bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

This 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

Introduction

Recently, 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.

Methods

Cells 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.

Results

CD38 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.

Conclusion

CD38 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 Information

Ruth 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 Information

This 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 Information

This 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 Information

NSF‐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 Information

This 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.
Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response
Authors: Kumar Akhilesh , Birnbaum Michael D , Patel Devang M , Morgan William M , Singh Jayanti , Barrientos Antoni , Zhang Fangliang
Published: November 2023

Arginylation is a protein posttranslational modification (PTM) which leads to addition of an extra arginine at the N‐terminus of the target proteins and peptides. This process is evolutionarily conserved, and is mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. Here we report that arginylation promotes growth arrest and cell death, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events under stressing conditions such as oxidative stress, heat shock, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild‐type cells with normal stress response, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found that Ate1 is required to suppress mutation frequency in yeast and mammalian cells during DNA‐damaging conditions such as ultraviolet irradiation. Our data thereby explain the basis for the involvement of arginylation in physiological and diseased conditions by its regulation of cell death and/or mutagenesis. We also uncover a novel mechanism by which a posttranslational modification exerts a global effect on mutagenesis.
Probing the chromophore of the green fluorescent protein with 4‐cyano‐L‐phenylalanine
Authors: Piacentini Juliana , Olenginski Gregory M. , Brewer Scott H. , Phillips‐Piro Christine M.
Published: November 2023

The unnatural amino acid (UAA) 4‐cyano‐L‐phenylalanine (pCNF) has been shown previously to be an effective vibrational reporter of local protein environments. Here, pCNF was genetically incorporated into the 66‐site of superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl‐tRNA synthetase to probe the local environment around the GFP chromophore. The 66‐site is in the internal chromophore of the protein, which is responsible for the photo‐physical properties of sfGFP. Vibrational spectroscopy coupled with isotopomers of this UAA was utilized to probe the local solvation state of this site. This spectroscopic data was coupled with the X‐ray protein crystal structure of this construct. The spectroscopic and crystallographic data will be presented.
Phosphorylation regulates apoptotic caspase function through diverse molecular mechanisms
Authors: Hardy Jeanne A , Serrano Banyuhay P , Eron Scott J
Published: November 2023

Caspases, the cysteine proteases that execute apoptosis, are tightly regulated via phosphorylation by a series of kinases. Although all apoptotic caspases work in concert to promote apoptosis, different kinases regulate individual caspases. Several sites of caspase‐7 and −9 phosphorylation have been reported, but without knowing the molecular details, it has been impossible to exploit or control these complex interactions, which normally prevent unwanted proliferation. This work provides new molecular mechanisms for phosphorylation‐based control of both caspase‐7 and caspase‐9 function.

During dysregulation, PAK2 kinase plays an anti‐apoptotic role, phosphorylating caspase‐7 and promoting unfettered cell growth and chemotherapeutic resistance. PAK2 phosphorylates caspase‐7 at two sites, inhibiting activity using two different molecular mechanisms, before and during apoptosis. Phosphorylation of caspase‐7 S30 allosterically obstructs its interaction with caspase‐9, preventing intersubunit linker processing, slowing or preventing caspase‐7 activation. S239 phosphorylation renders active caspase‐7 incapable of binding substrate, blocking later events in apoptosis. In parallel and in response to cAMP, Protein Kinase A (PKA) phosphorylates caspase‐9 at three sites preventing caspase‐9 activation and suppressing apoptosis progression. Phosphorylation of caspase‐9 by PKA at the functionally relevant site, S183, acts as an upstream block of the apoptotic cascade, inactivating caspase‐9 by a two‐stage mechanism. First, S183 phosphorylation prevents caspase‐9 self‐processing and directly blocks substrate binding. In addition, S183 phosphorylation breaks the fundamental interactions within the caspase‐9 core, promoting disassembly of the large and small subunits. This occurs despite S183 being a surface residue distal from the large: small interface. This phosphorylation‐induced disassembly promotes the formation of ordered aggregates around 20 nm in diameter. Similar aggregates of caspase‐9 have not been previously reported. This two‐stage regulatory mechanism for caspase‐9 has not been reported previously but may be conserved across the caspases. Each of these mechanisms of regulation for caspase‐7 and caspase‐7 is novel, representing new opportunities for synergistic control of caspases and their counterpart kinases.

