bluuebunny/crossref_metadata_2025 huggingface dataset:
Common variants of Drosophila melanogaster Cyp6d2 cause camptothecin sensitivity and synergize with loss of Brca2
Authors: Thomas Adam Michael , Hui Carrie , South Adam , McVey Mitch
Published: June 2021

Many chemotherapeutic agents selectively target rapidly dividing cells, including cancer cells, by causing DNA damage that leads to genome instability and cell death. We utilize Drosophila melanogaster to study how mutations in key DNA repair genes affect an organism's response to chemotherapeutic drugs. In this study, we focused on camptothecin and its derivatives, topotecan and irinotecan, which are type I topoisomerase inhibitors that create DNA double strand breaks in rapidly dividing cells. Here, we describe two polymorphisms in Drosophila Cyp6d2 that result in extreme sensitivity to camptothecin, but not topotecan or irinotecan. We confirmed that the sensitivity was due to mutations in Cyp6d2 by rescuing the defect with a wild‐type copy of Cyp6d2. Additionally, we showed that combining a Cyp6d2 mutation with mutations in Drosophila BRCA2 results in extreme sensitivity to camptothecin. Given the frequency of the Cyp6d2 polymorphisms in publicly available Drosophila stocks, our study demonstrates the need for caution when interpreting results from drug sensitivity screens in Drosophila and other model organisms. Furthermore, our findings illustrate how genetic background effects can be important when determining the efficacy of chemotherapeutic agents in various DNA repair mutants.
Association of Lin28 Expression and Tumorigenesis in Human Wilms’ Tumor Cell Lines
Authors: Nguyen Sang , Urbach Achia , Daley George
Published: June 2021

LIN28 is an evolutionarily conserved RNA binding protein that plays an essential role in regulating developmental timing in Caenorhabditis elegans. Recently, many reports have shown the effect of LIN28 in stem cell function, cell differentiation, cell proliferation and tumorigenesis. In mammals, LIN28 is highly expressed in embryonic stem cells and in early embryogenesis. One of the major targets of LIN28 is the Let7 microRNA family. By binding to the pri/pre Let‐7 microRNA genes, LIN28 prevents the maturation of this miRNA family and thus enables the translation of genes that are suppressed by Let‐7 miRNAs. Therefore, LIN28 is capable of regulating translation and stability of oncogenes including K‐Ras, C‐Mys and many others. In cancer cells, LIN28 is up‐regulated as an oncogene, which promotes transformation and tumor progression. It has been shown by our lab that ~15% of human cancers are linked to the expression of LIN28A or its paralog LIN28B. In addition, some reports show that knocking‐down LIN28A reduces cancer cell viability and cell growth in vitro. We have shown that LIN28B is expressed in samples of Wilms’ tumor cells. In Wilms’ tumor cell lines, in vitro LIN28B‐knockdown significantly decreased cell proliferation compared with control cells. Taken together, LIN28B might function in controlling cell survival and differentiation in Wilms’ tumor. To further study the role of LIN28 in Wilms tumor formation, we will transplant “normal” and knock‐down human Wilms’ tumor cell lines under the kidney capsule of immunodeficient mice and compare. We will also over‐express LIN28A/B during normal mouse kidney development.
Swarmer cells of Proteus mirabilis migrate faster than swimmer cells in viscous environments
Authors: Sacotte Ryan Michael , Weibel Douglas , Tuson Hannah , Copeland Matthew , Trevino‐Dopatka Sonia
Published: June 2021

It has been suggested that swarming, a type of surface motility that allows rapid migration, is involved in urinary tract colonization. During swarming, cells of P. mirabilis undergo a morphological change from ~5 μm long rods with 5–15 flagella to ~10–50 μm long rods with 50 to more than 100 flagella. Prior work has shown that increasing the number of flagella on a cell does not yield the expected increase in torque – i.e., a four‐fold increase in the number of flagella yields less than a two‐fold increase in torque. This result suggests that the dense flagellation of swarmer cells should not convey a motility advantage. However, mutant cells of Bacillus pseudiformis that express an increased number of flagella do swim faster than their wild‐type counterparts in viscous solutions, and it has been proposed that this increase in speed is due to altered flagellar bundling. We are examining whether this phenomenon also occurs with the different morphological forms of P. mirabilis. We have found that swarmer cells of wild‐type P. mirabilis are significantly faster (P<0.001) than all other cell types in media with a viscosity of 9 – 830 cP. In media with a viscosity of 8340 cP, swarmer cells are motile while all other cell types are amotile. These results suggest a possible role for the swarm cell morphology: allowing migration through viscous mucosal layers during infection.
Human PCID2 plays analogous roles to Centrin 2 outside of the TREX‐2 complex.
Authors: Cunningham Corey Nathaniel , Schmidt Casey Alexandra , Resendes Karen Kay
Published: June 2021

Human PCID2, is characterized by its PCI interaction domain and its involvement in the mammalian transcription export complex, TREX‐2. One component of the TREX‐2 complex, Centrin‐2, is known to be involved in protein export in addition to mRNA export and was originally identified as a component of the centrosome. We attempted to determine if PCID2 also performs these other functions in the cell. We have demonstrated that PCID2 colocalizes with γ‐tubulin and centrin‐2 at the centrosome of HeLa cells. Furthermore, knockdown of PCID2, while not affecting nuclear protein import, does lead to a delay in nuclear protein export. In contrast we have demonstrated that another TREX‐2 component, Eny2, is not present at the centrosome nor is it involved in nuclear protein transport. Therefore, our results indicate that PCID2 distinctly serves roles outside of the TREX‐2 complex that parallel the function of Centrin‐2.
Calcium mediated regulation of karyopherin nuclear transport receptors
Authors: Foltz Lauren Elizabeth , Cholewa Kelly , Grenet Ashley , Resendes Karen K
Published: June 2021

