bluuebunny/crossref_metadata_2025 huggingface dataset:
In‐silico Design of Safe and Effective Topoisomerase IB Inhibitors from Parent Compounds Isolated from Annona muricata
Authors: Adeyemi Emmanuel Tofunmi , James Ayorinde Babatunde , Odeyemi Oluwatimilehin Ademilolani , Magbagbeola Olubunmi Abiola
Published: June 2021

Rational drug design using in‐silico tools have helped improve the time and possibilities of the development of safer drugs in recent times. This study was designed to develop safer DNA topoisomerase 1B inhibitors from two parent compounds isolated.

The adsorption, distribution, excretion and toxicity (ADMET) properties of selected isoquinoline compounds were predicted using swiss ADME online tool. Analogues of these compounds were also created using ChemDraw professional Suite 16.0 by adding and removing functional groups based on the pharmacophoric structure of topotecan. 3D in‐silico docking was carried out between the designed compounds and the DNA topoisomerase 1B using AutoDock Vina software. Search space for ligand binding was defined and the program returned the binding affinities between the designed compounds and DNA topoisomerase 1B.

Five analogues were designed for each compound. Out of the analogues designed, parent compound 1‐analogue 3 proved likely to be effective and safe in cancer treatment by having a binding affinity of −9.9 kcal/mol for DNA topoisomerase 1B and good ADMET prediction. The other compound (parent‐2) analogues had very high binding affinities for DNA topoisomerase 1B but they generally had ADMET problems. They were either predicted to be P‐glycoprotein substrates or blood‐brain barrier permeants.

Rational drug design for anticancer agents from selected isoquinolines in Annona muricata predicted study compounds to be safe and effective in inhibiting DNA topoisomerase 1B.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selecting Pharmacogenomic Variables that influence Propofol Pharmacokinetic Mean Residence Times in Surgical Patients using Machine Learning
Authors: Eugene Andy Roger , Eugene Beata
Published: June 2021

BACKGROUND

Propofol is the most widely used intravenous anesthetic agent for surgery and outpatient procedures. We sought to test the hypothesis that CYP2B6, CYP2C9, and UGT1A9 pharmacogenes predicts prolonged Propofol Mean Residence Times (MRT), a pharmacokinetic parameter, or normal Propofol MRTs. The importance of this study will help further understanding of the pharmacogenomics of Propofol anesthesia to clinically selectively identify patients who would be at risk of delayed emergence or complications from Propofol.

METHODS

Propofol pharmacokinetic data was obtained from the Mikstacki et al 2017 study that genotyped patients for the following pharmacogenes: CYP2C9 1075 A>C, UGT1A9 98T>C, and CYP2B6 516G>T that included 85 Polish patients (53 males and 32 females) who were rated as a I or II according to the American Society of Anesthesiologists physical status classification system (1). The Random Forest and Adaptive Boost Machine Learning algorithms were used to classify patients as being either having a prolonged MRT or a normal MRT, relative to the population mean. All analyses were conducted using the R statistical programming language (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

After assigning the patients into the two dichotomous groups, the final machine learning model included CYP2B6, CYP2C9, UGT1A9, gender, and age (in decades) from a total of 43 patients, from the original 85. Propofol MRTs ranged from 8‐minutes to 504‐minutes, among the two groups. The Random Forest classifier resulted in best predictive performance with an ROC AUC of 0.98 with a variable importance (descending): CYP2B6, gender, CYP2C9, age (in decades), and UGT1A9.

CONCLUSION

Among the pharmacogenomics and demographic variables tested, the CYP2B6 gene and gender were identified as the most important variables predicting the Propofol pharmacokinetic parameter of Mean Residence Times in surgical patients.

Support or Funding Information

None.
Therapeutic effect of MC‐4 in combination with everolimus against progressive renal cell carcinoma by double‐targeting of PTEN/AKT/PKM2 and mTOR1 signaling pathways
Authors: Jiyeong Kim
Published: June 2021

The purpose of this study was to assess the antitumor effects of MC‐4, a novel compound extracted from Artemisia annua L., as a monotherapy or combined with everolimus against Caki‐1 (Von Hippel‐Lindau (VHL)+/+) and 786‐O (VHL−/−) human RCC cells in vitro and in vivo. RCC cells were treated with MC‐4 alone or combined with everolimus and were analyzed for viability, apoptosis, and autophagy to evaluate synergistic antitumor effects in vitro and in xenograft in vivo models. MC‐4 inhibited Akt and pyruvate kinase M2 (PKM2), and significantly increased growth inhibition and autophagic cell death in RCC cells. However, everolimus led to compensatory Akt activation by inhibiting only the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. In contrast to everolimus, MC‐4 enhanced phosphatase and tensin homolog (PTEN) expression and reduced its downstream effector, PKM2, leading to decreased expression of glucose transporter 1 (GLUT1) associated with cancer cell metabolism. Collectively, the synergistic cytotoxic effects induced by co‐administration of MC‐4 and everolimus involve cell growth inhibition and autophagic cell death via dual targeting of Akt/PKM2 and mTOR. This suggests that MC‐4 is a novel Akt/PKM2 inhibitor that could overcome the limitation of existing mTOR inhibitors used for mRCC therapy. The data herein encourage further clinical studies combining MC‐4 with established chemotherapeutics as a strategy to treat patients with rapidly progressing RCC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure Activity Relationship Study of CAPE and its Derivatives on Liver Carcinoma
Authors: Nguyen Julie , Bhave Vishakha , Hielscher Abigail , Wang Xinyu
Published: June 2021

