bluuebunny/crossref_metadata_2025 huggingface dataset:
Set4 Promotes Survival during Oxidative Stress through Regulation of Stress Response Genes
Authors: Tran Khoa , Jethmalani Yogita , Jaiswal Deepika , Green Erin M.
Published: June 2021

The yeast protein Set4 contains both a PHD finger and a SET domain, a common signature of chromatin regulatory proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET and the human protein MLL5. Although MLL5 has been implicated in diverse pathways such as hematopoiesis and spermatogenesis, as well as disease states including neurodevelopmental disorders and cancer, its cellular function is still largely unclear. The biological role for Set4 and its function in chromatin regulation has not been investigated. Set4 had previously been reported to be lowly expressed under normal growth conditions, and therefore not expected to have a substantial role in chromatin function or gene expression regulation. However, our data indicated that it plays a highly‐specialized role during stress responses, and that it is a key context‐dependent chromatin regulator. Specifically, we have determined that Set4 activity is critically‐balanced in the cell to promote survival specifically during oxidative stress. Gene expression analysis indicates that the role for Set4 during oxidative stress is to activate genes that promote survival in the presence of oxidative insults. Our data further indicate that Set4 interacts with chromatin in a highly‐regulated manner, and it directly associates with stress‐response genes upon oxidative damage. Our results suggest a model in which Set4 is a stress‐responsive chromatin protein that promotes the activation of gene expression programs required for cellular protection during oxidative stress. To our knowledge, this work is the first to define a biological role for Set4, a conserved SET‐domain protein in yeast that was predicted, although not yet characterized, to be a chromatin regulator. These results further our understanding of mechanisms that protect cells during oxidative stress, and will open new avenues for the investigation of the Set3‐Set4 subfamily of SET‐domain containing proteins and their roles in gene expression control in response to fluctuating environmental conditions.

Support or Funding Information

NIH R01GM124342

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histone chaperone Nap1 facilitates histone dynamics in the nucleosome
Authors: Lee Tae‐Hee , Lee Jaehyoun
Published: June 2021

We developed a single‐molecule FRET (smFRET) experimental system with which we can separately monitor histone H2A‐H2B dimer dynamics and DNA dynamics within a nucleosome particle. Utilizing the system, we monitored the nucleosome dynamics in the presence of histone chaperone Nap1 in order to investigate how the chaperone alters the dynamics of the nucleosome with and without histone acetylations at H3K56 and H4K16. We also compared the nucleosome dynamics in the presence of Nap1 to that in its absence at an elevated salt concentration. Our results indicate that DNA unwraps out of the nucleosome before histone dimer moves at an elevated salt concentration of 650 mM NaCl. On the contrary, we observe over 60 % of the nucleosomes show dimer motion prior to DNA unwrapping in the presence of Nap1. Upon monitoring the DNA unwrapping kinetics with histone acetylation at H3K56 or H4K16, we found that DNA unwrapping becomes facilitated mainly by destabilizing the fully wrapped nucleosomal state. Our results strongly suggest that Nap1 would have a direct impact on RNA polymerase II progression through the nucleosome, which we have also confirmed with our system, and that histone acetylations would amplify this effect in the presence of Nap1.

Support or Funding Information

NIH [GM097286]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1000 Ways to Die: Synthetic Lethality with an HDAC
Authors: Alexandre Karlbuto , Hodges Evan , Ciccaglione Kerri , Law Michael
Published: June 2021

Histones are proteins that can be post‐translationally modified and these modifications will determine the expression of genes. Many studies have shown that it is very common for cancers to have mutations in their histone genes. The concept of synthetic lethality states that if a cell has a single mutation it will be able to survive. But if a second mutation is applied, the cell will become synthetically sick and/or die. In our study, we are seeking to determine synthetically lethal interactions between the histone H3 gene and RPD3 histone deacetylase enzyme. In order to discover potential genetic targets to develop therapies against cancers. To achieve this we used a genetically engineered yeast strain that has the histone H3 gene and RPD3 gene deleted. The H3 gene deletion is covered by a wild type H3 plasmid. These yeast were then transformed with plasmids from the SHIMA library. The yeast were then grown on plates lacking uracil and tryptophan. The individual colonies were then patched and replica plated onto tryptophan deficient media. Finally, they were replica plated onto agar plate media containing 5‐ FOA. The DNA from yeast deemed synthetically lethal or sick were then put through “Library Prep” which is a high throughput method used to isolate the H3 gene within the plasmid with low error rate. The lethals were then put into Next Generation sequencing, aligned, and transferred in the IGV. Finally, we were able to estimate with high accuracy the frequency of H3 mutants in our lethal pool. The top three frequencies discovered were F67A, F84A, and Q93A. They represented 35%, 12% and 10% of our lethal pool respectively. In total we discovered ten new potential synthetically lethal interactions between histone H3 and RPD3. We also used this information in combination with previous knowledge to generate a genetic interaction map for RPD3 from our findings. Although still in the preliminary stages when verified with duplication studies our new potential synthetically lethal interactions can be targets to develop cancer therapies. Also, the knowledge gained from our study can be used to understand genetic interaction between histones and histone modification enzymes.