Support or Funding Information

This work was supported by the National Institutes of Health (NIH) GM 080532.
Creating Peptide Hydrazides via Intein Splicing for Native Chemical Ligation and Protein Labeling
Authors: Santoleri Dominic A , Liu Jun , Ekanayake Oshini , Rozovsky Sharon
Published: November 2023

Peptide hydrazides are powerful surrogates for the thioesters typically used in protein ligation as well as being an effective handle for labeling. Typical peptide hydrazide preparation relies on solid‐phase peptide synthesis, a technique limited by peptide size and solubility that also uses harsh chemicals. Here, a more effective and biochemically‐friendly means of generating these peptide hydrazides has been established involving split PhoRadA inteins, which only regain their splicing activity upon association with each other. This method also avoids several issues involved with full intein splicing, such as in vivo self‐cleavage and low expression levels. C‐Terminal peptide hydrazides are a powerful means of creating protein labels as we have shown them to react with small aldehyde‐containing molecules that can serve as a tag or a marker in analytical studies.

Support or Funding Information

Funding for this research project is provided through an NSF CAREER Award.
Role of SUMOylation in Alcohol‐induced Liver Fibrosis
Authors: Tomasi Maria Lauda , Cossu Carla , Ramani Komal
Published: November 2023

Background & Aim

Alcohol‐induced liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components, especially collagen in response to chronic hepatocellular damage. If alcohol consumption is continued, fibrosis may progress to cirrhosis which is a major cause of morbidity and mortality. Reactive oxygen species (ROS) generated in hepatocytes by alcohol metabolism plays an important role in producing liver damage and initiating hepatic fibrogenesis. ROS disrupts lipids, proteins and DNA inducing necrosis and apoptosis of hepatocytes and amplifies the inflammatory response via Kupffer cells that directly induces hepatic stellate cells (HSCs) activation, resulting in the initiation of fibrosis. SUMOylation is a post‐translational modification that plays an important role in a wide range of cellular processes. SUMOylation is oxidative stress inducible and involves the covalent attachment of a member of the SUMO (small ubiquitin‐like modifier) to the target protein. We previously demonstrated that the level of Ubiquitin‐conjugation enzyme 9 (Ubc9), a sole E2‐conjugating enzyme essential for SUMOylation, is induced in the livers of intragastric ethanol‐infusion (EI) treated mice and lipopolysaccharide‐induced inflammation promotes Ubc9 phosphorylation and its activity in Kupffer cells. Our aim was to examine whether the dysregulated SUMOylation could regulate the ethanol‐induced liver fibrosis and elucidate the molecular mechanism(s).

Methods

Studies were done using in vivo binge ethanol‐fed mice, primary mouse and human HSCs and LX‐2 cells. Oxidative stress markers were measured using commercial kits.

Results

We found that ethanol treatment for 48 hours (25mM) in LX‐2 and HSCs cells increased ROS production, Ubc9, α‐SMA and COL I mRNA levels, well known as activated HSCs markers, by 1.5‐fold (H2O2), 1.4‐fold (Ubc9), 1.3‐fold (α‐SMA) and 1.5‐fold (COL I), respectively. RNAi‐mediated knockdown of Ubc9 to 75% of control blocked ethanol‐induced ROS production and the increase in α‐SMA and COL I mRNA levels. Interestingly, we found that silencing SUMO‐1 in activated LX‐2 apoptosis was induced by 50%, while quiescent LX‐2 cells in matrigel were not affected. This finding confirmed that sumoylation is playing a key role on stellate cells activation. In addition, isolated mouse stellate cells from binge ethanol‐fed mice overexpressed high level of Ubc9 mRNA and protein levels compared with control mice.

Conclusion

This data is strongly suggesting that ethanol is inducing HSCs activation in human and mouse liver involving the oxidative stress‐induced sumoylation machinery. Identifying the molecular mechanisms will require knowing which proteins are affected and what the functional outcomes of a change in sumoylation status is on those proteins, which will pave the way for future exploring.