Globally, the sixth most common cancer and the second largest contributor to cancer mortality is malignancy of the liver. The most frequent form of liver cancer is hepatocellular carcinoma (HCC), a primary liver cancer of the parenchymal cells. Presently, there are no effective treatment options aside from liver resection and transplantation. For this reason, caffeic acid phenethyl ester (CAPE) has been proposed as a potential therapeutic candidate. CAPE, a phenolic compound found in bee propolis, is reported to have a plethora of bioactive properties including antitumor traits. The objective of this study is to elucidate the relationships between the structure of CAPE and its effect on liver cancer cell growth in addition to exploring the potential mechanism for this. Seven CAPE derivatives were examined including one novel derivative previously synthesized in our lab, fluorinated caffeic acid phenethyl ester (FCAPE). We evaluated the effect of CAPE and its derivatives on the viability and proliferation of a human HCC cell line, HepG2. Our preliminary data show that CAPE and some of its derivatives reduce the viability and inhibit the proliferation of HepG2 cells in a dose‐ and time‐dependent manner. Comparison of CAPE derivatives' structures versus their activity indicates that the catechol ring functional group is required for their inhibitory effect on HepG2 cell growth. To examine whether induction of apoptosis is responsible for the cell death, western blot was conducted to check protein expression of apoptosis‐related factors including caspase‐3 and poly‐(ADP‐ribose) polymerase (PARP). Our data show the breakdown of caspase‐3 and PARP by CAPE, suggesting that CAPE may induce apoptosis of HepG2 cells. In conclusion, our data suggest that CAPE inhibits the growth of HepG2 cells through induction of apoptosis, and this effect of CAPE requires the integrity of a catechol ring functional group in order to carry out its cytotoxic effects.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer Activity of A Silyl Andrographolide Analogue Mediated Through Wnt/β‐Catenin Signaling In Colon Cancer Cells
Authors: Reabroi Somrudee , Saeeng Rungnapha , Chairoungdua Arthit , Piyachaturawat Pawinee
Published: June 2021

Hyperactivation of Wnt/β‐catenin signaling implicated in oncogenesis of colorectal cancer (CRC) is a potential molecular target for chemotherapy. An andrographolide analogue, 3A.1 (19‐tert‐butyldiphenylsilyl‐8, 17‐epoxy andrographolide) has previously been reported to be potently cytotoxic toward cancer cells by unknown molecular mechanisms. The present study explored the anti‐cancer activity of analogue 3A.1 on Wnt/β‐catenin signaling in colon cancer cells (HT29 cells) which were more sensitive to the others (HCT116 and SW480 cells). Analogue 3A.1 inhibited viability of HT29 cells with IC50 value of 11.1 ± 1.4 μM at 24 h, which was more potent than that of the parent andrographolide. Analogue 3A.1 also suppressed the proliferation of HT29 cells and induced cell apoptosis in a dose‐dependent manner. Its apoptotic activity was accompanied with increased expressions of proteins related to DNA damages; PARP‐1 and γ‐H2AX. In addition, analogue 3A.1 significantly inhibited T‐cell factor and lymphoid enhancer factor (TCF/LEF) promoter activity of Wnt/β‐catenin signaling. Accordingly, the expressions of Wnt target genes and β‐catenin protein were suppressed. Moreover, analogue 3A.1 increased the activity of GSK‐3β kinase, which is a negative regulator responsible for degradation of intracellular β‐catenin. This mode of action was further supported by the absence of the effects after treatment with a GSK‐3β inhibitor, and over‐expression of a mutant β‐catenin (S33Y). Our findings reveal, for the first time, an insight into the molecular mechanism of the anti‐cancer activity of analogue 3A.1 through the inhibition of Wnt/β‐catenin/GSK‐3β pathway and provide a therapeutic potential of the andrographolide analogue 3A.1 in CRC treatment.