Support or Funding Information

Summer Medical Research Fellowship at Rowan University School of Osteopathic Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis for H2B Monoubiquitylation by Bre1 and Rad6
Authors: Hyun Kwangbeom , Su Xiaonan , Li Haitao , Kim Jaehoon
Published: June 2021

Histone ubiquitylation is implicated in many cellular processes that include transcriptional regulation and DNA damage repair. In yeast, monoubiquitylation on lysine 123 residue of histone H2B is mediated by Rad6 E2 ubiquitin conjugating enzyme and Bre1 E3 ubiquitin ligase. Interestingly, our biochemical analyses previously showed that Rad6 binds to the N‐terminal coiled‐coil motif (CCM) of Bre1 while most E2 enzymes interact with RING‐finger domains within their cognate E3 enzymes. To understand detailed structural basis for Rad6‐Bre1 interaction, we solved the crystal structure of Rad6‐Bre1 CCM complex at 2.1Å resolution and deployed in vitro protein interaction and H2B ubiquitylation analyses with purified factors to validate obtained structural model. We found that Bre1 CCM anchors to the backside of Rad6 and this unique Rad6‐Bre1 recognition mode directs histone H2B monoubiquitylation rather than polyubiquitylation. In addition, two Bre1 CCM monomers form a homodimer in an asymmetric manner with each adopting a distinct conformation. This explains why two Bre1 homologs, Bre1A and Bre1B, are evolved to form a heterodimeric E3 enzyme in higher eukaryotes. Our structural study reveals a novel RING‐finger independent interaction of Rad6 and Bre1 and establishes the molecular mechanism of H2B monoubiquitylation.

Support or Funding Information

National Research Foundation (NRF) of Korea (2015R1A1A1A05001593)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of SOCS5 as a gene target of miR‐9 in inflammatory monocytes
Authors: Stander Matthew , Rabquer Bradley
Published: June 2021

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammatory joint destruction. The inflammation is caused in part by monocyte migration into the joints and subsequent macrophage differentiation. MicroRNA (miRNA) are short, single‐stranded, noncoding RNA that bind to mRNA to regulate gene expression. Our lab has previously found that miR‐9 was upregulated in monocytes isolated from patients with RA. SOCS5 is part of the suppressor of cytokine signaling (SOCS) family which downregulates cytokine expression such as epidermal growth factor receptor and interleukin‐6. We hypothesize that SOCS5 is a gene target of miR‐9 and propose a luciferase assay to confirm binding.

Using microRNA.org, SOCS5 was determined as a target gene of miR‐9 in silico with a mirSVR predicted binding score of −2.17. microRNA.org uses a combination of miRanda, an algorithm to predict miRNA binding sites, and mirSVR, a support vector regression that provides a predicted binding score. PCR primers that amplify the miR‐9 3′ UTR binding site of SOCS5 were designed using primer‐BLAST. U937 monocytes were grown, mRNA was isolated, and cDNA was produced. The PCR product and pmirGLO vector were digested with Sac1 and Sal1 restriction enzymes and then ligated. The ligated vector was transformed into E. coli cells. Plasmid vector was isolated from single colonies. We determined the ligation and transformation were successful by performing PCR using sequencing primers that amplify the multiple cloning site. Gel electrophoresis was used to separate and view the ligated plasmid PCR product. The SOCS5 insert was calculated to be 576 bp, whereas the non‐ligated plasmid came out to be 380 bp. The ligated plasmid was sequenced to confirm ligation.

In conclusion, we have determined that SOCS5 is expressed by U937 monocytes. We have also successfully cloned the 3′ UTR miR‐9 binding site of SOCS5 into the pmirGLO vector for use in luciferase assays to assess binding between miR‐9 and SOCS5.

Support or Funding Information

Albion College Biology Department and The Foundation for Undergraduate Research, Scholarship, and Creative Activity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection and comparison of circular RNAs in mouse striatum and retina
Authors: Chen Jian‐Huan
Published: June 2021

Purpose

Circular RNAs (circRNAs) are a type of non‐coding RNAs (ncRNAs), and could be potential clinical biomarkers in complex disorders of the nervous system. Expression of circRNAs possess spatio‐temporal properties. Herein we used RNA sequencing (RNA‐Seq) to detect and compare circRNAs and their expression pattern in the mouse striatum and retina.