Support or Funding Information

Supported by NIH grant 5 K01 AA022372‐04, maria lauda tomasi
Thermodynamics of the Gamma B Crystallin Protein Demonstrated by T1/T2 NMR Experiments
Authors: Umphred‐Wilson Katharine , Fadden Aaron , Zanet John , Mathews Kaylee , Thurston George , Mills Jeffrey , Michel Lea Vacca
Published: November 2023

Gamma B Crystallin proteins are soluble proteins found in the eye lens which help maintain its transparency; however, over time their structure may mutate due to UV radiation and other environmental stresses causing the proteins to aggregate and develop into cataracts. Using light scattering, nuclear magnetic resonance (NMR) spectroscopy and other protein modeling techniques, our team is attempting to understand the intermolecular interactions of gamma B crystallin proteins that are responsible for this aggregation. We expect to learn the structural and chemical properties that dictate how the proteins interact, as well as the effect of the thermodynamic properties in the fluid mixtures of the eye lens on the crystallin interactions. We expressed the gamma B crystallins in Escherichia coli and isolated them using size exclusion and ion exchange chromatography. The protein was analyzed using various types of NMR experiments, including T1/T2 experiments. The Tau C values, which were derived from these experiments, correlate with a decreased tumbling time; this means that lower Tau C values indicate increased aggregation. Our preliminary results suggest that cold temperatures and high concentrations of our protein increase aggregation levels and the number of attractive intermolecular interactions.

Support or Funding Information

NIH EY018249 (G. Thurston)
Probing solvation environments in Tt H‐NOX by site‐specific incorporation of 4‐cyano‐L‐phenylalanine
Authors: Kearney Caroline , Olenginski Lukasz T. , Tariq Daniyal , Hirn Trexler D. , Brewer Scott H. , Phillips‐Piro Christine M.
Published: November 2023

Heme Nitric Oxide and/or Oxygen (H‐NOX) binding proteins are a diverse group of hemoproteins that bind diatomic gases like nitric oxide (NO), carbon monoxide (CO), and/or oxygen (O2). H‐NOX proteins, which can be found in both prokaryotic and eukaryotic cells, contain two distinct domains: a heme‐containing sensor domain that binds gaseous ligands and an effector domain that generates a cascade of signals for cells to function. Here local protein environments in Thermoanaerobacter tengcongensis H‐NOX (Tt H‐NOX) were investigated using the vibrational reporter unnatural amino acid (UAA) 4‐cyano‐L‐phenylalanine (pCNF). This UAA was genetically site‐specifically incorporated into Tt H‐NOX at multiple positions individually including near the heme pocket using amber codon suppression technology. Infrared analysis of the constructs probing the position of the nitrile symmetric stretch of pCNF revealed a myriad of local environments in the protein. These results highlighting solvated or buried positions in the protein will be presented.

Support or Funding Information

NIH R15GM1219842CMPP and the Fred A. Snavely Research Award
Labilization of the Cobalt‐carbon Bond in Vitamin B12 Bound to Adenosyltransferase
Authors: Campanello Gregory Cook , Twahir Umar , Brunold Thomas , Warncke Kurt , Banerjee Ruma
Published: November 2023

Vitamin B12 is an essential, scarce, and reactive cofactor that is acquired by mammals from the diet. Studies on patients with inborn errors of cobalamin metabolism have led to the discovery of nine genes involved in the tailoring, chaperoning, and utilization of cobalamin as a cofactor. One of these genes encodes adenosyltransferase (ATR), a bifunctional enzyme that is responsible for the synthesis of 5′‐deoxyadenosylcobalamin (AdoCbl) and for its delivery to the client enzyme, methylmalonyl CoA mutase (MCM). Mutations in ATR and MCM result in methylmalonic aciduria, a devastating metabolic disorder, which can result in infant mortality. ATR catalyzes the synthesis of AdoCbl via transfer of the adenosyl group of ATP to the supernucleophile cob(I)alamin, resulting in the release of inorganic tripolyphosphate (PPPi). PPPi binds to the ATR•AdoCbl complex with an apparent Kd≤ 1 μM. Binding of PPPi to ATR•AdoCbl under anaerobic conditions induces a unique UV‐visible spectroscopic signature with an absorption maximum at 439 nm. Exposure of this complex to O2 results in cleavage of the cobalt‐carbon bond and formation of an ATR‐bound 4‐coordinate cob(II)alamin species with a λmax of 464 nm. The resulting spectrum is virtually identical to that of neopentylcobalamin, a synthetic B12 derivative with a weak cobalt‐carbon bond. Under aerobic conditions, neopentylcobalamin also undergoes spontaneous decomposition to cob(II)alamin (1). We are using multiple spectroscopic approaches to characterize this unusual reaction and to place it in a functional context. We hypothesize that binding of PPPi to ATR‐bound AdoCbl results in weakening of the cobalt‐carbon leading to the formation of tightly bound cob(II)alamin, thus prioritizing sequestration of the cofactor under conditions where the acceptor MCM, might be limiting.

