bluuebunny/crossref_metadata_2025 huggingface dataset:
CKD712 promotes wound closure by producing VEGF through AMPK‐dependent HO‐1 induction
Authors: Jang Hwajin , Park Eunjung , Kim Hyejung , Chang Kichurl
Published: June 2021

Given the importance of vascular endothelial growth factor (VEGF) and heme oxygenase (HO)‐1 in wound healing, the present study was tested our hypothesis that CKD712, a strong HO‐1 inducer, produces VEGF through HO‐1 induction for stimulating wound healing. Results indicated that CKD712 significantly increased VEGF production and accelerated migration of human dermal fibroblast (HDF) where addition of anti‐VEGF antibody antagonized the effect of promoting cell migration. Both AMPK inhibitor (compound C) and HO‐1 inhibitor (SnPPIX) but not inhibitors of MAPKs, PI3K, and PKC significantly reduced the production of VEGF by CKD712. Interestingly, SnPPIX inhibited HO‐1 expression but not p‐AMPK by CKD712. By contrast, compound C inhibited both p‐AMPK and HO‐1 induction by CKD712, indicating that AMPK controls HO‐1 induction. Moreover, CKD712 decreased HO‐1 induction without affecting the expression of p‐AMPK in siHO‐1 transfection but it failed to induce HO‐1 induction in siAMPKƒÑ1‐ transfected cells, supporting that AMPK is involved in HO‐1 induction by CKD712. In addition, administration of CKD712 significantly shortened the time of wound closure in SnPP IX‐sensitive manner in full‐thickness skin wound mice model. Taken together, we concluded that CKD712 accelerates cutaneous wound healing, at least, by production of VEGF through AMPK‐dependent HO‐1 induction.

Grant Funding Source: NRF (03‐2010‐0298)
Liganded thyroid hormone receptor induces nucleosome removal and histone modifications to activate transcription during adult stem cell development
Authors: Shi Yun-Bo
Published: June 2021

Thyroid hormone (T3) plays important roles in regulating vertebrate development and pathogenesis. We are using T3‐ dependent Xenopus metamorphosis as a model to study how T3 regulates transcription during development. T3 exerts its metamorphic effects through T3 receptors (TRs). TRs recruit, in a T3‐dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during development is largely unknown. Here, we analyzed histone modifications at T3 target genes during intestinal metamorphosis, a process that involves de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.
Alternative splicing of key survival genes during adipogenesis
Authors: Patel Niketa A , Apostolatos Andre , Li Pengfei , Watson James , Cooper Denise
Published: June 2021

Increased proliferation and differentiation of pre‐adipocytes to mature adipocytes (adipogenesis) within the fat tissues are central to obesity. 3T3‐L1 cell differentiation provides a model parallel to mouse adipogenesis. Pre‐adipocytes undergo apoptosis while mature adipocytes are resistant to apoptosis. The basis for this is poorly understood. Here we demonstrate that terminal adipocyte differentiation is accompanied by alternative expression of apoptosis genes. Our data demonstrate that during adipocyte differentiation between days 4–6, expression of Bcl2 and Bcl‐x, caspase 9 and PKCδ alternatively spliced variants change to its pro‐survival variants along with increases in pBAD, pPTEN and pAKT. Using overexpression and knockdown experiments, we elucidate the link between these genes, adipogenesis and apoptosis. PKCδII, a serine/threonine protein kinase promotes cell survival. Our data points to PKCδII as the signaling kinase upstream of Bcl2‐mediated survival pathway. Natural compounds such as resveratrol and curcumin exhibit anti‐adipogenic activities and promote apoptosis in adipocytes. Our data shows that they inhibit PKCδII expression. Hence, we have identified the intracellular target which is central to pro‐survival pathways in adipocytes that may be modulated to induce anti‐adipogenic effects via splicing. Supported by VA Merit Review Grant (NAP)
Functional linkage of Retinoblastoma protein family turnover and potency during mouse embryonic stem cell development
Authors: Henry Ronald William , Arnosti David N. , Knott Jason G. , Sengupta Satyaki
Published: June 2021

In embryonic stem (ES) cells, the Retinoblastoma family of proteins is held in a biochemically inactive state through robust cyclin/cdk‐mediated phosphorylation. However, at the onset of differentiation, diminished cyclin/cdk activity contributes to the establishment of functional RB/p107/p130 pathways and regulated cell cycle control. We report that the invocation of pocket protein function during ES cell differentiation is also correlated with decreased repressor abundance, suggesting that repressor potency is linked to turnover. Indeed, wild type p107 levels were diminished by pharmacological inhibition of cyclin/cdk activity while mutant p107 lacking an evolutionarily conserved instability element within its C‐terminal regulatory domain was refractory to cyclin/cdk modulation. Select amino acid substitutions within this instability element increased p107 abundance and phosphorylation, but crippled p107 repressor function. The instability elements of both p107 and p130 can direct proteasome‐mediated turnover when fused to heterologous reporter proteins indicating that these elements can function as autonomous degrons. Importantly, mutations within the p130 degron have been observed in human lung carcinoma patients indicating an important role for the regulation of degron function in human disease. This research was supported by the National Institutes of Health.
Identification of human Myofibril‐inducing RNA
Authors: Kochegarov Andrei , Moses Ashley , Lian Will , Hanna Michael C , Lemanski Larry F
Published: June 2021