Methods

Whole striatum and retina was collected from adult mice, and total RNA was extracted using TRIzol followed by phenol/chloroform processing. RNA‐Seq libraries were prepared from total RNA using the ribosomal RNA depletion method. RNA‐Seq was performed for three striatum and three retina samples on the Illumina HiSeq X10 platform with a configuration of 90 million 150 bp pair‐end reads per sample. Reads was aligned to the mouse full genome (UCSC version mm10) and splice junctions were identified by using RNA STAR version 2.5. circRNAs were detected and quantified using DCC version 0.4.4, and difference of circRNA expression between the striatum and retina was analyzed using CircTest. Functional analysis including gene ontology (GO) enrichment and KEGG pathway was performed using DAVID bioinformatics resources 6.8.

Results

In total 781 circRNAs were detected and passed quality control in the striatum and retina, among which 342 showed significant expression difference between the two types of neural tissue. Among the differentially expressed circRNAs, 5 were only detected in the striatum and 29 only in the retina. Functional Annotation Clustering analysis using DAVID showed that a total of 70 host genes of differentially expressed circRNAs were enriched in K48‐linked ubiquitination and positive regulation of cell cycle, and 39 of these genes had a high expression level in the brain.

Conclusion

In the current study, our results demonstrated remarkable difference in circRNA patterns between two types of neural tissue including the striatum and retina, suggesting the potential importance of circRNAs in development and differentiation of nerve system.

Support or Funding Information

The Natural Science Foundation of China (No. 31671311, 81371033 and 81000397), the “Six Talent Peak” Project of Jiangsu Province (SWYY‐127), the Guandong High‐level Personnel of Special Support Program, the Yangfan Plan of Talents, and the Fundamental Research Funds for the Central Universities (JUSRP51712B).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LncRNA GAS8‐AS1 directs epigenetic modulation and activation of the tumor suppressor GAS8 via MLL1/WDR5 to suppress hepatocarcinogenesis
Authors: Yang Ming
Published: June 2021

Long noncoding RNAs (lncRNAs) are vital players in hepatocellular carcinoma (HCC). We previously identified a significantly mutated lncRNA GAS8‐AS1 locating in intron 2 of GAS8. However, its involvement in HCC is still largely unknown. In this study, we thoroughly functional evaluated its critical role in regulating chromatin dynamics, gene expression, cancer development and sorafenib multi‐kinase target therapy in HCC. We for the first time declare that GAS8‐AS1 and its host gene GAS8 act as novel HCC tumor suppressors employing a series of in vitro and in vivo assays. Consistent with this notion, a significantly decreased GAS8‐AS1 or GAS8 expression exists in HCC tissues and is associated with poor prognosis of HCC patients. Interestingly, lncRNA GAS8‐AS1 could evidently promote GAS8 transcription. Although GAS8‐ AS1 does not impact promoter DNA methylation of GAS8, we successfully annotated two lncRNA interacting proteins, Mixed Lineage Leukemia 1 (MLL1), a histone 3 Lys4 (H3K4) methyltransferase, and its partner WD‐40 Repeat Protein 5 (WDR5) based on bioinformatics methodology. Chromatin immunoprecipitation and RNA immunoprecipitation assays elucidate that lncRNA GAS8‐AS1 is required to maintain the GAS8 promoter in an open chromatin state. In accord with this, knockdown of GAS8‐AS1 results in reduced MLL1/WDR5 binding, decreased H3K4me3 levels, diminished RNA Pol II functions and gene silencing of GAS8. GAS8‐AS1 may function as part of a surveillance mechanism that keep activation of GAS8 promoter and transcription, which thus prevents hepatocarcinogenesis. Our results highlight the prevalent involvement of regulatory lncRNAs in cancers and provide pathogenic insights into HCC development and treatment.

Support or Funding Information

National Natural Science Foundation of China (31671300); Taishan Scholars Program of Shandong Province (tsqn20161060)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutation W209R in Human D‐amino acid oxidase protein
Authors: Birdsong Garrick Leif , Jonsson Amanda L.
Published: June 2021

Human D‐amino acid oxidase (hDAAO) is an enzyme found in the brain that is responsible for breaking down the signaling molecule D‐serine. hDAAO is composed of two identical subunits that each bind FAD as a cofactor. Increases in hDAAO activity will lead to too much hydrogen peroxide being produced as a product and a decrease in activity will lead to high levels of D‐serine. Because of this, mutations in hDAAO are linked to several disorders such as schizophrenia or amyotrophic lateral sclerosis. One particular mutation involves residue 209, which is located at the dimer interface, far from FAD and the active site. In the wild‐type, residue 209 is a tryptophan and in the mutant it has changed to an arginine; this W209R mutation increases the activity of the enzyme. Molecular dynamics simulations were used to analyze how the protein structure changes over time for both the wild‐type and W209R mutant hDAAO enzyme to help determine how changing a single residue far from the active site can impact protein function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteasomal ATPases Hard at Work: The Inner Workings of a Protein Destruction Machine
Authors: Snoberger Aaron M. , Smith David M.
Published: June 2021