Oxidative stress disrupts the normal function of cells and severe stress situations can result in apoptosis. A specific effect of oxidant stress is an increase in cellular calcium levels, which can be mimicked by treatment with the drug thapsigargin, which inhibits Ca2+ uptake into the endoplasmic reticulum. We sought to determine if changes in cellular calcium levels induced by thapsigargin disrupted nuclear transport by alteration of karyopherin transport receptors and/or the Ran gradient. Altered Ca2+ levels were monitored using a fluorescence based assay. We found that thapsigargin lead to an accumulation of Ran in the cytoplasm, while prolonged exposure led to cell death. However, the disruption of the Ran gradient could be reversed before the induction of apoptosis and rescue was enhanced by the presence of the calcium chelator BAPTA. Thapsigargin treated cells also exhibited altered location of several karyopherin transport receptors including Transportin, Importin alpha, Importin beta, Crm1, and its cofactor RanBP3. These results suggest that alteration of cellular calcium levels causes disruption of the Ran gradient and variation in karyopherin localization, suggesting that there is a role for calcium as a regulator of Ran‐mediated nuclear transport pathways. Therefore, cellular oxidative stress may result in apoptosis due to the calcium induced alteration of nuclear transport pathways.
Triiodothyronine increases adiponectin mRNA by indirect action in adipocytes cell culture, 3T3‐L1.
Authors: de Oliveira Mirane , De Sibio Maria Teresa , Olimpio Regiane Marques Castro , De Azevedo Melo Luvizotto Renata , de Paula Lopes da Costa Lucas , Conde Sandro Jose , Moretto Fernanda Cristina Fontes , Nogueira Celia Regina
Published: June 2021

The aim of this study was to examine the effects of thyroid hormone (TH), triiodothyronine (T3), in the modulation of adiponectin mRNA expression and the involvement of signaling pathway phosphatidyl inositol 3 kinase (PI3K) in adipocytes, 3T3‐L1. We examined the involvement of these pathways in mediating TH effects by treating adipocytes with T3 (1000 nM), incubation for one hour, in the absence or presence of either the PI3K inhibitor (LY294002‐LY). To examine whether adiponectin is directly or indirectly induced, we used the translation inhibitor cycloheximide (CHX). For mRNA expression analyzes was used RT‐qPCR. Statistical analysis was performed using ANOVA, complemmented with Tukey's test and P<0.05 was considered significant. T3 increased the adiponectin mRNA expression of 1 (± 0.22) to 2.86 (±0.011, P<0.001). This increase was preservated by LY294002 (3.55, ±0.48, P<0.05), but abrogated in the presence de CHX (0,68, ±0.08, P<0.05). However in the presence of LY without the addition of T3 showed an increase of adiponectin from 1 (±0.22) to 7.2 (±0.024, P<0.001). These results demonstrate that activation of PI3K cytosolic signaling pathway no is necessary to indirect T3 action on the adiponectin gene expression of the into adipocytes, 3T3‐L1. However PI3K pathway is necessary to modulate normal levels adiponectin in this cells. Financial Support: FAPESP (2010/16911–4).
Role of HDAC1 complexes in gene regulation at the nuclear lamina
Authors: Bushman Gregory , Milon Beatrice C , Nurminsky Dmitry I
Published: June 2021

In Drosophila, Lamin B has been shown to be involved in the transcriptional repression of multigenic regions located at the nuclear periphery through histone deacetylation by HDAC1. In vivo, HDAC1 is found in a variety of complexes that are thought to add specificity and efficacy to its function. In order to further clarify the molecular mechanisms by which HDAC1 elicits the transcriptional repression of gene clusters found at the nuclear lamina, it is important to identify the specific HDAC1 complex involved in the repression. To do this, we decided to analyze the derepression of two testis‐specific gene‐clusters in somatic cells (Schneider 2 cells) after systematic knockdown by RNA interference of proteins known to complex with HDAC1. Of the proteins that we have tested, the knockdown of one has induced re‐expression of the testis‐specific genes. The 90% knockdown of NAP1 resulted in the derepression of the testis‐specific genes in somatic cells, indicating that the Sin3A HDAC1‐containing complex may be involved in the transcriptional repression of gene‐clusters found at the nuclear periphery.
Determination of VraR Binding Activity on fmtA Promoter
Authors: Yang Zhifeng , Golemi‐Kotra Dasantila
Published: June 2021

In S. aureus, the VraSR two‐component system can sense cell wall stress and coordinate the response to antibiotics that inhibit cell wall peptidoglycan biosynthesis. fmtA encodes a low‐affinity penicillin binding protein and is part of the core cell wall stimulon. fmtA is described to be under the regulation of VraSR.

In this study, we investigated binding of VraR to the fmtA promoter region. Two VraR binding regions in fmtA promoter have been found by EMSA and DNase I footprinting assays. The VraR binding motifs on these regions were predicted, and single or double nucleotide mutations were generated to investigate, the recognition or binding elements for VraR. We are using runoff in vitro transcription assays to investigate further the binding mode of VraR. The qRT‐PCR showed that the fmtA expression level was upregulated by VraS/VraR under oxacillin treatment. Our study suggests that VraR coordinates fmtA expression in vivo.