Support or Funding Information

We are very grateful for NIH grants awarded to G.C.C. (5 F32 GM113405‐02) and R.B. (DK45776) which supported this research.
Structural Changes in the Nickel‐Responsive Regulator NikR from Helicobacter pylori
Authors: Baksh Karina Amelia , Wong Jeremy , Pham Tamara , Zamble Deborah Beth
Published: November 2023

Helicobacter pylori is a pathogenic bacterium estimated to infect 50% of the global population and causes peptic ulcers, gastritis, and gastric cancer. H. pylori is able to survive the fluctuating pH levels of the stomach mainly due to expression of two enzymes – urease and [NiFe]‐hydrogenase – that require nickel as an essential cofactor. Therefore, regulation of nickel concentration is crucial in order to avoid toxicity while maintaining an adequate supply. This is achieved by the nickel‐responsive transcription factor NikR (HpNikR), which maintains intracellular nickel homeostasis through binding the promoters of various genes, including those encoding nickel storage and import factors, and activating or repressing their transcription. HpNikR also plays a role in acid acclimation and there is evidence that its DNA‐binding activity can be modulated by changes in pH. It is therefore likely that there are promoter specific conformations exhibited by HpNikR that are sensitive to both nickel concentration and pH. However, the mechanisms by which nickel and pH activate DNA binding remain unclear. In this work, a series of mutants at key secondary structural elements were created as targets for labeling with a 19F probe and 19F‐NMR was used to uncover how nickel and pH influence the HpNikR conformational changes required for binding to different promoters.

Support or Funding Information

This work was supported in part by funding from the Canadian Institutes of Health Research.
Biochemical and biophysical comparison of the human and Drosophila melanogaster I kappa B kinase protein complexes suggest structural conservation
Authors: Rogers W Eric , Huxford Tom
Published: November 2023

The IMD signaling pathway in Drosophila melanogaster is responsible for the expression of antimicrobial peptide genes in response to detection and binding of products of Gram‐negative bacterial infection at the cell membrane. Several macromolecular components of the IMD signaling pathway have close structural and/or functional homologs within the canonical NF‐κB signal transduction pathway that regulates inflammation and cell survival in mammals. Chief among these is the IκB kinase complex (IKK), which functions in both humans and flies to activate transcription factor NF‐κB. In this study, we compare the biophysical and biochemical properties of the Drosophila melanogaster IKK complex with those of its human homolog. We find striking similarities in both solution properties and intra‐complex interactions. Size exclusion chromatography measurements indicate similar hydrodynamic radii for the enzymes. Multi‐angle light scattering reveals a similar shape independent molecular weight. Hydrogen/deuterium exchange‐mass spectrometry studies identify that the region that is most protected upon binding is at the C‐terminus of the human and Drosophila IKKbeta subunit. Interestingly, residues that map to theoretical ubiquitin‐like domain are also protected upon interaction with IKKgamma. These experiments suggest overall structural conservation between the human and Drosophila IKKbeta:IKKgamma complexes despite considerable amino acid sequence variation in the individual polypeptide subunits.
New ABCG2 homology model reveals importance of C‐terminus in protein trafficking
Authors: Cui Jinming , Sayed Hiba , Huang Jonathan , Woodward Owen M.
Published: November 2023