Support or Funding Information

This research project was supported by TRF through the RGJ‐Ph.D. Program (PHD/0193/2553 to PP and SR) and the research grant IRN‐58W0004.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A unique method for antibody to possess the catalytic function (3rd report)
Authors: Uda Taizo , Akiyoshi Yuko , Taguchi Hiroaki , Hifumi Emi
Published: June 2021

Since a natural catalytic antibody found at 1989, many mouse‐ and human‐type of catalytic antibodies cleaving the targeting antigens such as peptides, nucleotides, viruses and bacterial proteins, molecules such as factor VIII, CCR5, TNFα etc. have been produced so far (1, 2). In contrast, thousands of monoclonal antibodies (mAbs) have been produced over past 40 years. However, not all monoclonal antibodies have catalytic activity. It is considered that the proportion possessing a catalytic function is about several percentages or lower. Thus, the authors have continued to explore the technology how to put the catalytic function to those normal (non‐catalytic) mAbs. Recently, we have made a significant progress in part with respect to the technology.

After the genes of human antibody light chain were inserted into pET20b (+), E. coli was transformed and each gene was expressed by inducing with IPTG. After recovering the soluble fraction of the culture supernatant, it was purified by Ni‐NTA‐, cation‐, and/or size exclusion‐chromatography. The enzyme activity was measured using Arg(R)‐pNA as a synthetic substrate and/or the antigens such as amyloid‐beta (Aβ) peptide etc. Since

We found interesting two light chains, #7TR and #7GY. The former could degrade both FRET‐Aβ substrate and Aβ1–40 full peptide. In contrast, #7GY, whose sequence is identical to that of #7TR except for the amino acids of the positions at 29th and 30th, did not degrade the FRET‐Aβ substrate and Aβ1–40 full peptide at all. The chemical features of the two light chains were investigated in detail by using a synthetic substrate, Arg‐pNA. #7TR showed higher hydrolytic activity for Arg‐pNA substrate than that of #7GY by a factor of one and half. In addition, the presence of Zn (II) ion hugely enhanced the catalytic activity of #7TR light chain but not #7GY. Fluorescence spectroscopy clearly exhibited the difference of the two antibody light chains. Through these facts and discussion, we will propose a clue how to we can put the catalytic function to a normal (non‐catalytic) antibody.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the food components and physiological activities of Brassica juncea from Jeongseon
Authors: Sim Wan‐Sup , Choi Sun‐Il , Jung Tae‐Dong , Cho Bong‐Yeon , Choi Seung‐Hyun , Han Xionggao , Kim Young‐Cheul , Lee Ok‐Hwan
Published: June 2021

This study was conducted to provide basic data of Brassica juncea from Jeongseon (BJJ) which is the food components and physiological activities. The moisture, crude protein, crude fat, crude ash and carbohydrate content of BJJ were 91.58±0.08%, 2.29±0.26%, 0.07±0.00%, 1.17±0.00% and 4.89±0.03%, respectively. The sugar content was 0.29±0.02 g/100 g of glucose and 0.10±0.00 g/100 g of fructose. Major fatty acids were palmitic acid(C16:0), octadecenoic acid(C18:1n9c) and stearic acid(C18:0), especially the composition of the palmitic acid was the highest at 31.22%. Major minerals were K, Ca, P, Na, Mg, especially the K content was highest at 464.31±6.33 mg/100 g. Vitamin was mainly composed of Vitamin B1, B2, B6, C, E. The primary organic acid was 305.26±0.90 mg/100 g of acetic acid. Total amino acid was mainly composed of L‐Proline at 774.30±119.06 mg/100 g. DPPH radical scavenging activity and FRAP value increased depending upon the concentrations of BJJ extract (0.1, 1.0, 5.0 mg/mL). Total phenolic contents and total flavonoid contents were 7.88±0.06 mg GAE/g, 37.31±0.39 mg RE/g. In addition, we assessed the lipid accumulation and the antioxidant effect of BJJ extract on 3T3‐L1 and Human Dermal Fibroblasts (HDFs), respectively. After we confirmed no significant difference of cell viability between control group and BJJ extract of 25, 50, 100 μg/mL through XTT assay, we applied this concentration of BJJ extract on the continuous experiments. Oil Red O (ORO) staining showed that the lipid accumulation was inhibited by 25% at a concentration of 100 μg/mL compared to control. Through NBT assay, the reactive oxygen species (ROS) production decreased in all experiment groups treated by BJJ extract, especially the ROS production decreased about 23% at a concentration of 100 μg/mL compared to control. However, at low concentrations (25, 50 μg/mL), the protective effect of BJJ extract was no significant effect but 100 μg/mL. These results suggested BJJ has a potential as a functional food material.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coupling Fluorescence‐Activated Cell Sorting and Targeted LC‐MS/MS for Epi‐Proteomic Analysis of Normal Leukocytes
Authors: Camarillo Jeannie , Swaminathan Suchitra , Abshiru Nebiyu , Sikora Jacek , Morris Juliette , Kelleher Neil , Thomas Paul
Published: June 2021