Source of research support: CIHR Canadian Institutes of Health Research.
Role of HSF1 phopshorylation in heat shock response
Authors: Budzyñski Marek Andrzej , Puustien Mikael , Joutsen Jenny , Anckar Julis , Sistonen Lea
Published: June 2021

Highly conserved, molecular chaperones, known as Heat Shock Proteins (HSPs), assist in protein folding and perform quality‐control process. Impaired activity of HSPs results in uncontrolled protein aggregation. Inducible transcription of HSPs is driven by Heat Shock Response, mechanism strictly regulated by Heat Shock Factor 1 (HSF1). Upon proteotoxic insult, HSF1 undergoes complex post‐translational modifications and multistep activation process, involving trimerization, gain of DNA‐binding activity and transactivation capacity. Past studies have taken for granted that the phosphorylation of HSF1 is required for its transcriptional activity, but experimental evidence is still missing. Phosphorylation of HSF1 mainly occurs within the regulatory domain (RD), a region of HSF1 shown to be a master regulator of HSF1 transactivation capacity. The goal of this project is to elucidate the role of HSF1 phosphorylation. Using phosphorylation‐deficient HSF1 mutant, where all known phosphorylation sites residing within the RD are mutated to alanines. Generated mutant, not only is stress inducible transcription factor, but also more potent than wild‐type version of HSF1 in terms of gene expression. This results indicate that phosphorylation within regulatory domain is not required for stress inducible activation of HSF1 but also is an inhibitory mechanism upon stress stimuli.
Comprehensive Characterization of Posttranslational Modifications of Histones Using a Novel High‐Throughput Middle‐Down Proteomics Approach
Authors: Hess Sonja , Kalli Anastasia , Moradian Annie , Sweredoski Michael J
Published: June 2021

Until recently, histones have mostly been characterized by either bottom‐up or top‐down mass spectrometric analysis. While the bottom‐up approach is more sensitive, the important connectivity of the posttranslational modifications of the N‐terminus is often lost. This connectivity is maintained in top‐down proteomics, but the sensitivity of this approach is lacking. Middle‐down proteomics is a compromise between bottom‐up and top‐down analysis using a limited digestion that creates peptides large enough to maintain the important connectivity in the N‐terminal tail of histones. To achieve a high‐throughput method, middle‐down proteomics was coupled with on‐line chromatographic separation using electron capture dissociation (ECD), a fragmentation technique that preserves the posttranslational modifications. Taking advantage of the high mass accuracy in the FTICR mass analyzer, several histone variants were identified and modification sites were successfully localized, including single, multiple and positional isomeric PTM sites. In general, protein sequence coverage was well over 50%, reaching 100% in many instances.

Middle‐down nano‐LC‐ECD MS/MS approach enables the comprehensive, high‐throughput analysis of unfractionated histones and reveals global modification profiles and localization of PTMs.
Translational Repression Mediated By the Genotoxic Agent Sodium Dichromate Results in P‐body Formation in S. cerevisiae
Authors: Hopfauf Whitney Leigh , Bullard Chris , Segner Sara , Rathmann Blaine , Koch Tony , Wilson Ted , Segal Scott P
Published: June 2021

Mechanisms to cope with oxidative stress are conserved across eukaryotes. For instance, exposure to oxidation can result in lipid and nucleic acid damage and elicit a stress response. The genotoxic agent, sodium dichromate oxidizes guanines resulting in OxoG bases, induces double strand breaks, or generates abasic sites. Therefore, an oxidative stress response is crucial to maintain proper cellular function. In response to stress, cells control gene expression by repressing translation. Repressed mRNA may localize to P‐bodies or stress granules; cytoplasmic structures where non‐translating mRNA are found. While P‐bodies contain mRNA degradation factors, stress granules contain translation initiation factors, allowing for storage of mRNA until removal of the stress.

Exposure of yeast to sodium dichromate caused an increase in P‐bodies, whereas stress granules failed to form. The response occurs within 10 min. of exposure and is dependent on translational repression. Interestingly, use of the antioxidant, quercetin, abrogated the increase in P‐body formation during co‐treatment with sodium dichromate. Our data suggests a model in which damaged mRNA or mRNA transcribed from damaged genes are degraded rather than stored. We suspect oxidative damage may halt translating ribosomes on these mRNA, activating the no‐go decay pathway.
MicroRNA in Leishmania RNA Virus 1
Authors: Odera Joab Otieno , Porter‐Kelley Johanna
Published: June 2021

Leishmania RNA Virus 1 (LRV1) is a viral like particle found only in Leishmania species of the Amazon basin. microRNAs (miRNAs) are a large family of small endogenous RNAs, approximately 22 nucleotides long that regulate gene expression post‐transcriptionally in a complex process of binding to mRNA. Our hypothesis is that microRNAs are found in Leishmania RNA virus 1. The genome of Leishmania RNA Virus 1 Genome was retrieved from the National Center for Biotechnology Information Website. Sanger Institute‐miRBase Prediction software and subsequently, we chose five to further analyze based on E‐value and probable strand matches. The potential miRNAs data were consigned to the mFOLD software to determine their stem‐loop structures. Sanger Institute‐miRBase Prediction software revealed 500 potential miRNAs. Three of five selected miRNA sequences successfully showed miRNA stem loop structures that satisfied acceptable miRNA characteristics.
The Effect of Aldosterone on the miRNA Content of a Murine Inner Medullary Collecting Duct Cell
Authors: Jacobs Mollie Elizabeth , Jeffers Lauren A. , Welch Amanda K. , Wingo Charles S. , Cain Brian D.
Published: June 2021

MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension‐related renal disease.

Aldosterone is the major known hormone responsible for sodium retention in the kidney collecting duct. Our working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption.