Recent genetic studies have implicated ABCG transporters in human disease and identified potential causal variants. However interpretation of the resulting molecular defect is often limited by the lack of high‐resolution crystal structures. This has been the case for ABCG2. Here we present a new homology model of ABCG2 using a recent ABCG5/G8 crystal structure (PDB:5do7) as a template in an effort to better understanding the implications of human ABCG2 gout causing mutations. Our homology model is composed of the ABCG2 NBD domains (residues 29–288) and TMD domains (residues 289–655) and was built using the Swiss‐Model server. To avoid forcing the ABCG2 into a heterodimer, the ABCG5 served as template for both ABCG2 copies because its NBD structure is closer to the canonical NBD structure. The loop regions of the model were further refined by GalaxyLoop. The final model complied with the modular multi‐domain architecture of ABC proteins, and had RMSD values with ABCG5 NBD, ABCG5 TMD, ABCG8 NBD, ABCG8 TMD of 0.2Å, 0.5Å, 1.7Å, and 2.4Å, respectively. The new ABCG2 homology model allows for the visualization of parts of the protein previously undefined. One intriguing finding was that the loop between the first and second β‐strands of the NBD (residues 42–63) is in close proximity with the TMD C‐terminus S655. Notably, the corresponding loop (residues 47–65) and the C‐terminus R651 were not resolved in the ABCG5/G8 crystal structure, suggesting a dynamic interaction in this region. We explored the significance of the C‐terminal tail (resides 652–655; KKYS) on protein abundance and trafficking. The addition of a C‐terminal Flag‐tag or the deletion of the last 4 residues (651X) led to a profound decrease in protein abundance. The 651X protein is not glycosylated and localizes to the ER only. Interestingly, a YS653AA (KKAA) substitution is less deleterious and results in protein that reaches the Golgi compartment, highlighting the dilysine motif, a potential COPI target, as key in trafficking ABCG2. Finally, we found the S655A substitution (KKYA) results is significantly greater protein abundance than wild type and partially rescues the abundance of the Q141K human gout mutation, implicating the Y654 as a key trafficking residue. In conclusion, our new ABCG2 homology model suggests the C‐terminus is in close proximity to the NBD and may be a critical feature in regulating the trafficking of the ABCG2 protein.

Support or Funding Information

American Heart Association 14SDG18060004
Insights Into The Mechanism Of Protein Functional Loss Upon Covalent Modification By Homocysteine Thiolactone
Authors: Sharma Gurumayum Suraj , Singh Laishram Rajendrakumar
Published: November 2023

Increased level of homocysteine (Hcy), associated with homocystinuria (or hyperhomocysteinemia), and is known to result in various clinical manifestations including cardiovascular complications and neurodegeneration. One likely mechanism underlying harmful effects of Hcy is the covalent post‐translational modification of protein by homocysteine thiolactone (HTL), a highly reactive cyclic thioester of Hcy formed by methionyl‐tRNA synthetase in an error editing reaction. Modification of proteins by HTL (a process termed N‐homocysteinylation) is known to result in protein functional loss. In the present study, the effects of HTL on structural and functional activity of three proteins RNase‐A, Lysozyme and Carbonic anhydrase were studied. We observed that RNase‐A and Lysozyme did not exhibit any functional loss upon overnight incubation with HTL. However, these enzymes required several days incubation to come to complete loss of function. On the other hand, CA was rendered non‐functional upon overnight incubation. The results indicate that certain protein requires exceptional longer incubation to bring about functional loss upon modification by HTL. Analyses of the mechanisms for the loss of enzyme function revealed that CA loses its enzyme function due to certain conformational alterations leading to aggregates formation. On the other hand, loss of activity in case of RNase‐A and Lysozyme was due to generation of exceptionally higher oligomeric spherical ensembles with varying sizes. The study revealed two independent mechanisms occurring for protein functional loss upon post‐translational modification by HTL.
Structure of hRpn13 at the Proteasome
Authors: Lu Xiuxiu , Liu Fen , Nowicka Urszula , Sridharan Vinidhra , Dyba Marzena , Tarasov Sergey G. , Walters Kylie J.
Published: November 2023