Across all domains of life, the proteasome is responsible for the majority of targeted protein degradation in the cell. Despite its crucial role in virtually every cellular process, our understanding of how the proteasome operates from a mechanistic perspective is still highly limited. The proteasomal ATPases are the epicenter of the 26S proteasome, as they constitute the primary machinery that catalyze the rate limiting step in substrate degradation: Protein translocation into the proteolytic particle. In order to understand how these complex molecular motors function, we study the archaeal proteasomal ATPases (PAN). In both the 26S ATPases and PAN, we have previously shown that energy from ATP hydrolysis is tightly coupled to unfolding, and we have proposed that such tight regulation is the result of highly coordinated ATP binding and hydrolysis. We could determine the stoichiometry of ATP binding (2 high and 2 low affinity sites per hexamer) as well as their position relative to one another (neighboring subunits), but since PAN is a homohexamer we were unable to distinguish which subunits were responsible for ATP binding. In the present study, we utilized an extensive disulfide crosslinking approach to determine that PAN's N‐domains exist in distinct conformations that we were able to capture. This allowed us to correlate specific N‐terminal conformations with functional activities with minimal structural perturbations. This approach is also allowing us to correlate N‐terminal conformations with substrate recruitment and regulation of ATP‐binding. These studies further provide direct evidence to support our previously reported trans‐arginine finger “sensing” model that describes how the ATPases translocate protein substrates.

Support or Funding Information

NIH‐R01GM107129 F31GM115171

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional characterization of glutamate carboxypeptidase II: a multifunctional Zn‐metalloprotease
Authors: Holderby Kyrsten Faith , Gamage Hashi E. , Derham Jessica M. , Greiner Brooke R. , Mendoza Jocelyne
Published: June 2021

Glutamate Carboxypeptidase II (GCPII) is a dimeric, transmembrane, zinc metallopeptidase produced in many organisms including humans. Human GCPII (hGCPII) is expressed in the brain, prostate, intestine, and kidney, and is therefor predicted to exhibit varying tissue‐specific hydrolase activities. The only documented GCPII hydrolase activities are the breakdown of N‐acetyl‐aspartyl‐glutamate (NAAG) – GCPII is involved in the development of neurotoxicity due to excess glutamate – and folyl‐poly‐γ‐glutamate – GCPII is involved in folate uptake across intestinal mucosal membrane. However, functions in other tissues have not been reported. A comprehensive understanding of potentially diverse tissue‐specific proteolytic and other biochemical functions of GCPII requires detailed molecular level in vivo and in vitro studies using the full‐length GCPII and suitable model organism respectively.

Homology modeling and protein structure analyses identified a hGCPII ortholog in the roundworm Caenorhabditis elegans, (hereafter referred as cGCPII), thus C. elegans has been chosen for the in vivo studies. The hGCPII and the cGCPII share all essential protein structural features. The cGCPII is encoded as three paralogs (gcp‐2.1, gcp‐2.2, gcp‐2.3) compared to five paralogs in humans. Whole animal phenotypic studies carried out using the wild‐type (N2) and the mutant RB1055, which lacks the gcp‐2.1, showed a larger brood size for RB1005 than N2 and longevity assays showed similar lifespans for both strains. The N2 and RB1055 strains raised in the presence of the folate hydrolase inhibitor sulfasalazine (SSZ) and GCPII specific inhibitors 2‐(Phosphonomethyl)pentane‐1,5‐dioicacid (PMPA) or 2‐(3‐Mercaptopropyl)pentanedioic acid (2‐MPPA) and the showed significantly reduced brood size for RB1055 compared to N2. The functional roles of individual cGCPII paralogs were investigated by RNAi mediated knockout experiments. Collective results indicate that the all three cGCPII paralogs may be involved in functional complementation in C. elegans. For the in vitro studies, the full‐length hGCPII was cloned and overexpression was carried out in Pichia pastoris. Several hGCPII expressing transformants have been identified and expression optimization is underway. Parallel cloning in insect cells in process to compare the functionally important glycosylation levels in Pichia versus the insect cells in order to choose an optimal expression system. Together, the in vivo and in vitro findings will aid in further understanding of the physiological functions of GCPII.

Support or Funding Information

Eastern Illinois University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidation of a 2.3 Å Resolution Norovirus GII.4 Protease Structure by X‐Ray Crystallography
Authors: Muzzarelli Kendall Marie , Kuiper Benjamin , Spellmon Nicholas , Brunzelle Joseph , Hackett Justin , Kovari Iulia , Yang Zhe , Kovari Ladislau
Published: June 2021

According to a current report from the Centers for Disease Control (CDC), it is estimated that noroviruses are responsible for 685 million cases of acute gastroenteritis each year with an estimated $60 billion medically and socioeconomically. This overwhelming global burden drives the need for identification and characterization of potential norovirus drug targets to exploit for design and development. Specifically, the GII.4 norovirus genotype accounts for more than 50% of all outbreaks worldwide. Up until now, the only GII.4 model was based on an alignment of the GI.1 and GII.4 protease sequences which resulted in a sequence identity of only 67%. This high genetic diversity among human noroviruses enhances the need for an improved model of the GII.4 protease. We report the crystallization and structure elucidation of a 2.3Å resolution structure of the GII.4 norovirus protease (GII.4 NoV PR) through X‐ray crystallography. Additionally, extensive investigation and characterization of the GII.4 norovirus protease structure and mechanism were carried out through computational studies and enzyme kinetics. With the elucidation of the GII.4 norovirus protease structure, we hope to significantly improve the structure‐based design of both potent therapeutic inhibitors and potential vaccine strategies for future studies.

