bluuebunny/crossref_metadata_2025_split huggingface dataset:
IL‐6 modulates cardiac muscle proteostasis and anti‐oxidative capacity post‐burn
Authors: El Ayadi Amina , Wang Ye , Prasai Anesh , Wetzel Michael , Goullry Ashley , Porter Craig , Herndon David N , Finnerty Celeste 
Published: November 2023

Heart failure is the number one cause of death in severely burned patients. Burn‐induced cardiac dysfunction includes increased cardiac work, tachycardia and increased energy expenditure. Burn injury induces a surge in catecholamine and cytokines levels that lasts up to 3 years post injury. While cardiac dysfunction has been associated with stimulation of the β‐adrenergic receptors (β‐ARs) by catecholamines, the role of inflammatory cytokines in the cardiac muscle function is still unclear. We have reported that burn injury alters the beta‐adrenergic signaling in the heart at 7 days post‐burn. In another study, we have shown that burn injury induces atrophy of the diaphragm muscle through activation of the IL‐6/JAK/Stat pathway.Male wild‐type and interleukin 6 knockout mice (IL‐6 KO) were subjected to a 30% total body surface area full‐thickness burn. Animals were sacrificed 10 days post injury. The hearts were isolated and western blotting performed to analyze the role of JAK/Stat signaling pathways and antioxidant enzymes in cardiac muscle proteostasis post‐burn.Burn injury significantly increases Stat‐3 phosphorylation at Ser 727 in the WT (p<0.05) but not in the IL‐6‐KO animals. Stat‐3 phosphorylation at tyrosine705 (Y705) was activated in the cardiac muscle of both the WT (p<0.05) and IL‐6 KO (p<0.05) mice post‐burn. At basal levels, p‐Stat‐3(Y705) was higher in the control IL‐6 KO than the control WT animals (p< 0.05). P‐Stat‐3 (Y705) levels were also higher in L‐6 KO burned animals compared to WT burned animals (p<0.01). The protein levels of antioxidant enzymes SOD‐2 and catalase were significantly decreased post‐burn in the WT (both p<0.05) but not in the IL‐6 KO animals.These data suggest that the molecular mechanisms regulating cardiac muscle proteostasis and anti‐oxidative capacity following burn injury are altered in the IL‐6‐KO mice supporting the role of IL‐6 as a critical mediator in these processes.Support or Funding InformationThis study was supported by grants from the National Institute of Health (P50‐GM60338, R01‐GM56687, and T32‐GM8256, NIH R01 GM 112936‐01, UL1TR000071), Shriners Hospitals for Children (71008, 71001 and 84080), and the Shriners Hospital for Children fellowship for AE (SHC 84202).
Characterization of a Short Motif Capable of Enhancing Human Cap‐Independent Translation
Authors: Juba Amber N. , Hansen Allison , Chaput John C. , Wellensiek Brian P. 
Published: November 2023

The classical model for eukaryotic translation initiation involves linear scanning, a cap‐dependent process in which the ribosome recognizes a 7‐methylguanosinecap at the 5′ end of the RNA message and scans the 5′ untranslated region for an appropriate initiation site. Alternatively, a growing body of evidence suggests that cap‐independent mechanisms play an important role during many cellular events where cap‐dependent scanning is inhibited. However despite this evidence, the mechanism by which this cap‐independent mode of translation occurs remains largely unknown. In a previous study, we began to address this by combining messenger RNA display with next‐generation deep sequencing to profile the human genome for RNA leader sequences that can enhance the translation of uncapped mRNA transcripts. Analysis of these sequences revealeda short, 13 nucleotide motif that was present in many of the RNAs isolated via this method. Given these findings, we propose that the identified 13 nucleotidemotif, when present within the leader sequence of an open reading frame, has an integral role in determining the increased translational activity driven by the larger leader sequence. To explore this role we examined the translation enhancing activity of several human in vitro selected translation enhancing elements (TEEs), which either contained or did not contain the 13 nucleotide motif. A series of reporter vectors containing the TEEs were used to assay the effect of the presence and absence of the motif on overall translation efficiency. Functional analysis studies performed in vitro and in cultured human and mammalian cells indicate that the 13 nucleotide motif, when present within a larger TEE, increased protein translation levels by several fold. Additional studies have identified distance constraints for translation enhancement with respect to the 13 nucleotide motif and the downstream open reading frame. Taken together these results provide evidence that the identified short 13 nucleotide motif plays an important role in the mechanism of cap‐independent translation from the human genome. This observation provides a new opportunity to explore the mechanistic details of human translation enhancing elements and their contribution to the human proteome.Support or Funding InformationFunding for this research was provided by Midwestern University intramural funds awarded to BPW, a Midwestern University College of Veterinary Medicine research fellowship awarded to AH, and an NIH grant awarded to JCC
Protein Synthesis Regulation by Soy Isoflavones Metabolite Equol in Metastatic Breast Cancer Cells
Authors: Cruz‐Collazo Ailed Marie , de la Parra Columba , Schneider Robert J , Dharmawardhane Suranganie Marie 
Published: November 2023

