasar Arfat T.; Arva, Akshaya; Sajan, Christin; Manzano, Jasmin; Hennes, Andrew; Haynes, Jacy; Brower, Christopher S.Neurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.Item Beta-amyloid induces apoptosis of neuronal cells by inhibition of the Arg/N-end rule pathway proteolytic activit(Impact Journals, 2019) Kechko, Olga I.; Petrushanko, Irina Yu; Brower, Christopher S.; Adzhubei, Alexei A.; Moskalev, Alexey A.; Piatkov, Konstantin I.; Mitkevich, Vladimir A.; Makarov, Alexander A.Alzheimer’s disease (AD) is accompanied by the dysfunction of intracellular protein homeostasis systems, in particular the ubiquitin-proteasome system (UPS). Beta-amyloid peptide (Aβ), which is involved in the processes of neurodegeneration in AD, is a substrate of this system, however its effect on UPS activity is still poorly explored. Here we found that Aβ peptides inhibited the proteolytic activity of the antiapoptotic Arg/Nend rule pathway that is a part of UPS. We identified arginyltransferase Ate1 as a specific component of the Arg/N-end rule pathway targeted by Aβs. Aβ bearing the familial English H6R mutation, known to cause earlyonset AD, had an even greater inhibitory effect on protein degradation through the Arg/N-end rule pathway than intact Aβ. This effect was associated with a significant decrease in Ate1-1 and Ate1-3 catalytic activity. We also found that the loss of Ate1 in neuroblastoma Neuro-2a cells eliminated the apoptosis-inducing effects of Aβ peptides. Together, our results show that the apoptotic effect of Aβ peptides is linked to their impairment of Ate1 catalytic activity leading to suppression of the Arg/N-end rule pathway proteolytic activity and ultimately cell death.Item Both acute and consecutive days of formoterol stimulation influence myogenic, mitochondrial, and myomir gene expression in human skeletal muscle cells(MDPI, 2023) Gordon, Ryan A.; Zumbro, Emily L.; Guerin, Gena D.; Sokoloski, Matthew L.; Ben-Ezra, Vic; Brower, Christopher S.; Rigby, Rhett B.; Duplanty, Anthony A.Skeletal muscle physiology is regulated by microRNA that are localized within skeletal muscle (myomiRs). This study investigated how the expression of myomiRs and genes regulating skeletal muscle mass and myogenesis are influenced in response to acute and consecutive days of exercise-related signaling using the exercise mimetic, formoterol, in vitro. Human skeletal muscle cells were proliferated and differentiated for 6 days. Experimental conditions included: (a) control, (b) acute formoterol stimulation (AFS), and (c) consecutive days of formoterol stimulation (CFS). For AFS, myotubes were treated with 30 nM of formoterol for three hours on day 6 of differentiation, and this was immediately followed by RNA extraction. For CFS, myotubes were treated with 30 nM of formoterol for three hours on two or three consecutive days, with RNA extracted immediately following the final three-hour formoterol treatment. We observed increased myomiR expression for both AFS and CFS. AFS appeared to promote myogenesis, but this effect was lost with CFS. Additionally, we observed increased expression of genes involved in metabolism, mitochondrial biogenesis, and muscle protein degradation in response to AFS. myomiR and gene expression appear to be sensitive to acute and long-term exercise-related stimuli, and this likely contributes to the regulation of skeletal muscle mass.Item Caenorhabditis elegans egg-laying and brood-size changes upon exposure to Serratia marcescens and Staphylococcus epidermidis are independent of DBL-1 signaling(Caltech Library, 2019-04) Madhu, Bhoomi; Salazar, Aileen E.; Gumienny, Tina L.Effects of S. marcescens and S. epidermidis on egg laying and brood size in wild-type and dbl-1(nk3) populations. (A) Comparison of mean eggs laid between wild-type (WT) and dbl-1(nk3) populations fed on E. coli OP50, S. marcescens, or S. epidermidis. The counts of number of eggs laid were reported only for plates with live animals. Error bars represent SEM. n = 7-10 surviving animals each. (B) Comparison of total brood size between wild-type and dbl-1(nk3) populations