Support or Funding Information

Wayne State University School of Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rationally designed mutations of E. coli alkaline phosphatase confer selective purine derivative binding
Authors: Malecha Mitchell Richard , Weaver Todd Michael , Grilley Daniel Patrick
Published: June 2021

Small organic molecules, like phenylalanine and theophylline, are effective inhibitors of mammalian alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIAP). However, organic compounds do not hamper E. coli alkaline phosphatase (EcAP) activity. Sequence and structural analysis of alkaline phosphatase isozymes revealed a lack of conservation at EcAP residues that may be important for organic inhibition, thereby providing a potential explanation for the contrary inhibition. By mutating these EcAP residues to mimic analogous residues in mammalian APases, uncompetitive inhibition of EcAP by a class of aromatic molecules was conferred. While variants with single mutations are unaffected by organic effectors, variants expressing multiple mutations are inhibited, suggesting a synergistic relationship essential for organic binding. Circular dichroism was utilized to verify similar stability and kinetics for each variant. Michaelis‐Menten experiments were used to identify inhibition and confirm similar pH and cofactor dependence. The importance of key residues was determined based on their ability to confer organic inhibition. Furthermore, utilization of a set of purine derivatives allowed determination of the structure‐activity relationship for inhibitor binding. Specifically, this analysis enabled identification of hydrogen bonding requirements for organic inhibition. In total, designed substitutions altering EcAP residues near the active site to mimic analogous residues in mammalian APases confers uncompetitive inhibition by specific derivatives of a class of aromatic organic molecules.

Support or Funding Information

Research generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity grant (MRM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genotype and Phenotype of Caffeine Metabolism in Identical Twins
Authors: Passang Tenzin , Ye Jiayu , Kline Gabriel M. , Millard Julie T.
Published: June 2021

A wide range of individual variability to caffeine's effects have been reported in the literature, with factors including gene variants, gender, and personal habits modulating the response to caffeine. For example, the literature suggests that a single nucleotide polymorphism (SNP) in intron 1 of the CYP1A2 gene is associated with “fast” versus “slow” caffeine metabolism. The goal of our study was to genotype and phenotype identical twins to investigate environmental versus genetic influences on caffeine metabolism. For genotyping, we are using PCR‐RFLP to determine C/A at rs762551. For phenotyping, we are using a micro‐scale solid phase extraction of caffeine from saliva, determining the concentration of caffeine via GC‐MS, and using the loss of caffeine over time to monitor the activity of CYP1A2, the principal enzyme for caffeine metabolism. Initial results suggest a good correlation of caffeine half‐lives within identical twin pairs. However, it was not possible to predict this half‐life based on rs762551 genotype. Our findings indicate a strong influence of behavioral factors such as daily caffeine consumption on CYP1A2 activity. These findings could have significance for optimizing the ergogenic effects of caffeine dosing.

Support or Funding Information

Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondria‐targeting peptide from Hibiscus sabdariffa
Authors: Loo Shining , Kam Antony , Tam James P.
Published: June 2021

Cysteine‐rich peptides (CRPs) are hyperstable, multi‐disulfide‐constrained peptides. Structurally, CRPs contain evolutionary conserved disulfided scaffolds, often with functional intercysteine loops which contribute to their multiple biological activities. Previously, we have identified Hibiscus sabdariffa as a rich source of CRPs. Hibiscus sabdariffa, commonly known as roselle, is a South‐east Asian plant. It is used as a folk medicine for treating age‐related conditions and diseases. The prototypic CRP derived from roselle is roseltide rT1, a 27‐residue cysteine‐rich peptide with six cysteine forming four intercycteine loops. We showed that Loop 1 contributes to the inhibitory activities against human neutrophil elastase. However the functions of other loops, individually or in combination have not been determined. Here we report that roseltide rT1 is a mitochondria‐targeting peptide with bioenergetic properties. We showed that roseltide rT1 interacts with the mitochondrial import receptor TOM20 for its localization to the mitochondria. We prepared the linear precursor of roseltide rT1 using a stepwise solid‐phase method followed by oxidative folding to its native state. The conformation of synthetic rT1 was confirmed using reversed‐phase high‐pressure liquid chromatography and nuclear magnetic resonance spectroscopy. Using N‐hydroxysuccinimide ester, N‐terminal biotin‐ and Cy3‐labeled roseltide rT1 were generated for biological assays. Our results showed that mitochondrial depolarization induced by mitochondria uncoupler carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone does not abolish the mitochondrial localization of Cy3‐rT1. In silico docking suggested that the leucine‐rich loop 2 of roseltide rT1 interacts with TOM20. Using pull‐down assay, biotin‐rT1 was shown to interact with TOM20. Using live‐cell confocal microscopy, Cy3‐rT1 was shown to co‐localize with TOM20‐GFP in stably‐transfected HEK293 cells. Additionally, the localization of Cy3‐rT1 with MitoTracker Green was reduced in TOM20‐knocked‐down HepG2 cells compared to control. Taken together, this study shows that the mitochondrial import receptor TOM20 plays an important role for the localization of roseltide rT1 to the mitochondria.