Fluorescence‐activated cell sorting (FACS) is widely used to isolate subtypes of various immune cells from human blood. Most recently, these subpopulations have been subjected to next generation sequencing to elucidate changes in gene expression associated with differentiation. Histone modifications are essential for regulating chromatin and maintaining gene expression, and few studies have investigated global alterations in histone modifications required for differentiation. Here, we describe an approach for integrating FACS and targeted mass spectrometry to define a global “epiproteomic” signature for white blood cells from normal individuals. Using our multiple‐reaction monitoring LC‐MS/MS method, which can measure 90+ histone modifications on all core histones, we assessed changes in histone modifications from different collection methods and cell types. First, collection buffer conditions were optimized to allow for sorting directly into histone extraction buffer, thereby reducing the likelihood of changes in histone modifications that could occur during long sorts. Next, we sorted neutrophils, monocytes, B cells, and T cells and identified differences in histones modifications across these broad cell types. Finally, FACS was used to sort naïve, TH1, and TH2 T cells from human peripheral blood for histone analysis, revealing little differences in histone marks in these closely‐related subtypes. This method allows for interrogation of global changes in histone modifications in distinct cell types, defining an “epi‐proteomic” signature that can be used to more fully understand differentiation in hematopoiesis and disease development.

Support or Funding Information

This work was carried out with financial support from the National Resource for Translational and Developmental Proteomics (P41GM108569).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Revisiting the Pharyngeal Apparatus: Arches, Pouches, & Grooves—Oh My!
Authors: Dennis Jennifer
Published: June 2021

The vertebrate face is the product of growth and differentiation of structures arising from the pharyngeal apparatus. The arches, pouches, grooves, and membranes that comprise the pharyngeal apparatus undergo morphogenetic movements allowing for a typically seamless integration of tissues and cell types that orchestrate craniofacial development. The differentiation of these structures is critical for facial and skull bones, muscle, connective tissue, skin, and the central and peripheral nervous systems of the head, face, and neck to develop correctly. Hence, it is not surprising that without the proper integration of these tissues during development that craniofacial defects arise, accounting for one‐third of all congenital malformations within human populations. Of central importance, is a thorough understanding of the specific tissue interactions, movements, and fusions that occur between the craniofacial primordia in regulating craniofacial morphogenesis. In this master class, we will discuss the embryology of the head and neck, taking a holistic approach to the components of the pharyngeal apparatus, to review the various elements that come together to form the vertebrate face.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatocyte specific ablation of β‐ and γ‐catenin contributes to loss of hepatocyte polarity and EMT induction by activation of the TGFβ pathway
Authors: Pradhan‐Sundd Tirthadipa , Singh Sucha , Molina Laura , Podder Minakshi , Bell Aaron , Monga Satdarshan Pal
Published: June 2021

A hallmark of epithelial cells is polarization. Like any other epithelial cell, hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell. Polarization of hepatocytes is necessary for many functions and requires carefully orchestrated cooperation between cell adhesion molecules, extracellular matrix, cell junctions, cytoskeleton, and intracellular trafficking machinery. Loss of hepatocyte polarity is associated with various liver diseases including cholestasis. Previously, we discovered that simultaneous deletion of adherens junction protein β and γ catenin in the mice liver led to cholestasis in the double knockouts (DKO). However, the molecular mechanisms that led to cholestasis in DKO were only partially elucidated. Here, we characterize the phenotype further. Electron microscopy revealed dilation of bile canaliculi with significant reduction in microvilli in the DKO livers. IHC, protein and RT‐PCR analysis revealed loss of several hepatocyte polarity markers including CD10, ZO‐2, BSEP, and MRP2, increased fibrosis and epithelial‐mesenchymal transition in the liver. Remarkably, RNA‐seq analysis revealed over activation of TGFβ pathway in double knock out liver and blocking TGFβ pathway led to partial rescue of tight‐junctional proteins and cholestasis. Taken together, our findings suggest that β; γ‐catenin are critical to the maintenance of hepatocyte polarity and cholestasis development by regulating TGFβ signaling in hepatocytes. Profound understanding of the regulation of hepatocyte polarity by both β ‐and γ‐catenin will be useful not only in understanding cholestasis but also in the context of liver development and pathophysiology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quality Control During 40S Ribosome Assembly
Authors: Karbstein Katrin
Published: June 2021

While much is known about how ribosomes faithfully translate mRNAs into protein, how ribosomes are assembled within cells remains poorly understood. With ~ 2,000 ribosomes synthesized every minute ribosome assembly is the major metabolic activity of a growing cell. Given its profound energy requirements, ribosome assembly is tightly regulated in response to extracellular stimuli. In addition to providing sufficient numbers of ribosomes, cells must also ensure that the produced ribosomes are correctly assembled. This is important to safeguard their catalytic activity and extraordinary fidelity. We are exploring cellular mechanisms to ensure proper assembly of nascent 40S subunits, as well as the consequences of defects in assembly.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteoform Dynamics in Steady‐State
Authors: Zecha Jana , Meng Chen , Kuster Bernhard
Published: June 2021

Utilizing a classical dynamic SILAC (stable isotope labeling of amino acids in cell culture) approach, mass spectrometry‐based technologies nowadays allow for the determination of native protein turnover on a proteome‐wide scale. Obtaining proteoform resolution, however, is often still challenging due to incomplete quantitative information of peptides across pulse time‐points. To address this shortcoming, we evaluated the merits of (a) combining dynamic SILAC with TMT (tandem mass tag)‐labeling of ten pulse time‐points to compute absolute peptide turnover rates, and (b) utilizing solely single time‐point pulses to obtain relative differences in peptide half‐lives.