fed on E. coli OP50, S. marcescens, or S. epidermidis. Total brood sizes of animals that desiccated were censored. Error bars represent SEM. n = 7-10 surviving animals each. Within each genetic background, significant differences between the E. coli OP50-fed control and the pathogen-fed populations are marked with asterisks (*, p< 0.05, **, p< 0.001).Item Degradation of the separase-cleaved REC8, a meiotic cohesin subunit, by the N-end rule pathway(Elsevier, 2016) Liu, Yu-Jiao; Chang, ZeNan; Wadas, Brandon; Brower, Christopher S.; Song, Zhen-Hua; Xu, Zhi-Liang; Shang, Yong-Liang; Liu, Wei-Xiao; Wang, Li-Na; Dong, Wen; Varshavsky, Alexander; Hu, Rong-Gui; Li, WeiThe Ate1 arginyltransferase (R-transferase) is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N-terminal Asp, Glu, or (oxidized) Cys. The resulting N-terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeast Saccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/Mcd1 cohesin subunit generates a C-terminal fragment that bears N-terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N-terminal Glu, a substrate of the Ate1 R-transferase. Here we constructed and used a germ cell-confined Ate1−/− mouse strain to analyze the separase-generated C-terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N-terminal arginylation, and that male Ate1−/− mice are nearly infertile, due to massive apoptotic death of Ate1−/− spermatocytes during the metaphase of meiosis I. These effects of Ate1 ablation are inferred to be caused, at least in part, by the failure to destroy the C-terminal fragment of Rec8 in the absence of N-terminal arginylation.Item Editorial: Cytokine-mediated organ dysfunction and tissue damage induced by viruses(Frontiers, 2020-01-22) Spencer, Juliet; Religa, Piotr; Lehmann, MichaelCytokines are small proteins, mostly secreted into the extracellular environment, that bind to specific cell surface receptors, which mediate cell differentiation, migration, growth, and death. Gene expression and cellular release of cytokines are strictly regulated to assure proper function of cells, tissues, and organs. Upon virus infection, a cell starts producing type I interferons (IFN) and inflammatory cytokines (ICs) to restrict spread and replication of the respective virus. Ideally, the virus is completely eliminated by the immune system and the antiviral mechanisms are turned off within a reasonable time frame. However, there are different scenarios where this process does not work efficiently or does not happen at all, leading to cytokine-mediated organ dysfunction and tissue damage.Item Effects of 17β-Estradiol on growth, primary metabolism, phenylpropanoid- flavonoid pathways and pathogen resistance in Arabidopsis thaliana(Scientific Research Publishing, Inc., 2016-10) Upadhyay, Pallavi; Maier, CameliaMammalian sex hormones are spread in the environment from natural and anthropogenic sources. In the present study, the effect of estradiol on Arabidopsis thaliana growth primary metabolism, phenylpropanoid and flavonoid pathways and pathogen resistance were investigated. Treatments of Arabidopsis plants with 10 and 100 nM 17β -estradiol resulted in enhanced root growth and shoot biomass. In addition, treated plants had an increased rate of photosynthesis with a concomitant increase in carbohydrate and protein accumulation. Plants exposed to higher concentrations of 17β -estradiol (10 μM) had significantly lower root growth, biomass, photosynthesis rate, primary metabolite and phenylpropanoid and flavonoid contents indicating a toxic effect of estradiol. Treatments with increasing estradiol concentrations (10 nM, 100 nM and 10 μM) resulted in the downregulation of phenylpropanoid-flavonoid pathway genes (PAL1, PAL4, CHI and ANS) and subsequent decreased accumulation of phenolics, flavonoids and anthocyanins. Estradiol-treated plants were inoculated with Pseudomonas syringae pv. Tomato DC3000 and basal resistance was determined. Estradiol treatments rendered plants susceptible to the pathogen, thus compromising the plant defense mechanisms. These results indicate that at low