Support or Funding Information

This research was supported in part by Nanyang Technological University Internal Funding ‐ Synzyme and Natural Products (SYNC) and the AcRF Tier 3 funding (MOE2016‐T3‐1‐003).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemical tools for engineering glycan interactions at the stem cell‐matrix interface to promote germ layer specification
Authors: Godula Kamil
Published: June 2021

Glycans (also known as carbohydrates, saccharides or, simply, sugars) are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells' physiological state, but also regulate cellular communication and responses by engaging receptors on neighboring cells and in the extracellular matrix. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell‐surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organized network with specialized functions, called the glycocalyx. In my presentation, I will describe our recent efforts in this area, placing emphasis on the applications of glycomaterials to manipulate growth factor signaling at the surfaces of embryonic stem cells to promote differentiation into the neruoectodermal and mesodermal germ layer subtypes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Transcriptional Changes Induced by an Ovarian Cancer Targeting Peptide
Authors: Weintraub Lindsey , Rudy James , Hum‐Musser Susan , Musser Richard , Soendergaard Mette
Published: June 2021

The fifth leading cause of cancer‐related deaths among women is ovarian cancer. Current standard treatment involve cytoreductive surgery, as well as platinum and taxane based chemotherapy. While most patients initially respond to therapy, the majority relapses with drug resistant malignancy, and ultimately succumbs to the disease. Bacteriophage (phage) display technology was previously used to discover a 15‐mer peptide (J18) with specific binding to human ovarian adenocarcinoma (SKOV‐3) cells. Based on the ovarian cancer binding properties of J18, the cytotoxic properties of the peptide was investigated using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. In short, 0.25 mg/mL peptide J18, peptide p30‐1 (non‐cytotoxic), or DMSO were incubated with 103 SKOV‐3 cells for 48 h, after which MTT reagent was added. The cell viability was quantified spectrophotometrically at 560 nm. These results showed that J18 significantly (p<0.01) decreased SKOV‐3 cell viability compared to peptide p30‐1 and DMSO. Next, the mechanism behind the loss of cell viability was sought elucidated by investigation of transcriptional changes after addition of peptide J18. Specifically, genes involved in cancer progression (myc, elk‐1, smad4, hspb1, and tp53), and apoptosis (fadd, casp3, and cflar) as well as housekeeping (sdha) was chosen for analysis. In brief, SKOV‐3 cells (105) were incubated with 0.25 mg/mL J18 for 48 h, after which total RNA was extracted using the TRIzol method. Total RNA was used for cDNA synthesis with oligodT primers. Gene specific primers for selected genes were designed using Primer3 software. qPCR reactions were carried out using a SYBR Green Master Mix in optical 96‐well plates on an Applied Biosystems StepOnePlus Real‐Time qPCR System. Sdha was used as the endogenous control to correct for sample variation. The comparative quantification method deltadelta Ct (ΔΔCt) was used, and data were transformed to absolute values with 2‐ΔΔCt to obtain fold changes between treatments. Each treatment consisted of five biological replicates. Data were analyzed with ANOVA then Fisher's LSD at p<0.05. Results showed that expression of myc, elk1, smad4, hspb1, tp53, fadd, casp3, and cflar were not significantly different for peptide J18 compared to DMSO. These results indicate that the loss of cell viability mediated by peptide J18 is not associated with changes to the transcriptional levels of myc, elk‐1, smad4, hspb1, tp53, fadd, casp3, and cflar. Next, microarray analysis will be performed to investigate the changes in gene expression on the human transcriptome.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Inhibitors by Ligand‐based Virtual High Throughput Screening
Authors: Chelliah Selvam , Mock Charlotta D. , Mathew Omana P. , Ranganna Kasturi
Published: June 2021