Here we report the miRNA landscape in mIMCD‐3 cells using Toray 3D‐GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 35 highly expressed miRNAs, as well as several miRNAs whose levels appear to be affected by the presence of aldosterone. Two aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1and sodium/potassium‐transporting ATPase subunit beta‐1 mRNA. Argonaute pull‐down experiments also demonstrated that endothelin‐1 mRNA is a target of the RNA‐induced silencing complex indicating miRNA action on this transcript.

This work was supported by NIH NIDDK 1RO1DK082680 to B.D.C and C.S.W. and a 5T32DK076541–04 pre‐doctoral fellowship to M.E.J. Additional funding support and miRNA microarrays were provided by Toray Industries, Inc.
Next‐generation sequencing analysis of rat heart left ventricle: novel microRNAs, novel isomiRs and transmural distribution of microRNA expression
Authors: Collins Anthony , McGahon Mary K , Yarham Janet M , Daly Aideen , Guduric‐Fuchs Jasenka , Simpson David A
Published: June 2021

MicroRNAs (miRNAs) have a significant role in regulating gene expression in heart disease. They can exist as isoforms (isomiRs) that are potentially (patho)physiologically important. The heterogeneity of cardiomyocyte physiology across the left ventricular wall has an important influence on heart (patho)physiology, and may well be affected by transmural miRNA expression gradients. With the aims of determining rat heart isomiR profiles and left ventricular transmural miRNA expression we investigated epi/midmyo/endocardial tissue samples using Illumina next‐generation sequencing. We detected significant quantities of 146 known rat miRNAs and 68 novel orthologs of known miRNAs. Many novel isomiRs were detected at a higher frequency than the corresponding mature sequence, and have predicted targets different from those of the corresponding canonical miRNA. We identified a novel rat miR‐1 analogue from a second miR‐1 gene and a novel rat miRNA similar to miR‐676. We also cloned and sequenced the rat miR‐486 gene. Several important cardiac intracellular signalling pathways are predicted targets of the most highly detected miRNAs. Most miRNAs are not expressed in a gradient across the ventricular wall; the exceptions include miR‐10b, miR‐21, miR‐99b and miR‐486. These findings suggest new mechanisms of cardiac gene regulation in health and disease. Funded by British Heart Foundation PG110/039/28365.
Cavin‐1/PTRF as a new substrate of the SOCS3 E3 ubiquitin ligase complex
Authors: PALMER TIMOTHY MARTIN , WILLIAMS JAMIE , BAILLIE GEORGE , PILCH PAUL
Published: June 2021

Suppressor of cytokine signalling 3 (SOCS3) is an inducible specificity factor for a multi‐subunit E3 ubiquitin ligase complex. However the full spectrum of ubiquitylated SOCS3 substrates is unknown. By performing an unbiased SILAC‐facilitated comparison of tandem affinity purified ubiquitinomes from WT and SOCS3‐null fibroblasts, we have identified cavin‐1/PTRF as a protein whose ubiquitylation status is significantly enhanced in SOCS3‐expressing versus SOCS3‐null cells. Importantly, studies of endogenous cavin‐1 expressed in adipocytes and fibroblasts have shown it to be ubiquitylated. Co‐immunoprecipitations and overlapping peptide array overlays confirmed that SOCS3 interacted directly with two distinct regions within cavin‐1 in an interaction that did not require tyrosine phosphorylation. Cavin‐1 ubiquitylation was also found to be enhanced in the presence of SOCS3 and its cognate Cul‐Rbx‐elongin B/C E3 ligase scaffold. In summary, our data suggest a potentially novel role for SOCS3 in regulating caveolae assembly via cavin‐1 ubiquitylation. This provides a novel mechanism by which SOCS3‐inducing stimuli may influence caveolar architecture and function.
Mechanism for recruitment of the endosome‐associated deubiquitinating enzyme, AMSH
Authors: Davies Christopher W , Paul Lake N , Das Chittaranjan
Published: June 2021

The endosomal‐sorting complexes required for transport (ESCRT) is a ubiquitin‐mediated process that is implicated in endosomal sorting, trafficking, and viral budding. The ESCRT machinery regulates signaling events by internalizing cell surface receptors and targets them for degradation by the lysosome. Two deubiquitinating enzymes (DUBs) regulate the activity of the ESCRT complex, STAM‐binding protein (AMSH), and ubiquitin specific protease Y (UBPY), by counteracting the ubiquitination of the cargo. Recently, we characterized AMSH's mode of substrate recognition through mutational analysis and found that AMSH's recognition of distal ubiquitin is significantly different from the homologous, but ESCRT‐independent protein, AMSH‐like protein (AMSH‐LP). In the current study, we are aiming to understand how AMSH's mode of substrate recognition in conjunction with AMSH's recruitment to the initial ESCRT complex affects the enzyme's activity using a combination of biochemical and biophysical analysis. We have recapitulated AMSH recruitment to the initial ESCRT complex using the minimal domains and have shown that the enzyme's activity is enhanced. This result supports AMSH's role as a major regulator of ubiquitin mediated degradation by the ESCRT machinery. Results from our studies could further provide insight into defining a clear role for AMSH within the ESCRT machinery.

Support: NIH (1R01RR026273) and American Heart Association (12PRE12060249)
J‐protein requirements of multiple prion variants in two distinct yeast strains reveal functional specificity in yeast prion propagation
Authors: Harris Julia , Patel Milan , Hines Justin K
Published: June 2021

The objective of this study was to determine the importance of prion variant and yeast strain variation on prion‐chaperone requirements. The J‐protein Sis1 and the prions [PSI+] and [RNQ+] were used as a model system to explore the impact of amyloid conformation (prion variants) and yeast strain polymorphisms (yeast genetic background) on chaperone requirements. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast Saccharomyces cerevisiae.