concentrations, estradiol functions as a biostimulant of growth, yield and primary metabolism of Arabidopsis. However, estradiol functions as a potential transcriptional regulator of the phenylpropanoid pathway genes in Arabidopsis, having a negative effect on the phenylpropanoid and flavonoid biosynthetic pathways.Item The effects of nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2) activation in preclinical models of peripheral neuropathic pain(MDPI, 2022) Basu, Paramita; Averitt, Dayna L.; Maier, Camelia; Basu, ArpitaOxidative stress, resulting from an imbalance between the formation of damaging free radicals and availability of protective antioxidants, can contribute to peripheral neuropathic pain conditions. Reactive oxygen and nitrogen species, as well as products of the mitochondrial metabolism such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are common free radicals. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor encoded by the NFE2L2 gene and is a member of the cap ‘n’ collar subfamily of basic region leucine zipper transcription factors. Under normal physiological conditions, Nrf2 remains bound to Kelch-like ECH-associated protein 1 in the cytoplasm that ultimately leads to proteasomal degradation. During peripheral neuropathy, Nrf2 can translocate to the nucleus, where it heterodimerizes with muscle aponeurosis fibromatosis proteins and binds to antioxidant response elements (AREs). It is becoming increasingly clear that the Nrf2 interaction with ARE leads to the transcription of several antioxidative enzymes that can ameliorate neuropathy and neuropathic pain in rodent models. Current evidence indicates that the antinociceptive effects of Nrf2 occur via reducing oxidative stress, neuroinflammation, and mitochondrial dysfunction. Here, we will summarize the preclinical evidence supporting the role of Nrf2 signaling pathways and Nrf2 inducers in alleviating peripheral neuropathic pain.Item The elongin B ubiquitin homology domain(Elsevier, 1999) Brower, Christopher S.; Shilatifard, Ali; Mather, Timothy; Kamura, Takumi; Takagi, Yuichiro; Haque, Dewan; Treharne, Annemarie; Foundling, Stephen I.; Conaway, Joan Weliky; Conaway, Ronald C.Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.Item Estrogen modulation of the pronociceptive effects of serotonin on female rat trigeminal sensory neurons is timing dependent and dosage dependent and requires estrogen receptor alpha(Lippincott, 2022) Kaur, Sukhbir; Hickman, Taylor M.; Lopez-Ramirez, Angela; McDonald, Hanna; Lockhart, Lauren M.; Darwish, Omar; Averitt, Dayna L.The role of the major estrogen estradiol (E2) on orofacial pain conditions remains controversial with studies reporting both a pronociceptive and antinociceptive role of E2. E2 modulation of peripheral serotonergic activity may be one mechanism underlying the female prevalence of orofacial pain disorders. We recently reported that female rats in proestrus and estrus exhibit greater serotonin (5HT)-evoked orofacial nocifensive behaviors compared with diestrus and male rats. Further coexpression of 5HT2A receptor mRNA in nociceptive trigeminal sensory neurons that express transient receptor potential vanilloid 1 ion channels contributes to pain sensitization. E2 may exacerbate orofacial pain through 5HT-sensitive trigeminal nociceptors, but whether low or high E2 contributes to orofacial pain and by what mechanism remains unclear. We hypothesized that steady-state exposure to a proestrus level of E2 exacerbates 5HT-evoked orofacial nocifensive behaviors in female rats, explored the transcriptome of E2-treated female rats, and determined which E2 receptor contributes to sensitization of female trigeminal sensory neurons. We report that a diestrus level of E2 is protective against 5HT-evoked orofacial pain behaviors, which increase with increasing E2 concentrations, and that E2 differentially alters several pain genes in the trigeminal ganglia. Furthermore, E2 receptors coexpressed with 5HT2A and transient receptor potential vanilloid 1 and enhanced capsaicin-evoked signaling in the trigeminal ganglia through