Equol, one of the most studied metabolites of the soy isoflavone daidzein, is associated with both anticancer and pro‐cancer effects in breast cancer. These contradictory results have been shown to be concentration dependent, where high equol concentrations reduced non‐metastatic breast cancer cell growth and proliferation, and reduced breast cancer risk. However, we have shown that at low physiological dietary concentrations, equol increases cell proliferation, tumor growth and metastasis in metastatic breast cancer cell models. Also, equol increases the expression of the eukaryotic initiation factor 4G (eIF4G), increasing the cap‐independent protein synthesis of pro‐cancer molecules (de la Parra et al., J Biol Chem. 2012, 287(50):41640–50 and J Biol Chem. 2015, 290(10):6047–57). To elucidate the role of equol in the regulation of protein synthesis in metastatic breast cancer cells, a polysome profile assay using an Affymetrix microarray was conducted in cells treated with the following experimental conditions: vehicle control, equol, eIF4G knockdown (kd), or both equol + eIF4G kd. The following equation was used to identify the mRNAs that were actively translated for each experimental condition: [(mRNA associated with heavy or light polysome fraction/total mRNA)/(control mRNA associated with heavy or light polysome fraction/total control mRNA)]. This analysis using a false discovery rate (FDR) of <0.05 demonstrated that >1000 protein coding and non‐coding RNAs were differentially associated with polysomes in response to equol or eIF4G kd, but not for the combined treatment of equol + eIF4G kd. Ingenuity Pathway Analysis for mRNAs with a log2 fold change >2, demonstrated significant changes in mRNAs of cancer invasion proteins like MMP1, chromatin remodeling proteins like Histones, and cancer‐associated microRNAs (miRNAs) like miR21 and miR590. Therefore, our data has identified critical targets of equol involved in breast cancer metastasis regulation that are differentially expressed via translational control.Support or Funding InformationThis research was supported by grants from NIH/NIGMS SC3GM094824 to SD, RCMI G12RR03051 to UPR MSC, and MBRS RISE (R25GM061838) to UPR MSC (AC).
Characterization of CD38 mRNA, Protein, and Enzyme Activity in the Cell Types of the Heart
Authors: Boslett James , Zweier Jay L. 
Published: November 2023

IntroductionRecently, we showed that NAD(P)+‐hydrolyzing CD38 is activated in myocardial ischemia/reperfusion (I/R) injury, leading to depletion of its enzymatic substrate NADP+ and depletion of the NADP(H) pool. This in turn decreases nitric oxide (NO) production from endothelial NO synthase (eNOS) and triggers postischemic endothelial dysfunction (1). CD38 inhibition affords protection to the heart against I/R injury with increased recovery of left ventricle function and decreased infarction (1). CD38 has an important role in I/R injury; however, questions remain regarding which cardiac cells express CD38 and the levels of endothelial, fibroblast, and myocyte expression. Therefore, we characterize CD38 in the cell types of the heart including cardiac myocytes, endothelial cells, and cardiac fibroblasts. To fully evaluate CD38 in these cells, we measure levels of transcription (mRNA), translation (protein), and enzymatic activity.MethodsCells were derived from Sprague Dawley rats. Rat aortic endothelial cells (ECs), and rat cardiac fibroblasts (CFs) were obtained from Innoprot and cultured. Rat cardiac myocytes (CMs) were prepared by collagenase II digestion. mRNA was extracted from each cell type by trizol/chloroform extraction and analyzed for CD38 mRNA by RT‐qPCR. Total cellular proteins were extracted from each with RIPA buffer and analyzed by immunoblotting. CD38 activity assays were performed using whole cell homogenate incubated with CD38 substrate etheno‐NAD, a fluorescent NAD+‐analog allowing highly sensitive measurements of CD38 activity.ResultsCD38 mRNA, normalized by the ornithine decarboxylase antizyme 1 (OAZ1) housekeeping gene, was highest in ECs, with lower levels in CFs, and trace levels in CMs (Table). These trends were consistent when CD38 protein was measured, with highest levels in ECs, lower levels in CFs, and trace levels of CD38 in CMs. Levels of CD38 protein expression in ECs were 6‐fold, and >25‐fold higher than in CFs, and CMs, respectively. Lastly, CD38 activity measurements were consistent with levels of CD38 protein as expected, with high levels in ECs, low levels in CFs, and trace or no activity in CMs.ConclusionCD38 mRNA, protein, and activity measurements, show that ECs are rich in CD38, with lower levels in CFs, and low levels of message and almost no CD38 protein or activity in CMs. Thus, CD38 is abundant in the ECs with lower expression and activity in CFs. It is effectively absent from the CMs. The primary effects of CD38 would thus be on the endothelium; however, CD38 activation in ECs can modulate eNOS function and NO production (1, 2), in turn affecting downstream cardiac myocyte function and salvage.