Summary of Results

Yeast strain background had a small but noticeable effect on chaperone requirements; this effect is likely due primarily to altered chaperone protein levels between strains. While many prion variants shared similar chaperone requirements, some significant differences were found. Strikingly, despite these variations, our investigation revealed that individual prions have distinct, non‐overlapping Sis1 domain specificity, an observation which implies that Sis1 has prion‐specific functionality.
Thermal manipulation during chicken embryogenesis results in enhanced HSP70 gene expression and the acquisition of thermotolerance
Authors: Al‐Zghoul Mohammad Borhan
Published: June 2021

SUMMARY

The aim of this study was to evaluate the effect of thermal manipulation (TM) at 38.5°C for 6 h or 18 h during embryonic days (ED) 10–18 on the muscle mRNA levels of heat shock protein 70 (HSP‐70) and the chick's ability to cope with the thermal challenge (TC; 41°C for 6h) at day 3, 7 and 42 of age. 750 fertile chicken eggs were obtained and divided randomly into 3 different treatment groups: control group was maintained at 37.8°C and 56% relative humidity (RH), TM1 was subjected to TM at 38.8°C for 6 h and 65% RH during ED 10–18 and TM2 was subjected to TM at 38.8°C for 18 h and 65% RH during ED 10–18. After hatching, the chicks from each treatment groups were randomly divided into 2 sub‐treatment groups (Naive and challenged). Chick's response to thermal challenged (41°C fro 6 hrs) was evaluated by determination of body temperature, plasma thyroid hormone levels (T3 and T4), plasma HSP70 and muscle mRNA levels of Hsp70. Our results indicated a long‐term improvement in the gene expression of HSP70 in the thermal manipulated chicks when compared with control, as manifested by a significant increase in the muscle mRNA levels of Hsp70 in thermally manipulated chicks during embryogenesis and during thermal challenge in post‐hatch chickens.
Systematic analyses of all possible ubiquitin mutants
Authors: Bolon Dan
Published: June 2021

We quantified the affects of all point mutations in ubiquitin on yeast growth. These fitness analyses delineated the physical requirements at each amino acid position in ubiquitin. We identified a stability window where many ubiquitin mutants were capable of populating a natively folded state, but unable to support yeast growth. Many mutants within this stability window accumulated as high molecular‐weight species in cells indicating that they were efficiently attached to substrates but inefficiently targeted to the proteasome. In addition, we identified a founding member of a new class of ubiquitin mutants that failed to accumulate in cells unless substrate conjugation was blocked with a second mutant in the C‐terminal tail. Heteronuclear NMR indicated that this destabilizing mutation reduces the unfolding temperature while retaining a ground‐state structure similar to wild‐type that enables binding to proteasome receptors. Based on these results, we propose that dynamics within the native ubiquitin structure are critical for proteasome targeting and that ubiquitin stability to unfolding is required for efficient recycling during proteasome‐mediated substrate degradation.
Discovery of pan‐deubiquitinating enzyme inhibitors
Authors: Long Marcus John Curtis , Hedstrom Liz
Published: June 2021

The ubiquitin system is the linchpin in maintenance of cellular fitness. While many studies have focused on the ubiquitylation pathways, comparatively little is known about the deubquitination proteins (DUBs). Recent research suggests that DUB inhibition is a promising strategy to treat diseases such as cancer, Alzheimer's, and Creutzfeldt‐Jakob disease. Thus development of new methods to improve understanding of deubiquitylation pathways is paramount for basic science and drug development. Known pan‐DUB inhibitors G5 and NSC632839 contain 2–3 highly reactive electrophillic groups that suggest these compounds may be non‐specific cross linkers. G5 and NSC632839 cause an accumulation of high molecular weight ubiquitin species as previously reported by others, but also cause the formation of large protein aggregates in vitro. We observe a similar pattern of accumulation of high molecular weight ubiquitin species, and protein aggregates, with non‐specific cross linkers such as phthalaldehyde and glutaraldehyde in vitro. Thus many pan DUB inhibitors may act through a random cross linking mechanism. We identified several compounds that cause the accumulation of high molecular weight ubiquitin species but not the formation of protein aggregates. These compounds do not contain highly reactive electrophiles. We suggest that these compounds represent a new class of pan DUB inhibitors.
Mind the gap: long range charge transfer across the periplasm of Shewanella oneidensis
Authors: Elliott Sean Joseph , Firer‐Sherwood Mackenzie , Ando Nozomi , Bewley Kathryn , Mock Jee Young , Drennan Catherine
Published: June 2021

Shewanella oneidensis a gram negative proteobacteria, is capable of the dissimilatory metal reduction (DMR) of insoluble iron and manganese oxides, through the deployment of multiple strategies, including using multiple multi‐heme c‐type cytochromes that electrochemically wire the cellular interior to the exterior. In order to harness the remarkably biological chemistry of Shewanella in bioenergy and microbial fuel cells, we examined the molecular details of long‐range electron transfer within and between Shewanella multi‐heme cytochromes that are implicated in the DMR pathway. To probe the model of Mtr cytochrome interactions, we have developed new analytical methods to quantify the directionality and stoichiometry of redox reactions in the Shewanella Mtr cytochromes. We have modified traditional PFE methods to probe the protein:protein interactions that may be at work in the Shewanella DMR pathway, revealing an unforeseen unidirectionality for electron transfer. These data, coupled to structural studies of MtrA using small angle x‐ray scattering (SAXS) and analytical ultracentrifugation (AUC), elucidate a model of long‐range electron transfer that occurs from the quinol pool of Shewanella to the cell surface, as mediated by a series of multi‐heme cytochromes c. For each multi‐heme cytochrome, the high number of redox cofactors and their similar reduction potentials, convolutes the routine assignment of specific macroscopic reduction potentials to individual hemes. Here, novel Mtr cytochrome mutants where predicted heme ligands have been mutated from His to Met/Ala mutagenically “map” the nature of each heme center. The electrochemical, spectroscopic and physiological impact of such mutations will be presented.
Dihydrodipicolinate Synthase from E. coli: Characterization of Arginine 138 by Site‐Directed Mutagenesis
Authors: Vann Tyler , Karsten William E. , Chooback Lilian
Published: June 2021