estrogen receptor α. Overall, our data indicate that low, but not high, physiological levels of E2 protect against orofacial pain, and we provide evidence that estrogen receptor α receptor activation, but not others, contributes to sensitization of nociceptive signaling in trigeminal sensory neurons.Item Euphorbia bicolor (Euphorbiaceae) latex extract reduces inflammatory cytokines and oxidative stress in a rat model of orofacial pain(Hindawi, 2019) Basu, Paramita; Hornung, Rebecca S.; Averitt, Dayna L.; Maier, CameliaRecent studies have reported that the transient receptor potential V1 ion channel (TRPV1), a pain generator on sensory neurons, is activated and potentiated by NADPH oxidase-generated reactive oxygen species (ROS). ROS are increased by advanced oxidation protein products (AOPPs), which activate NADPH oxidase by upregulating Nox4 expression. Our previous studies reported that Euphorbia bicolor (Euphorbiaceae) latex extract induced peripheral analgesia, partly via TRPV1, in hindpaw-inflamed male and female rats. The present study reports that E. bicolor latex extract also can evoke analgesia via reduction of oxidative stress biomarkers and proinflammatory cytokines/chemokines in a rat model of orofacial pain. Male and female rats were injected with complete Freund’s adjuvant (CFA) into the left vibrissal pad to induce orofacial inflammation, and mechanical allodynia was measured by the von Frey method. Twenty-four hours later, rats received one injection of E. bicolor latex extract or vehicle into the inflamed vibrissal pad. Mechanical sensitivity was reassessed at 1, 6, 24, and/or 72 hours. Trigeminal ganglia and trunk blood were collected at each time point. In the trigeminal ganglia, ROS were quantified using 2,7-dichlorodihydrofluorescein diacetate dye, Nox4 protein was quantified by Western blots, and cytokines/chemokines were quantified using a cytokine array. AOPPs were quantified in trunk blood using a spectrophotometric assay. E. bicolor latex extract significantly reduced orofacial mechanical allodynia in male and female rats at 24 and 72 hours, respectively. ROS, Nox4, and proinflammatory cytokines/chemokines were significantly reduced in the trigeminal ganglia, and plasma AOPP was significantly reduced in the trunk blood of extract-treated compared to vehicle-treated rats. In vitro assays indicate that E. bicolor latex extract possessed antioxidant activities by scavenging free radicals. Together our data indicate that the phytochemicals in E. bicolor latex may serve as novel therapeutics for treating oxidative stress-induced pain conditions.Item Euphorbia bicolor (Euphorbiaceae) latex phytochemicals induce long-lasting non-opioid peripheral analgesia in a rat model of inflammatory pain(Frontiers Media, 2019-09-03) Basu, Paramita; Tongkhuya, Sirima A.; Harris, Taylor L.; Riley, Angela R.; Maier, Camelia; Granger, John; Wojtaszek, Jennie; Averitt, Dayna L.The negative side effects of opioid-based narcotics underscore the search for alternative non-opioid bioactive compounds that act on the peripheral nervous system to avoid central nervous system-mediated side effects. The transient receptor potential V1 ion channel (TRPV1) is a peripheral pain generator activated and sensitized by heat, capsaicin, and a variety of endogenous ligands. TRPV1 contributes to peripheral sensitization and hyperalgesia, in part, via triggering the release of proinflammatory peptides, such as calcitonin gene-related peptide (CGRP), both locally and at the dorsal horn of the spinal cord. Ultrapotent exogenous TRPV1 agonists, such as resiniferatoxin identified in the latex of the exotic Euphorbia resinifera, trigger hyperalgesia followed by long lasting, peripheral analgesia. The present study reports on the analgesic properties of Euphorbia bicolor, a relative of E. resinifera, native to the Southern United States. The study hypothesized that E. bicolor latex extract induces long-lasting, non-opioid peripheral analgesia in a rat model of inflammatory pain. Both inflamed and non-inflamed adult male and female rats were injected with the methanolic