Cell Type
CD38 mRNA (CD38/OAZ1)*
Protein expression (relative densitometric units)**
CD38 activity (nmol/min/mg protein)

ECs
1.00 ± 0.15
1.00 ± 0.03
5.00 ± 0.25

CMs
0.06 ± 0.002
0.037 ± 0.02
0.079 ± 0.01

CFs
0.39 ± 0.02
0.16 ± 0.01
0.99 ± 0.14

ECs normalized to 1.
30 mg total cell protein loaded per sample. ECs normalized to 1.
Differentiation of human keratinocytes requires translational control by the eIF2 kinase GCN2
Authors: Collier Ann , Wek Ronald , Spandau Dan 
Published: November 2023

In the human epidermis, differentiation of keratinocytes is essential to form a barrier to protect against a variety of environmental insults. This differentiation process can become dysregulated in many skin diseases, including psoriasis, eczema, and non‐melanoma skin cancer. Although changes in transcriptional networks during keratinocyte differentiation are well characterized, little is known about the role of translational control. A major mechanism of translational control involves phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2‐P). In response to environmental stresses, induced eIF2‐P represses translation initiation, allowing cells to conserve resources and trigger cytoprotective gene expression. In addition to repression of global protein synthesis, eIF2‐P induces preferential translation of specific mRNAs that promote stress adaptation, including the transcription factors ATF4 and CHOP. Since keratinocytes undergo a massive change in morphology and gene expression during differentiation, we hypothesized that translational control plays a major role. To investigate this hypothesis we measured both genome‐wide and gene‐specific levels of translation. We observed a global reduction in translation initiation as measured by polysome profiling during differentiation in both monolayer and 3D organotypic keratinocyte cultures. Expression of eIF2‐P, ATF4, and CHOP was limited to suprabasal (differentiated) keratinocytes in vivo as visualized histologically in human skin tissue. Keratinocyte differentiation in vitro induced eIF2‐P to levels similar to that seen following endoplasmic reticulum stress, but eIF2‐P alone was not sufficient to induce differentiation. Of interest, the epidermal differentiation transcript involucrin (IVL) was preferentially bound to large polysomes following differentiation, indicative of preferential translation, which was similar to that seen for ATF4 and CHOP. Inhibition of eIF2‐P blocked both general and gene‐specific translational control during differentiation. Loss of either eIF2‐P or the upstream protein kinase GCN2 abrogated both IVL protein and mRNA expression. Strikingly, loss of GCN2 altered epidermal tissue formation in a 3D organotypic model as characterized by disorganized keratinocyte layers and a decreased number of cornified cells. These results provide novel evidence that a whole‐cell repression of protein synthesis alongside gene‐specific translational control occur during normal epidermal differentiation. These findings suggest that translational control through GCN2/eIF2‐P is important for proper epidermal stratification, likely though translational regulation of differentiation genes such as IVL. The GCN2/eIF2‐P pathway in keratinocyte biology provides for novel targets for intervention in dermatological diseases characterized by abnormal differentiation.Support or Funding InformationRuth L. Kirschstein National Research Service Award (1F31ES026517 to AEC)
The N‐terminus of SECIS Binding Protein 2 is Required for Processive Selenocystine Incorporation in Selenoprotein P
Authors: Pinkerton Mark Hurst , Vetick Michael , Shetty Sumangala P , Copeland Paul R 
Published: November 2023