The proteasome has a 20S core particle (CP) that is capped at either end by a 19S regulatory particle (RP). An ATPase ring from the RP directly contacts the CP and extends out to three ubiquitin receptors (Rpn13/Adrm1, Rpn10/S5a and Rpn1/S2/PSMD2) that recognize ubiquitin and ubiquitin folds of substrate shuttle factors. Here, we solve the solution structure of the ubiquitin‐binding hRpn13 Pru (Pleckstrin‐like receptor for ubiquitin) domain with the C‐terminal 14 amino acids of hRpn2 that is used to dock hRpn13 into the proteasome by NMR. We find that the hRpn13:hRpn2 interaction surface is proline rich, with hRpn2 extended across a shallow canyon of hRpn13 Pru domain. Our structure imposes an orientation to hRpn13 in available cryoEM density maps of the yeast 26S proteasome, and provides atomic level details for how hRpn13 assembles into the proteasome. Moreover, hRpn13 has been reported to be a promising therapeutic target. Cell permeable bis‐benzylidine piperidone RA190 restricts cancer growth in mice xenograft models of multiple myeloma and ovarian cancer and conjugates to hRpn13. We present structure‐guided biochemical assays that provide novel insights into the mechanism of action for RA190.

Support or Funding Information

Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research
Structural Insights into Oxalate Biosynthetic Component
Authors: Rhee Sangkee , Oh Juntaek , Kim Sanghoon
Published: November 2023

Bacterial quorum sensing (QS), a cell‐to‐cell communication process in many Proteobacteria, controls the gene expression for bacterial population‐wide characteristics, including bioluminescence, motility, and virulence‐related factors. Recently, QS has been recognized to provide further benefits at the population level by regulating the production of public goods, the function of which could be beneficial to all members of the group. In Burkholderia species, oxalic acid was recently identified as an excreted public good for the QS‐dependent growth. In these species, QS‐mediated oxalogenesis via the oxalate biosynthetic component (obc) is a cellular event indispensable for the survival of bacteria in the stationary phase. In B. glumae, obc consists of two genes encoding ObcA and ObcB for coordinating the production of oxalic acid, as well as acetoacetate and CoA, by using oxaloacetate and acetyl‐CoA as substrates. Unlike B. glumae, there is a bi‐functional enzyme Obc1 from B. thailandensis. Sequence analysis indicates that Obc1 has an ObcA‐like N‐terminal domain and shows ObcB activity in its C‐terminal domain despite no sequence homology with ObcB. We will present our recent progress on structural and functional analyses of ObcA and Obc1. In ObcA, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr322 as a general base, which then attacks the thioester carbonyl carbon of acetyl‐CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal‐coordination shell and absence of general acid(s) likely produces an unusual tetrahedral CoA adduct as a stable product. In Obc1, the C‐terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His‐934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis.

Support or Funding Information

This work was supported by Next Generation BioGreen 21 program of Rural Development Administration (Plant Molecular Breeding Center) of Republic of KOREA.
Heat Shock Protein 90 is Critical for Plasmodium Parasite Liver Stage Development
Authors: Keim Allison I , Posfai Dora , Haystead Timothy A , Derbyshire Emily R
Published: November 2023

Malaria remains a severe global health burden, and there is an urgent need to develop effective strategies to combat the Plasmodium parasites responsible for the disease. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood stage Plasmodium parasites, but its role during malaria's elusive liver stages remain unclear. Studies on protein function throughout the infection are challenging as Hsp90 is highly conserved between the parasite and human host. To address this issue, we used target‐based screens to identify compounds that bind to Plasmodium (Pf) and human Hsp90 with varying species selectivity and used them as chemical probes. All identified Hsp90‐binding compounds were potent blood and liver stage Plasmodium inhibitors in cell‐based malaria assays. Importantly, Hsp90 inhibitors synergistically reduced liver stage parasite viability when used in combination with phosphatidylinositol 3‐kinase inhibitors. Most notably, liver stage parasite inhibition strongly correlated with binding to PfHsp90, but not the human host protein. Analysis of Hsp90 protein and gene expression throughout the liver stage coupled with time course inhibition studies further support a critical role of Plasmodium Hsp90 in early liver stage parasite development. Our results suggest that PfHsp90, but not human Hsp90, is essential to malaria's liver stage and highlight the potential to selectively inhibit PfHsp90 in combination therapies for malaria treatment and prevention.

Support or Funding Information

This work was supported by the NIH for laboratory support (GM099796 to E.R.D.) and the Duke Pharmacological Sciences Training Program for fellowship support (2T32GM007105‐41 to A.I.K.).