Dihydrodipicolinate synthase catalyzes the formation of dihydropicolinate from pyruvate and L‐aspartate‐β‐semialdehyde (ASA). The enzyme catalyzes the first committed step for the biosynthesis of L‐lysine in bacteria and plants. The enzyme from Escherichia coli is feedback inhibited by lysine, the end product of the pathway. A study of the pH dependence of the kinetic parameters was done to elucidate the acid‐base chemical mechanism of the enzyme. The kinetic mechanism is ping pong with pyruvate binding to free enzyme. The ε‐amino group of lysine 161 attacks the carbonyl of pyruvate and forms a Schiff base intermediate. The loss of a proton from this intermediate leads to the formation of an enamine intermediate, with the loss of the proton accounting for the irreversible step and the ping pong kinetic mechanism. ASA binds to the enzyme:enamine covalent intermediate. Site‐directed mutagenesis was done to investigate the role of the active site arginine 138. The R138A and R138K mutants were created and the identity of the mutants was confirmed. Kinetic studies will be done to characterize the mutant enzymes.

This work was supported by grant P20RR016478 from the National Center for Research Resources a component of the National Institutes of Health and a grant from the University of Central Oklahoma office of Research and Grants to L.C.
The Y392F further stabilizes the pre‐decarboxylation intermediate on 1‐deoxy‐D‐xylulose 5‐phosphate synthase
Authors: Patel hetalben , Brammer Leighanne , Meyers Caren L Freel , Jordan Frank
Published: June 2021

The thiamin diphosphate (ThDP)‐dependent enzyme 1‐deoxy‐D‐xylulose 5‐phosphate (DXP) synthase catalyzes the condensation of D‐glyceraldehyde‐3‐phosphate (D‐GAP) and pyruvate to form DXP, a precursor in the biosynthesis of vitamins B1, B6 and isoprenoids. Our groups recently reported (J.Am.Chem.Soc. 2012, 134, 18374–18379) stabilization of the ThDP‐bound predecarboxylation intermediate C2α‐lactylThDP on DXP synthase and an at least 600‐fold rate acceleration of the decarboxylation by D‐GAP. Model building and analogy with similar enzymes suggested that the residue Y392 could be responsible for this finding, perhaps by hydrogen bonding to the carboxylate anion of C2α‐lactylThDP. To explore this hypothesis, we have carried out steady state and pre‐steady state experiments with the DXP synthase Y392F variant. The results suggest that C2α‐lactylThDP is even more stable on the variant than on DXP synthase. An NMR experiment of the reaction mixture containing Y392F and pyruvate after 50s acid quench also supports the idea that Y392F stabilizes C2α‐lactylThDP. In the presence of D‐GAP the rate of decarboxylation was similar to that on DXP synthase, suggesting that Y392 might be holding the carboxylate group of C2α‐lactylThDP and addition of acceptor substrate D‐GAP immediately rescues the slow rate of decarboxylation. Supported by NIHGM050380 (RU) and NIH‐GM084998 (JHU).
Mutagenesis in Epulopiscium sp. B 1,3‐β‐glucanase
Authors: Boyce Samantha C , Piefer Andrew J
Published: June 2021

The purpose of this experiment is to study the changes in the enzyme kinetics of the Epulopiscium sp B. 1,3‐β‐glucanase when the amino acid sequence is mutated. Epulopiscium is a bacterium found in the gut of the surgeonfish where it produces a 1,3‐β‐glucanase to catalyze the breakdown of 1,3‐β‐glucans, found in the fish's herbivorous diet. To further understand the enzyme, two separate amino acid residues will be mutated; a conserved glutamate within the active site and a conserved tryptophan outside of the active site. Each residue will be mutated with a nonconservative and/or conservative amino acid substitution. The enzyme will be recombinantly expressed and purified from Escherichia coli. The activity of each mutated enzyme will be measured using a fixed time point kinetic assay to ascertain kinetic constants. Interestingly, we have previously noted thermostability in the glucanase with a temperature optimum near 60 °C. This research is funded by the Hartwick College Department of Chemistry.
Solute Studies on E. Coli Alkaline Phosphatase
Authors: Lownik Joseph , Gries Theodore
Published: June 2021

Alkaline phosphatase catalyzes the hydrolysis of phosphate ester bonds, which results in the removal of phosphate from many biologically relevant molecules (i.e. DNA, proteins, carbohydrates). This enzyme functions in a wide variety of organisms ranging from bacteria to mammals and has in vitro applications for genetic engineering. Numerous kinetic and crystal structure studies over the past fifty years have revealed many details of the alkaline phosphatase catalytic mechanism. However, few studies have investigated the solution‐phase conformational changes that accompany/drive this catalytic mechanism. Urea has long been used as a denaturant of proteins at concentrations above 4M. At low concentrations (0 to 3 M), urea can be used as a probe to detect changes in the amount of solution‐exposed amide surface. The activity of the alkaline phosphatase was examined at various, low concentrations of urea. We present the first quantitative analysis of solution‐phase conformational changes during the E. coli alkaline phosphatase mechanism.
Protease Sensitivity Map of the Highly Structured Heterodimerization Domain (HD) of the Human Notch 2 Receptor in the Presence and Absence of the Furin Cleavage Loop
Authors: Cheng Catherine Yanan , Cabral Kimberly S , Khan Aliya , Vardar‐Ulu Didem
Published: June 2021