A UGA stop codon is recoded to accommodate the incorporation of the 21st amino acid selenocysteine (Sec). For UGA to be recoded a specialized set of cis and trans factors are required and consist of: an mRNA with an in frame UGA codon, a selenocysteine insertion sequence (SECIS) in the 3′ untranslated region, a SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), and a selenocysteine tRNA. The N‐terminus of SBP2 is believed to have no direct role in Sec incorporation because the C‐terminus of SBP2 is sufficient for the incorporation of Sec into selenoproteins that have one Sec codon. Having recently developed an in vitro translation system in wheat germ requiring the addition of all Sec‐incorporation factors, we found that translation of SELENOP, which contains 10 Sec codons, is defective in that only early termination products are made. This contrasts with mammalian systems where full length protein is predominantly found. This observation, combined with the fact that neither SELENOP nor the N‐terminus of SBP2 are found in invertebrates, points to a specialized function for the N‐terminus of SBP2 related to SELENOP synthesis. To validate the relationship between the N‐terminus of SBP2 and SELENOP, C‐terminal and full length SBP2 were compared for in vitro translation of SELENOP. We found that the production of full length SELENOP is heavily dependent on the presence of the SBP2 N‐terminus. Our results suggest that the N‐terminus of SBP2 was appended during evolution to allow for processive incorporation of multiple Sec residues into SELENOP.Support or Funding InformationThis work was supported by National Institutes of Health Grant GM077073
Regulation of Ankyrin‐repeat and SOCS‐box protein 9 (ASB9) in ovarian follicles and identification of binding partners
Authors: Benoit Gabriel , Lussier Jacques G , Ndiaye Kalidou 
Published: November 2023

Ankyrin‐repeat and SOCS‐box protein 9 (ASB9) is a member of the large SOCS‐box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. ASB9 is known to interact with creatine kinase B and negatively regulates cell growth. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation and to identify its binding partners in GC of bovine ovulatory follicles. GC were obtained from small follicles (SF: 2–4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). RT‐PCR analyses showed a 104‐fold induction of ASB9 expression in GC of OF as compared to DF (P < 0.0001). Steady‐state mRNA levels of ASB9 in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 h after hCG injection showed a significant induction of ASB9 expression at 12 and 18 h (P<0.001), reaching a maximum induction at 24 h post‐hCG (P<0.0001) as compared to 0 h. These results were confirmed in western blot analysis showing highest ASB9 protein amounts in OF. Yeast two‐hybrid screening of OF‐cDNAs library resulted in the identification of five potential ASB9 binding partners in GC, confirmed by in vitro co‐immunoprecipitation analyses. Overall, these results support a physiologically relevant role of ASB9 in the ovulation process, and provide, for the first time, insights into the regulation and mode of action of ASB9 in GC.Support or Funding InformationThis work was supported in part by internal funds from Université de Montréal to KN and by a Discovery Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) to JGL.
Determination of Pair‐wise Protein Interactions between Spt7 and Drosophila melanogaster SAGA Subunits
Authors: Colon Acosta Edwin E. , Weake Vikki 
Published: November 2023

The association between spliceosomal components and the transcriptional machinery offers an interesting interconnection between the mechanisms of pre‐mRNA splicing and genetic transcription. Recent findings demonstrate the existence of two novel protein subunits in the D. melanogaster Spt‐Ada‐Gcn5‐Acetyltransferase (SAGA) transcriptional co‐activator protein complex, splicing factor 3B subunits 3 and 5 (SF3B3 and SF3B5), which are known to be part of the SF3B complex in the U2 small nuclear ribonucleoprotein particle (U2snRNP). Previous work has demonstrated that SF3B3 and SF3B5 interact in a yeast two‐hybrid system with both Spt7 and Sgf29 subunits in SAGA, thus Spt7 and Sgf29 provide the potential candidates to incorporate SF3B3 and SF3B5 into the SAGA complex. Recent findings show that SF3B5 is necessary for the expression of a subset of SAGA‐regulated genes independent of splicing, although not necessary for SAGA's histone‐modifying activities (Stegeman et al, 2016). However, the functions of both SF3B3 and SF3B5 within the SAGA complex are not completely characterized. In response to this, an Spt7 mutant allele library was developed to screen for mutations that disrupt protein‐protein interactions with SF3B3 and SF3B5 but retain the interactions that wild type Spt7 has with the SAGA complex. Finding specific mutants will potentially allow us to obtain a SAGA‐specific phenotype that determines the functions of SF3B3 and SF3B5 within the SAGA complex. To screen this Spt7 mutant allele library it is necessary to determine, beforehand, the protein subunits that wild type Spt7 interacts with in SAGA. In a yeast two‐hybrid system, we tested the Spt7 subunit for protein‐protein interactions with most of the SAGA subunits. Here, we report the protein subunits that wild type Spt7 is known to interact with in the SAGA complex, including a previously undetermined interaction with the TBP‐associated factor 10b (TAF‐10B) subunit. These findings will allow us to screen for Spt7 mutants that do not interact with SF3B components, but still interact with the SAGA complex.Support or Funding InformationNSF‐REU for Molecular and Biochemical Analysis of Proteins at Purdue University 2016
The Impact of T Cell Receptor Docking Geometry on T Cell Signaling
Authors: Devlin Jason , Harris Dan , Singh Nishant , Smith Sheena , Kranz David , Baker Brian 
Published: November 2023