In total, four cell culture replicates of each of the two different methodologies provided turnover information for 80,000 endogenously modified peptides assigned to up to 7,700 protein groups. Protein degradation rates were comparable to those reported with the standard pulsed SILAC approach. Moreover, replicate analyses established that the same reproducibility of turnover determination can be obtained for peptides and proteins eventually facilitating the investigation of proteoform resolved regulation of protein stability. Our comprehensive dataset showed that splice variants can exhibit vastly different cellular protein stabilities. Moreover, it revealed changes in turnover upon post‐translational modifications such as protein phosphorylation, ubiquitination and acetylation on a global scale. Several examples of differentially turned over peptides and proteins were investigated further. For example, differential peptide turnover indicated a previously unknown mechanism of activity regulation via post‐translational destabilization of only the light chain of cathepsin D as well as a phosphorylated version of DNA helicase BLM. Further, a parallel reaction monitoring assay applied to rotenone treated cells suggested that the high protein turnover of members of the respiratory chain complex I is affected by oxidative stress.

Our data indicates that the original pulsed SILAC concept can be either expanded by employing TMT labeling of pulse time‐points to estimate absolute peptide turnover rates or simplified by using single time‐point pulses to evaluate relative differences in peptide half‐lives. Both strategies facilitated proteoform resolved turnover measurements. Collectively, they revealed a widely disregarded level of post‐transcriptionally and post‐translationally regulated protein stability which can have important implications for the study of disease mechanisms as well as the assessment of pharmacological interventions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence that Tip60 Induces the DDR & Cardiomyocyte Replicative Senescence in the Neonatal Heart
Authors: Lough John , Lupton Carri , Wang Xinrui , Wan Tina , Auchampach John
Published: June 2021

Tip60 (Tat interactive protein, 60 kD) is a tumor suppressor protein that acetylates histone and non‐histone proteins. Among the latter, ATM (Ataxia Telangiectasia Mutated), a kinase that induces the DNA damage response (DDR), is activated in cancer cells by Tip60‐mediated acetylation. We are investigating the function of Tip60 in the heart, using global and conditional knockout mouse models to disrupt the Kat5 gene encoding Tip60. Because global knockout (i.e. Tip60−/−) causes early embryonic lethality, we used Tip60+/− heterozygotes to show that, when under cardiac stress, modest Tip60 depletion correlates with re‐activation of the cardiomyocyte (CM) cell‐cycle in the adult heart. More recently, using a knockout model wherein Tip60 is constitutively and specifically depleted in CMs, we observed CM dropout and lethality in three‐month‐old mice, wherein hearts exhibited features of mitotic catastrophe. Because the latter phenotype was preceded by increased CM density, it was suggested that Tip60‐depletion may extend the duration of CM proliferation during neonatal stages of heart development. Moreover, considered in the context of recent reports that ATM is activated at birth to induce the DDR, which in turn causes replicative senescence of CMs by mid‐neonatal stages, these findings suggest that Tip60 induces these events. To examine these possibilities we are conditionally depleting Tip60 in CMs of Tip60F/F;Cre neonatal mice by injecting tamoxifen at postnatal day 0 (P0). To date, immunostaining of sections from neonatal hearts with antibodies that recognize 5′‐BrdU, phospho‐histone‐H3, and phospho‐ATM (pATM) has revealed that Tip60 depletion significantly increases CM proliferation as early as P7, by which time CM proliferation is normally diminished, and that this is accompanied by significantly decreased pATM levels, indicative of a diminished DDR. These findings provide further support for the notion that DDR activation at the time of birth induces replicative senescence of neonatal CMs, implicating Tip60 as the inducer of this process.

Support or Funding Information

NIH HL131788 (JL & JA), and a grant from the MCW Cardiovascular Center

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the cell signaling pathways for epithelial restitution of a novel probiotic
Authors: Owens Joshua , Saeedi Bejan , Naudin Crystal , Darby Trevor , Jones Rheinallt
Published: June 2021