In recent years, the inhibition of angiogenesis has become a target for anti‐cancer therapeutics. Angiogenesis' role in the cancerous cells is different from that when it is employed in the physiological homeostasis of cells. As the interest of angiogenesis inhibition has grown so has, the interest in the vascular endothelial growth factor receptor‐2 (VEGFR‐2) complex that is highly expressed in many cancers such as breast, kidney. The present study reports the successful application of ligand‐based virtual high throughput screening method for the discovery of novel VEGFR‐2 inhibitors. We generated a ligand query model with pharmacophore features from the reported VEGFR‐2 inhibitors using vROCS tool and performed virtual high throughput screening. Among the 2.4 million lead like molecules of ZINC database screened, nineteen prioritized compounds were purchased from Enamine and Chembridge screening libraries and tested for VEGFR‐2 inhibitory activity using Promega's ADP‐Glo™ kinase assay. Experimental validation led to the discovery of four compounds 3, 7, 10, and 13. Staurosporine, a known kinase inhibitor was used a standard for this assay. At the beginning, all the 19 compounds were tested for VEGFR2 inhibitory activity at 50μM. The results are displayed in figure 1. The compounds 3, 7, 10, and 13 showed more than 50% inhibition are shown in red bars. These compounds were then tested for their inhibitory activity at 10 μM. Figure 2 represents the VEGFR inhibitory activity for the selected compounds at 10 μM. Docking was performed for these compounds and the predicted binding modes guide to explore the possible structural changes to get more potent VEGFR‐2 inhibitors.

Support or Funding Information

This study was made possible, in part, by Molecular Biology research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH. We thank the Openeye Inc for providing the academic license to use the virtual screening tools.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanding the Druggable Proteome: Ligand and Target Discovery by Fragment‐Based Screening in Cells
Authors: Parker Christopher G.
Published: June 2021

Advances in DNA sequencing and editing technologies have revolutionized our understanding of the genetic basis of human disease. However, many disease‐relevant genes encode proteins that are poorly characterized and/or are considered “undruggable”, hindering our understanding of disease mechanisms and translating this knowledge into new therapies. Chemical probes offer a valuable way to directly interrogate the function and disease‐relevance of proteins and can also serve as valuable leads for drug development, yet most proteins in the human proteome lack small‐molecule ligands that can serve as probes. More generally, the boundaries, if any, on the ligandability, and therefore potential druggability, across the proteome remains poorly understood. In this seminar, I will describe a platform that integrates fragment‐based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule‐protein interactions directly in cells. Many of these interactions can be site‐specifically determined and involve proteins that fall outside of traditional druggable classes. We demonstrate that this knowledge can be advanced to furnish fragment‐based ligands that affect protein activity, including inhibitors of PTGR2 and SLC25A20, proteins that play important roles in human metabolism but lacked chemical probes to study their function. Furthermore, we integrated this platform with phenotypic screening to identify ligand‐protein interactions that regulate complex cellular processes. For example, we identified small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment‐based screening in cells provides an extensive proteome‐wide portrait of native protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

Support or Funding Information

American Cancer Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
31P NMR Analysis of Divalent Magnesium Ions with ATP, ADP, AMP, and PPi in Various Buffer Systems
Authors: Donels Jordan , Greenwood Brandon , Zea Corbin
Published: June 2021

The human body would not be able to function without the efficient relationship between the binding of adenine triphosphate (ATP) and magnesium ions. Two equivalents of magnesium ions are required per ATP for enzyme catalysis. The conformational changes that occur upon magnesium ion binding to the phosphate groups is not well understood. The metal ion binding of magnesium to ATP, ADP, AMP, and PPi are explored at various pH's (6.5, 7.0, and 7.7) and various buffers (Bicine, HEPES, and Bis‐Tris) using 31P NMR. The addition of magnesium in 0.5 equivalents to the phosphate caused a chemical shift in the 31P NPR peaks. 31P peaks broaden and decreased in intensity at half equivalence magnesium to ATP and as the magnesium concentration increased, the peaks sharpen and regain their intensity, but never reach original state or chemical shift. ADP, AMP, and PPi follow the same patterns. Chemical shifts due to buffer selection and pH was also seen and greatly impacted 31P chemical shifts.

Support or Funding Information

R.W. & Mary Nelson Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstitution of P‐glycoprotein in Nanodiscs using the MSP1D1 Scaffold Protein for Biochemical Inhibitor Screens
Authors: Ballou‐Crawford Jett J. , Chen Gang , Oliveira Maisa C. , Wise John G. , Vogel Pia D.
Published: June 2021