Notch receptors of the highly conserved signaling pathway regulate cell fate decisions in metazoans. Normal activation of the receptor depends on a tightly regulated, ligand dependent, proteolytic cleavage within the extracellular Heterodimerization Domain (HD). Aberrant Notch signaling is associated with a multitude of diseases. The HD consists of an intertwined α/β sandwich around a conserved hydrophobic core, a hotspot for many disease‐inducing point mutations. HD harbors two of the three Notch specific proteolytic cleavage sites: S1, cleaved by a furin‐like protease during receptor maturation, and S2, by an ADAM‐type metalloprotease rendering the receptor susceptible to the activating γ‐secretase cleavage in the membrane. Here, we present our study on the accessibility of specific protease sites within the human Notch2 HD in the presence and absence of the unstructured loop around the S1 site. We used limited proteolysis with trypsin and chymotrypsin followed by Matrix‐assisted Laser Desorption Ionization‐Time of Flight mass fingerprinting and C18 reverse phase Liquid Chromatography to create a relative protease susceptibility map for HD and compared the experimental results to the structure based predictions. Together with other stability data on specific HD mutants, the presented information provides further insight into the mechanism of Notch activation both in the normal and diseased states.
Edman sequencing and amino acid analysis in the proteomic age
Authors: Williams Nancy , Pineda F. , Lam T.T. , Bruce C. , Bingham J. , Hodsdon M. , Khatri A. , Loll P. , McNeill L. , Mootien S. , Nathan C. , Schatz D. , Sheptovitsky Y. , Yamakoshi Y. , Crawford J.
Published: June 2021

In an era when large scale gene expression analysis and MS/proteomics are playing increasingly important roles, our review of 148 samples sequenced last year reveals that Edman degradation has evolved from being used to elucidate complete protein sequences to fulfilling several roles for which it is uniquely well suited. These include determining N‐termini of limited cleavage products and constructs used in structure/function and biophysical studies, and of proteins whose start sequences are not well predicted; and for QC of recombinant proteins (e.g., of antigens used to produce vaccines against tuberculosis). The demand for Edman sequencing may expand as emphasis shifts from completing genome sequences to identifying and understanding protein functions, interactions, and post‐translational modifications. In addition, our review of 1,558 amino acid analysis (AAA) samples indicates that hydrolysis/AAA is used to determine the accurate protein/peptide concentrations needed for quantitative biophysical studies and to match protein amounts for comparative proteome analyses such as iTRAQ, to verify the frequency of incorporation of modifications, and the compositions of synthetic and naturally occurring peptides (e.g., differentiating between isobaric Ile/Leu in peptides sequenced by de novo MS/MS). We also will report on preliminary LC‐MRM AAA studies.
A click chemistry‐mediated approach to understanding survivin:caspase‐9 protein‐protein interactions
Authors: Bishop Stephanie , Lampe Jed N
Published: June 2021

Survivin, a 16.5 kDa mammalian cytosolic protein, is upregulated in multiple cancers and is a promising therapeutic target. As its name implies, survivin facilitates cell survival by inhibiting apoptosis. Survivin is thought to inhibit the apoptotic initiator caspase‐9, however, the specific mechanisms of and amino acids involved in caspase‐9 inhibition have yet to be fully elucidated. Identifying the amino acid residues involved in the interaction between survivin and caspase‐9 will facilitate the rational design of small molecule protein‐protein interaction inhibitors. Click chemistry provides a facile approach to covalently link disparate azide‐ and terminal alkyne‐ containing molecules, thereby allowing for site‐specific incorporation of a fluorescent tag into a polypeptide sequence. Using site‐directed mutagenesis with the unnatural amino acid para‐azidophenylalanine (paF), we synthesized an azide‐containing mutant survivin protein. We then used copper‐free click chemistry to covalently modify the mutant survivin azide with an alkyne‐containing fluorophore dibenzylcyclooctyne‐488 (DBCO488) to generate a triazole‐containing cycloadducted product. Using western blotting, UV illumination, and Coomassie blue staining, we demonstrated the ability to site‐specifically cycloadduct the para‐azidophenylalanine in the survivin protein to the fluorescent cyclooctyne, DBCO488. This technology will now enable us to perform fluorescence analyses of the survivin:caspase‐9 complex in order to identify the specific amino acid residues involved in this interaction. This novel approach to monitor protein‐protein interactions holds untapped potential to improve rational drug design for survivin:caspase‐9‐targeted anticancer therapeutic agents.

Funding: Environmental Toxicology Training Program 5T32ES007079–32 (SCB), COBRE P20 RR021940‐06 (JNL), R01 GM099959‐01 (PI, Collaborator John Karanicolas)
Engineering Stabilized Variants of the NEMO N‐terminal Domain Coiled‐Coil
Authors: Zhou Li , Whitty Adrian
Published: June 2021