Probiotics are a group of beneficial bacteria that when consumed in adequate amounts confer a health benefit. Recently, our group showed that a novel probiotic, Lactococcus lactis sp cremoris (LLC) can elicit strong intestinal cytoprotection in murine experimental models of intestinal damage, and potently enhance restitution of a damaged gut epithelium. The objective of this study was to elucidate the mechanism whereby LLC promotes epithelial restitution. Our goal was to first identify which host pattern recognition receptor that could recognize LLC. We gavaged LLC or vehicle control to mice and assessed transcript enrichment in the colon after 4 hours by RNA‐seq. We detected the specific upregulation of TLR2. We next investigated whether a soluble factor from LLC was able to be detected by TLR2. To test this, we cultured epithelial monolayers with supernatant from an LLC overnight culture and detected elevated TLR2 expression. Furthermore, we detected potent activation of the NF‐kB pathway, and the Nrf2 signaling pathway in epithelial cells following contact by LLC supernatant. Importantly, no negative physiological influences, or cytotoxicity were detected in these cells. Functionally, we found that LLC supernatant accelerated the closure of scratch wound inflicted on an epithelial monolayer in a TLR2‐dpendant manner. These and other data were evidence for us to conclude that LLC mediates epithelial restitution through a TLR2‐dependent pathway. We also propose that cross‐talk between the NF‐kB and Nrf2 cell signaling pathways function in the beneficial effect of LLC on intestinal health and restitution following disease or injury.

Support or Funding Information

This work was supported by the NIDDK‐ R01DK098391

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a Physical Model of HigB Toxin and its Endonuclease Cleavage Mechanism
Authors: Chabbra Alesa , Alzamora Carolina , Ghali Helana , Handal Marina , Kathiria Mahima , Martinek Alexander , Nguyen Jenny , Patel Chandni , Patel Shreja , Pham Antoine , Lavin Emily Schmitt
Published: June 2021

The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University created a physical model depicting the mechanism of endonuclease cleavage by the HigB protein as described in the literature. The proteins, toxin HigB and antitoxin HigA, are part of a type II toxin‐antitoxin system that cleaves ribosome‐bound mRNAs in bacteria. Chemical or physical stresses, such as extreme temperatures and lack of nutrients, lead to antitoxin degradation that releases HigB. Temporary inhibition of growth by HigB allows cells to more efficiently utilize current resources. Although HigB is referred to as a toxin, it is not inherently detrimental and can be beneficial to cellular survival. Bacteria that use this mechanism include Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris. As such, HigB is of clinical interest due to its contribution in prolonging bacterial infections through antibiotic tolerance (persistence). When cells are not in a stressed state, HigA binds to and inactivates HigB, allowing growth and translation to proceed. Details of HigB in complex with the ribosome as well as residues important for mRNA cleavage were found in the Protein Data Bank file, 4ZSN, and imported into Jmol, a protein visualization software. The 4ZSN file consisted of several nucleic acid and protein bundles; Bundle 1 was examined in more detail. Specific program commands were developed to manipulate the original file into a format that was later 3‐D printed to create an instructional molecular model, which was funded in part by NSF‐DUE 1725940 for the CREST Project. This model highlights the ribosomal proteins, ribosomal RNA, and HigB along with its residues that are essential for mRNA cleavage. The N and C termini of the HigB protein were also indicated. This molecular model can be used to explain how ribosome‐dependent proteins (HigB) target ribosome‐bound mRNAs in bacterial systems. Providing a 3‐D tool allows students to engage actively in understanding the complex process of endonuclease cleavage.

Support or Funding Information

The development of this 3‐D molecular model was funded in part by NSF‐DUE 1725940 for the CREST (Connecting Researchers, Educators, and STudents) Project.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Type‐ and Protein‐Specific Induction of LAMP3
Authors: Zhao Ming , Venosa Alessandro , Tomer Yaniv , Beers Michael F. , Mulugeta Surafel
Published: June 2021

RATIONALE

The family of lysosome‐associated membrane proteins (LAMP) includes LAMPs 1, 2, and 3. While LAMPs 1 and 2 are ubiquitously expressed and account for half of the proteins in the lysosomal membrane, LAMP3 (DC‐LAMP) is only expressed in distinct cell types. LAMP3 expression is associated with poor prognosis of certain cancers, Parkinson's disease, and is believed to contribute to the unfolded protein response by promoting protein degradation and cell survival during proteasome dysfunction. LAMP3 is constitutively expressed in and has been used as a subcellular marker for alveolar type 2 (AT2) cells primarily localizing in the lamellar bodies (the principal storage site of pulmonary surfactant). In this study, the expression of LAMP3 in various cell types and the mechanisms regulating its induction were evaluated.

METHODS

In vitro cell line models (including lung epithelial A549 and H441 cells, kidney epithelial HEK292 cells, and cervical cancer cell line HeLa cells) transiently expressing fluorescent‐tagged proteins were used for co‐expression studies with immunostained LAMP3 by confocal microscopy. Additionally, LAMP3 mRNA levels in cells prior to and following transient expression were assessed by real‐time PCR.