Many cancers become multidrug resistant due to overexpression of the transmembrane efflux pump P‐glycoprotein (P‐gp). Compounds inhibiting P‐gp function may therefore have the potential to improve the effectiveness of chemotherapeutic drugs. Using in silico methods, we have been able to successfully identify a number of putative P‐gp inhibitor compounds. In cell culture assays, many of these compounds were shown to re‐sensitize cancers to chemotherapeutics. However, when tested with purified P‐gp, some of these compounds showed little effect on the catalytic activity of the isolated protein. The relatively high concentrations of detergents needed to solubilize P‐gp likely reduced the potency of the inhibitor compounds. To overcome this issue, purified P‐gp was inserted into nanostructures (nanodiscs) which much more closely resemble natural cell membrane. We and others have observed that this stabilizes P‐gp and stimulates its catalytic activity. However, previous assays of inhibitor compounds with medium size MSP1E3D1 nanodiscs still did not show clear effects on catalytic activity of the protein despite the fact that the inhibitors were effective in cell culture. We hypothesize that the previously used MSP1E3D1 nanodiscs still may have been too large, leaving hydrophobic space into which the inhibitors could partition, again reducing the effective concentration of the inhibitors. To improve on this protocol, we here show our efforts to use a shorter membrane scaffold protein, known as MSP1D1, for the assembly of smaller nanodiscs. After optimization of conditions for assembly and insertion, and tests for biological activity, we will assay our potential inhibitor hit compounds for their ability to affect catalytic ATP hydrolysis activities of P‐gp.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars Program, SMU Undergraduate Research Assistantships, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Retinoblastoma‐derived peptides directly inhibit the CMG helicase and impair proliferation of cancer cells
Authors: Larratta Auston J. , Borysov Sergiy
Published: June 2021

The CMG helicase is the main replicative helicase. Circumstantial evidence suggests that interference with the function of this helicase is detrimental for cancer cells, while tolerable for normal healthy cells, pointing to the CMG helicase as an ideal novel target for cancer treatment. Previous studies identified Retinoblastoma (Rb) protein as a potent native inhibitor of the helicase activity. For the purpose of drug discovery, here we explore Rb‐derived peptides for their abilities to inhibit the CMG helicase and proliferation of cancer cells. For the first time, we showed that Rb‐derived peptides directly inhibited activity of human CMG helicase, as assessed in an in vitro helicase assay. Importantly, Rb‐derived peptides caused a substantial decrease in the proliferation of cancer cells in vitro. Our results further support the idea of targeting CMG helicase for cancer treatment and developing novel anti‐cancer therapeutic agents based on Rb‐derived peptides.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Purified Plant Extracts and Homologous Commercial Derivatives on Entamoeba histolytica growth
Authors: Hunt Sara , Santos Yoly , Seeram Navindra , Rowley David , Ma Hang , Espinsosa Avelina
Published: June 2021

Entamoeba histolytica is a parasitic protist which causes amoebiasis in humans. Amoebiasis causes about 100,000 deaths every year worldwide. Currently, the antibiotic, metronidazole is the only effective treatment for amoebiasis. Metronidazole is effective; however, it has a high toxicity and causes damage to the nervous system. Finding an alternative treatment is necessary to manage the disease better and reduce the risks associated with the current medication. In previous studies, purified plant extracts and homologous commercial derivatives have shown significant antimicrobial character. Previously, we identified pomegranate, rhubarb (rhein), and maple syrup (maplifa) extracts as potential inhibitors of E. histolytica trophozoite growth. The effectiveness of the extracts was assessed by growth inhibition assays monitored at 12, 24, and 48 hours using 60 μM or 120 μM concentrations of pure compounds. Rhein and maplifa, showed promising inhibitory activity on E. histolytica trophozoite growth. Testing of compounds of analogous structures is currently in progress. Future studies will be completed to determine the biochemical mechanism of action of each of these inhibitors and test their toxicity to mammalian tissue cultures in comparison to the current drug of choice, metronidazole.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Identification of the Minimal Pharmacophore of KDT‐11
Authors: McCartney Julia , Dickson Paige , Kodadek Thomas
Published: June 2021

The 26S proteasome is a key player in a number of cell processes that help to remove misfolded and downregulate targeted proteins. Rpn13, a subunit of the 19S regulatory particle of the proteasome, is a ubiquitin receptor and UCH37 recruiter and activator. Rpn13 can be knocked out of yeast and mouse models without the development of a new phenotype, yet in many cancer cells it is over expressed. In 2015, the peptoid KDT‐11 was shown to be highly selective towards Rpn13 and toxic to multiple myeloma cells. However, because of this compound's size and hydrophobicity, it is a poor candidate for further development as a drug. Preliminary lab data suggests that N‐methyl‐N‐(3‐fluorobenzylamine) in the terminal position of the 6‐mer peptoid is critical for selective toxicity. However, the necessity of the amines in all other positions is not known. To make the molecule more biologically viable, I synthesized ten derivatives using solid‐phase bead synthesis. Florescence polarization and cell viability assays were conducted to assess the potential improvement or detriment offered by each variation, using KDT‐11 and Bortezomib, a 20S core particle inhibitor, as control factors. I found that shortening the peptoid does not inherently result in a loss of selectivity or cytotoxicity, and in some cases, appears to lead to an increase. However, truncating past a 4‐mer length resulted in general cytotoxicity to control and cancerous cells. These data indicate that while a certain length must be maintained, only the terminal N‐methyl‐N‐(3‐fluorobenzylamine) is position dependent. This development is highly advantageous as it targets a different subunit of the proteasome than currently available drugs, and could potentially be used in parallel.

Support or Funding Information

We would like to thank the Scripps Summer Undergratuate Research Fellowship for funding this work

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.