NF‐κB essential modulator (NEMO) is a subunit of the Inhibitor of κB Kinase (IKK) complex that regulates NF‐κB signaling. Inhibiting the protein‐protein interaction between NEMO and its ligand IKKβ represents a potential approach to treating inflammatory diseases and certain types of cancer. Rushe. et. al. (Structure, 16:798, 2008) reported that the IKKα/β binding domain fragment NEMO44 111 was disordered in the absence of IKKβ. To understand the structure and stability of the NEMO N‐terminal domain we have prepared several variants of NEMO N‐terminal domain by mutating all cysteine residues to alanine, or retaining just Cys54, or retaining Cys54 plus an additional engineered cysteine at position 107. All variants were expressed as His‐SUMO fusions containing a TEV cleavage site, purified by Ni/NTA chromatography, and then cleaved with TEV protease. We evaluated the binding affinity of each construct for IKKβ in a fluorescence anisotropy binding assay, and their structure and stability by circular dichroism spectroscopy and thermal, and chemical denaturation, under reducing and nonreducing conditions. The results address whether the N‐terminal domain of NEMO intrinsically disordered, define the role of Cys54 in the stability of the NEMO dimer, and identify stabilized forms of NEMO N‐terminal domain for crystallography.
Structural and sequence analyses of an unusual bacteriophytochrome from R. palustris
Authors: Missaghian Nima , Hernandez Cynthia N , Varela Angela E. , Marcus Marcella M. , King Rachel , Schirmer Aaron E. , Stojkovic Emina A.
Published: June 2021

Bacteriophytochromes (Bphs) are bacterial red‐light photoreceptors found in photosynthetic and non‐photosynthetic bacteria. Bphs undergo reversible photoconversion between two distinct red (Pr) and far‐red (Pfr) light absorbing states. An organic cofactor, biliverdin (BV) is required for Pr/Pfr photoconversion. Bphs are composed of a photosensory core (PC) including PAS, GAF and PHY domains and an effector domain, usually a histidine kinase. We report on the preliminary crystal structure of the unusual Bph from Rhodopseudomonas palustris, RpBphP3 (P3) PC bound to BV in the Pr state. Unlike classical Bphs, P3 photoconverts between Pr and near‐red (Pnr) states. P3 crystals (30–60 □m in all dimensions) yielded diffraction at 100 K to a maximum resolution of 2.8 Å. P3 crystals are in P212121 space group with unit cell dimensions of a=155.92 Å, b=185.32 Å, and c=199.53 Å. The initial P3 structure was determined with the molecular replacement (MR) method implemented in HKL3000 using the crystal structure of P3 lacking PHY domain (PDB ID code 2OOL) as search model. P3 crystallized as a dimer, which is physiologically relevant oligomeric state of the protein. Informed by structural and sequence analyses, we identify amino acids in the BV‐binding pocket of P3 and at the dimer interface that play a role in the unusual photochemistry and fluorescence of this protein.
The Anti‐Proliferative Properties of Tradescantia zebrina
Authors: Moehring Danielle Marie
Published: June 2021

Tradescantia zebrina (Tz) is a common plant species that has a wide variety of medicinal purposes worldwide. Its historical uses to treat skin conditions and common colds along with its many other medicinal uses suggest that Tz extracts may express anticancerous properties that could possibly inhibit the growth of certain cancer cells. To test this hypothesis, two cancer cell lines, A‐549 lung carcinoma cells and SCC‐13y malignant keratinocyte cells, were treated with an aqueous and a methanol Tz extract, dissolved in either DMSO or sterile H2O. Cell numbers were counted three separate times over a five day period to determine whether or not there was any growth inhibition and to help create growth curves. A control test was also performed on each of the cell lines, where the cells were treated with sterile water or DMSO instead of extract, allowing their growth to continue uninterrupted. When comparing the results from both the control and experimental tests, it was found that cellular growth had decreased after the addition of the Tz treatment. A non‐cancerous cell line of HFF‐1 human foreskin fibroblasts cells was also tested on with the Tz treatment to determine the relative toxicity of the extract. This study shows the inhibitive effects of Tradescantia zebrina extracts on cancerous and non‐cancerous cells, allowing further, more in depth research to be conducted on a variety of cell lines.
Mitochondria‐targeted antioxidant mitoTEMPO inhibits glycolysis and induces melanoma cell death by blocking ROSsensitive survival and metabolic pathways
Authors: Nazarewicz Rafal R , Dikalova Anna E , Bikineyeva Alfiya T , Ivanov Sergey V , Dikalov Sergey I
Published: June 2021

Superoxide (O2• ) plays an important role in regulation of cell proliferation and cell survival. It also stimulates malignant transformation and glycolysis. We have hypothesized that scavenging of mitochondrial O2• in cancer cells selectively inhibits redox sensitive regulation of metabolic and cell survival pathways resulting in cell death. In order to test this hypothesis we have studied direct effect of mitochondria‐targeted superoxide scavenger mitoTEMPO on B16‐F0 mouse melanoma cells and tumor growth in nude mice model of human melanoma using A375 cells. In this work, for the first time, we show that mitochondria‐targeted SOD mimetic mitoTEMPO inhibited cell growth, reduced viability and induced apoptosis in melanoma cells but did not affect nonmalignant skin fibroblasts. Treatment of melanoma cells with mitoTEMPO significantly diminished mitochondrial ROS, inhibited redox dependent Akt and Erk, restored activity of mitochondrial pyruvate dehydrogenase, reduced HIF1‐α and lactate dehydrogenase expression. These changes in signaling events caused metabolic switch from glycolysis to mitochondrial metabolism. Suppression of glycolysis in mitoTEMPO treated melanoma cells resulted in significant drop of cellular ATP and induced cancer cell death. Scavenging of mitochondrial O2• effectively suppresses growth of established tumor in the mouse model of human melanoma. Our data therefore support hypothesis that anticancer activity of mitochondria‐targeted antioxidants is likely mediated by inhibition of redox‐sensitive cell signaling and metabolic changes leading to reduced cancer cell survival. We suggest that mitochondrial superoxide is a novel and promising pharmacological target to treat melanoma.