extract of E. bicolor latex into the hindpaw and changes in pain behaviors were reassessed at various time points up to 4 weeks. Primary sensory neuron cultures also were treated with the latex extract or vehicle for 15 min followed by stimulation with the TRPV1 agonist capsaicin. Results showed that E. bicolor latex extract evoked significant pain behaviors in both male and female rats at 20 min post-injection and lasting around 1–2 h. At 6 h post-injection, analgesia was observed in male rats that lasted up to 4 weeks, whereas in females the onset of analgesia was delayed to 72 h post-injection. In sensory neurons, latex extract significantly reduced capsaicin-evoked CGRP release. Blocking TRPV1, but not opioid receptors, attenuated the onset of analgesia and capsaicin-induced CGRP release. Latex was analyzed by mass spectrometry and eleven candidate compounds were identified and reported here. These findings indicate that phytochemicals in the E. bicolor latex induce hyperalgesia followed by peripheral, non-opioid analgesia in both male and female rats, which occurs in part via TRPV1 and may provide novel, non-opioid peripheral analgesics that warrant further examination.Item Feedback regulation of BMP signaling by Caenorhabditis elegans cuticle collagens(American Society for Cell Biology, 2020-03-31) Gumienny, Tina L.; Madaan, Uday; Faure, Lionel; Chowdhury, Albar; Ahmed, Shahrear; Ciccarelli, Emma J.; Savage-Dunn, CathyCellular responsiveness to environment, including changes in extracellular matrix (ECM), is critical for normal processes such as development and wound healing, but can go awry, as in oncogenesis and fibrosis. One type of molecular pathway contributing to this responsiveness is the BMP signaling pathway. Owing to their broad and potent functions, BMPs and their pathways are regulated at multiple levels. In Caenorhabditis elegans, the BMP ligand DBL-1 is a regulator of body size. We previously showed that DBL-1/BMP signaling determines body size through transcriptional regulation of cuticle collagen genes. We now identify feedback regulation of DBL-1/BMP through analysis of four DBL-1–regulated collagen genes. Inactivation of any of these genes reduces DBL-1/BMP signaling, measured by a pathway activity reporter. Furthermore, depletion of these collagens reduces GFP::DBL-1 fluorescence and acts unexpectedly at the level of dbl-1 transcription. We conclude that cuticle, a specialized ECM, impinges on DBL-1/BMP expression and signaling. Interestingly, the feedback regulation of DBL-1/BMP signaling by collagens is likely to be contact independent due to physical separation of the cuticle from DBL-1–expressing cells in the ventral nerve cord. Our results provide an entry point into a novel regulatory mechanism for BMP signaling, with broader implications for mechanical regulation of gene expression.Item A fluorescence-based reporter of Arginyltransferase 1 (ATE1)(Texas Society for Microscopy, 2020) Kasu, Yasar Arfat T.; Dasgupta, Rinki; Brower, Christopher S.Arginyltransferase 1 (ATE1) is an enzyme that catalyzes the transfer of arginine onto protein fragments with acidic N-termini. This is an essential step in the degradation of these fragments by the N-degron pathway of the ubiquitin proteasome system. Previous studies have shown that arginylation is required for the removal of specific fragments associated neurodegeneration, and that the loss of ATE1 activity leads to neurological problems. Interestingly, reduced ATE1 activity was also associated with fat loss and resistance to diet-induced obesity. Thus, the modulation of ATE1 holds promise for treating these increasingly common human diseases. To this end, we synthesized a cell-based reporter that employs direct fluorescence to monitor ATE1 activity. Using confocal microscopy and immunoblot analysis, we show that this reporter provides a robust readout of ATE1 activity in vivo. This reporter will be useful in screening approaches aimed to identify modulators of ATE1, which may ultimately have therapeutic potential.
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