RESULTS

We discovered that none of the cell lines studied constitutively expresses LAMP3 (mRNA or protein). However, upon stimulation of cells by transient expression of certain proteins, A549 and HeLa cells strongly and weakly induced LAMP3 expression, respectively, while no LAMP3 induction was observed in either H441 or HEK293 cells. In A549 cells, transient expression of the AT2 cell specific proteins (ABCA3 and SP‐C) and the huntingtin poly‐Q repeat peptides (Mtt‐25 and Mtt‐72) induced LAMP3 expression. Media‐transfer assays also showed that LAMP3 induction takes place via paracrine signaling. In contrast, LAMP3 protein expression was absent in cells expressing empty fluorescence vector constructs including GFPC1, DsRedC1, and pcDNA3, as well as fluorescent‐tagged proteins including mCherry/LC3, RFP/Rab7, and GFP/NEDD4‐2. LAMP3 mRNA levels in these cells were comparable to those that induced protein expression, but the proteins appeared to be rapidly degraded by the cell's quality control system as demonstrated by the appearance of LAMP3 protein expression following autophagy and proteasome inhibition assays.

CONCLUSION

These results confirm, in vitro, the specific nature of LAMP3 expression in certain cells types and demonstrate its dependency on stimulation by distinct proteins, paracrine signaling, and disruption of cell quality control.

Support or Funding Information

NIH HL 129150 (SM), NIH HL 119436 (MFB), VA Merit Award BX001176 (MFB)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism and stability of type II bacterial toxin‐antitoxin complexes and the role of regulated antitoxin proteolysis that releases toxin
Authors: Pavelich Ian , Dunham Christine M.
Published: June 2021

Antibiotic resistance is the most prominent threat facing modern medicine and is unlikely to be resolved by novel antibiotics or reduction of improper antibiotic use. New strategies to combat antibiotic resistance by targeting novel bacterial pathways are necessary. One emerging approach focuses on understanding how bacteria adapt to diverse stresses by the inhibition of growth and transition to a dormant state. These bacteria, termed ‘persisters’, tolerate antibiotics without genetic mutations. This phenotype is triggered by the stringent response whereby transcriptional reprogramming and translational inhibition occurs. A class of ubiquitous bacterial gene pairs called toxin‐antitoxin complexes (TAs) undergo changes during the stringent response that activate the toxin component. Normally antitoxins bind and sequester their cognate toxins to function as autorepressors. In stress conditions, antitoxins are degraded by cellular proteases which frees toxins to inhibit growth until stress passes. We and others demonstrated that proteases recognize C termini of antitoxins. Many studies have focused on identifying toxin targets, yet the stability of TAs and how this influences proteolysis is unknown. Given the structural diversity of antitoxins, it is also unclear how proteases recognize antitoxins or if a common mechanism exists to describe all antitoxin proteolysis. This is an important question as proteolysis is the main step in the release of toxin and the initiation of bacterial persistence. Here, we examine the regulation of E. colitype II TAs DinJ‐YafQ, RelB‐RelE, and YefM‐YoeB. The structures of these pairs are known and demonstrate differences in how DinJ and RelB antitoxins interact with their toxins as compared to YefM. Each pair also forms higher ordered complexes that are differentially regulated. We designed C‐terminal truncations and measured their impact on toxin release and antitoxin stability. We find that DinJ and RelB share common structural motifs at their C termini that are important for toxin sequestration. YefM lacks this motif and its C‐terminal plays no role in toxin sequestration. Further, we explore the role of proteases, these motifs, and effects of diverse stress in targeted antitoxin proteolysis. This work identifies features of antitoxins that may recruit proteases and how proteolysis is a major underlying component of stress allowing bacteria to transition to antibiotic persistent states.

Support or Funding Information

The Dunham laboratory thanks the following sources for funding: NSF CAREER MCB 0953714, NIH NIGMS R01GM072528. Dr. Dunham is a Burroughs Welcome Investigator in the Pathogenesis of Infectious Diseases. Ian Pavelich is an ARCS awardee.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2
Authors: Desbois Muriel , Crawley Oliver , Evans Paul R. , Baker Scott T. , Masuho Ikuo , Yasuda Ryohei , Grill Brock
Published: June 2021

PHR (PAM/Highwire/RPM‐1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F box (SCF) complex that contains the F‐box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Support or Funding Information

National Institutes of Health Grant R01 NS072129

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Interaction Between Centrosomal Protein Centriolin and the E3 Ubiquitin Ligase, HECTD1
Authors: Salas Jesus , Garcia Alexander , Zora Vancy , Gromley Adam , Gromley Zeynep
Published: June 2021

Uncontrolled cellular division is a central hallmark of cancer. Thus, studying the molecular functions and interactions of the proteins that regulate the complex processes of cell division is crucial. Two proteins are at the focal point of this study. One is centriolin, a 270 kD protein that has been demonstrated as essential for cytokinesis. The second is a 289 kD E3 ubiquitin ligase called HECTD1. Prior experiments utilizing a yeast two hybrid screen concluded that there is an interaction between centriolin and HECTD1. Furthermore, results of immunoprecipation and western blot from synchronized cells showed that centriolin was diminished in G0, whereas HECTD1 levels were elevated compared to other phases of the cell cycle. In contrast, centriolin concentrations were much more robust in S phase, G1, and M phases, compared to that of G0. In separate experiments, we have used CRISPR to eliminate either centriolin or HECTD1 in HeLa cells to observe the repercussions of the loss of these proteins and their global effects on the cell cycle. Interestingly, we observed significant cell death when we eliminated the HECTD1 gene. Our further experiments revealed that the cell death is due to robust induction of apoptosis. Currently we are investigating whether or not the established interaction between centriolin and HECTD1 plays a role in the cell death that we observed.