A T cell receptor (TCR) expressed on the exterior of a cytotoxic CD8+ T cell recognizes the composite surface of a peptide bound in the the class I major histocompatibility complex molecule (MHC). The peptide is derived from the breakdown of proteins in the proteosome, and can come from a protein of self, viral, bacterial, or even tumor origin. The engagement of a peptide/MHC by a TCR initiates a cytotoxic T cell response against that peptide. The main determinant of the strength of the T cell response is the affinity of the TCR‐peptide/MHC interaction. The dissociation kinetics of the complex, as well as structural features of the TCR docking the peptide/MHC have also been postulated to be involved in modulating T cell signaling.A large assembly of proteins needs to form around the TCR‐peptide/MHC interaction in cases where T cell activation is coreceptor‐dependent. The CD8 coreceptor binds to the peptide/MHC and recruits a kinase to the cytosolic domain of the TCR to generate an immune signal. The requirement for coreceptor binding suggests that a favorable docking geometry exists for the TCR engaging the peptide/MHC so as to allow for coreceptor binding. Indeed, with a few exceptions, all TCRs characterized to date exhibit a similar docking mode.Recently, directed evolution has produced a TCR (RD1‐MART1‐high) that binds a peptide/MHC in a different binding geometry than other TCRs that bind the same peptide/MHC. Despite the difference in docking geometry, T cells expressing RD1‐MART1‐high still produced IL‐2 upon activation by the peptide/MHC antigen. By analyzing the signal strength produced by TCRs which bind the same peptide/MHC antigen, we can address if T cell signaling varies with changes in docking geometry. Two T cell receptors will be matched for affinity and kinetics to eliminate any difference in T cell signal due to these factors.
Importance of the C‐terminal histidine residues of Helicobacter pylori GroES for Toll‐like receptor 4 binding and interleukin‐8 cytokine production
Authors: Chow Lu‐Ping , Lee Haur , Su Yu‐Lin , Huang Bo‐Shih , Hsieh Feng‐Tse 
Published: November 2023

Helicobacter pylori infection is associated with the development of gastric and duodenal ulcers as well as gastric cancer. GroES of H. pylori (HpGroES) was previously identified as a gastric cancer‐associated virulence factor. Our group showed that HpGroES induces interleukin‐8 (IL‐8) cytokine release via a Toll‐like receptor 4 (TLR4)‐dependent mechanism and domain B of the protein is crucial for interactions with TLR4. In the present study, we investigated the importance of the histidine residues in domain B. To this end, a series of point mutants were expressed in Escherichia coli, and the corresponding proteins purified. Interestingly, H96, H104 and H115 were not essential, whereas H100, H102, H108, H113 and H118 were crucial for IL‐8 production and TLR4 interactions in KATO‐III cells. These residues were involved in nickel binding. Four of five residues, H102, H108, H113 and H118 induced certain conformation changes in extended domain B structure, which is essential for interactions with TLR4 and consequent IL‐8 production. We conclude that interactions of nickel ions with histidine residues in domain B help to maintain the conformation of the C‐terminal region to conserve the integrity of the HpGroES structure and modulate IL‐8 release.Support or Funding InformationThis study was supported by Ministry of Science and Technology (MOST 104‐2320‐B‐002 ‐ 045), Liver Disease Prevention & Treatment Research Foundation, and Ministry of Education, Taiwan.