Support or Funding Information

This research was supported by Lincoln Memorial University‐DeBusk College of Osteopathic Medicine Intramural Grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating proteolytic activity of engineered trypsin variants using naturally occurring macromolecular substrates
Authors: Villa Rodolfo
Published: June 2021

Serine proteases constitute numerous physiological roles that include but are not limited to the breakdown of food, the participation in immune responses, and with the triggering of cell apoptosis Engineered serine proteases as therapeutic agents constitute a new class of drugs for potential clinical applications. However, one main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors in the human body that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. For a protease to be effective it must display resistance towards inhibitors. Previously engineered trypsin variants K60A, K60V, Y39A, Y39F have shown to diminish prime side interactions, affecting micromolecular inhibition through the disruption of hydrogen bond networks occurring at residue positions 39 and 60. These variants displayed resistance towards certain naturally occurring inhibitors, bovine pancreatic trypsin inhibitor and M84R ecotin. Kinetic data was performed using commercially available tripeptide Z‐GPR‐pNA, also indicating no change of specificity. In order to ascertain the viability of these engineered variants as potential therapeutics we must study the interactions between naturally available macro molecular substrates. Experimental methods required the design and utilization of a novel spectroscopicassay tomonitor the proteolytic activity of trypsin variants using chymotrypsinogen (Cg) as our naturally occurring macromolecular substrate. Within the body, trypsin cleaves triggering the activation of zymogen Cg into its active form. Fluorescence will be measured using chymotrypsin specific 4‐methylumbelliferyl p‐tri‐methylammoniocinnamate chloride (MUTMAC). Preliminary data has shown that a 10:1 concentration ratio (100nM Trypsin to 10μM Cg) proved most efficient for activation. However, only ~22% of chymotrypsinogen has been activated using wild‐type trypsin, suggesting that further optimization of this assay is still required. Future work will aim to elucidate the proteolytic rates of these trypsin variant K60A, K60V, Y39A, Y39F where we hope to obtain a better understanding of the interactions between these variants and naturally occurring substrates.

Support or Funding Information

NIH MS/PhD Bridge: R25‐GM048972

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Allosteric Regulation of BAM2 in Arabidopsis thaliana
Authors: Chandrasekharan Nithesh , Roy Ian R , Monroe Jonathan D , Berndsen Christopher
Published: June 2021

Starch is an important component of the human diet as it is broken down into glucose to provide energy for metabolic reactions in the body. The amount and sources of starch that humans consume greatly varies around the world depending on the plants that are indigenous to each diet. In plants, starch is hydrolyzed into the disaccharide maltose by β‐amylase enzymes, a process that is important night time metabolism and the response to stress in plants. By understanding starch regulation by β‐amylases, we could potentially improve the quality of food crops which will benefit those who suffer from food insecurity and nutritional deficiencies. Arabidopsis thaliana contains nine β‐amylases (BAMs) that show distinct catalytic activities and functional regulation. Starch is located in the chloroplast and there are 4 chloroplastic BAMs. We are interested in understanding the functional and regulatory differences in these enzymes. The focus of our study is BAM2, which was recently found to be a tetramer and contains a secondary starch binding site which regulates BAM2 activity. Since there are no structures of BAMs from Arabidopsis, we aimed to characterize the structural mechanism of BAM2 regulation by starch binding to the secondary binding site. We first modeled BAM2 as a tetramer using previous experimental information and then performed simulations of this model in several substrate and starch bound states to understand the role of the secondary starch binding site on the active structure. We found that starch binding influences protein dynamics near the active site, which we suggest may be the basis for allosteric regulation. We further identified an amino acid which appears to regulate the T to R transition of the enzyme. We supported these computational studies with small angle X‐ray scattering studies of BAM2 structure and with biochemical assays of amylase activity. These latter studies were aided by the development of a novel fluorescent biosensor assay of maltose production that allows for comparison of diverse substrates from small polysaccharides to soluble starch within a single assay method. These comparisons indicate that BAM2 strongly prefers starch‐like substrates over shorter polysaccharides, likely due to allosteric regulation by the secondary starch binding site. Current studies are focused on understanding the effects of this molecular mechanism on starch structure with the goal of elucidating the functional role of BAM2 in plants.

Support or Funding Information

NSF‐RUI MCB‐1616467

NSF REU CHE‐1757874

4‐VA Organization

Jeffrey E. Tickle Scholarship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.