The axolotl, Ambystoma mexicanum, carries a c recessive lethal mutation that prevents normal embryonic heart development. In the heart of mutant embryos, expression of tropomyosin is reduced, the development of cardiac sarcomeres fails and the heart does not contract. Myofibril‐Inducing RNA (MIR) from anterior endoderm of normal axolotl embryos restores expression of tropomysin, promotes the formation of sarcomeric myofibrils and induces heart contractions. RNA from fetal and adult human hearts also rescues the axolotl embryonic mutant hearts. To identify the active RNA(s) in humans, we cloned the human heart RNAs and created a RNA library. Two human RNA clones were found to promote mutant axolotl heart development. The hearts treated with these RNAs beat rhythmically, and immunocytochemistry reveals tropomyosin expression and myofibril organization. The RNAs were sequenced; and interestingly, showed no sequence homology to the axolotl MIR. However, two active portions of the secondary structure of the axolotl MIR revealed significant similarities in secondary structure to the human RNA. We hypothesize that the strikingly similar areas of secondary structure in the human and axolotl RNAs are responsible for the rescue phenomenon in the mutant axolotl hearts and may be essential for normal heart development and cardiac myogenesis in all vertebrates.
Expression of Eph Receptors and their Ligands in Palate
Authors: Serrano Maria J , Miguel Symone San , Sachar Ashneet , Svoboda Kathy , Benson M Douglas
Published: June 2021

Cleft palate is one of the most common birth defects. Understanding of the molecular events in palate development is a prerequisite to more effective treatment of this condition. The hard palate of humans and mice forms from shelves of mesenchyme covered with a layer of epithelial cells. These shelves adhere at the midline to form the midline epithelial seam (MES). It has been well understood that epithelial to mesenchymal transition (EMT) and apoptosis in the MES require Transforming Growth Factor β3 (TGFβ3). Eph receptor tyrosine kinases and their ephrin ligands are responsible for many contact‐mediated developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). We discovered that ephrin‐B reverse signaling is required for palate fusion and is sufficient to do so in the absence of TGFβ3 in chicken palates. This leads us to propose that ephrin reverse signaling directs palatal EMT and fusion. To find out, we dissected palatal shelves from chicken and mice at different embryonic stages of fusion. We isolated RNA from these tissues and performed reverse transcription‐PCR for a panel of Ephs and ephrins. We found that all but a few of those genes were present in both chicken and mouse palatal tissues. Our future work will spatially locate the mRNA to specific cell layers.

Grant Funding Source : March of Dimes 6‐FY06‐321
Role of long noncoding RNAs in Polycomb‐mediated gene silencing
Authors: Zhao Crystal , Manakov Sergei
Published: June 2021

Recent evidence suggests that long non‐protein coding RNA (lncRNA) acts as a flexible scaffold to bind and guide the right combination of proteins to a specific genomic location to regulate gene expression. Previously, we have identified lncRNAs as key cofactors for histone methyltransferase, Polycomb Repressive Complex 2 (PRC2), in transcriptionally suppressing genes functioning in X‐inactivation. Furthermore, we discovered over 9000 PRC2 interacting RNAs in mouse embryonic stem cells (mESCs). Interestingly, highly enriched RNA peaks often locate at the 5′end non‐coding regions, including introns and 5′‐UTRs, of protein coding genes. Genes with such peaks are often highly expressed in mESCs but decrease significantly in fibroblasts, suggesting these genes are not silenced but are “poised” for silencing. This observation suggests PRC2‐RNA interaction may be important for silencing gene expression upon mESC differentiation but is not essential for maintaining mESC in an undifferentiated state. Indeed, PRC2 deficient mESCs can self‐renewal and stay undifferentiated. However, these cells cannot be differentiated into specific lineages, such as neuronal precursors. We conclude noncoding regions of a coding RNA play an essential role in PRC2‐mediated gene silencing upon mESC differentiation, providing a novel mechanism of gene regulation by noncoding elements during development.
Kinetics of tRNA Folding Monitored by Aminoacylation
Authors: Bhaskaran Hari , Rodriguez-Hernandez Annia , Perona John J
Published: June 2021

We describe a strategy for tracking Mg2+‐initiated folding of 32P‐labeled tRNA molecules to their native structures based on capacity for aminoacylation by the cognate aminoacyl‐tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by addition of tRNA synthetase and further incubation, yields a rapid burst of aminoacyl‐tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. Application of the approach to study the influence of post‐transcriptional modifications in folding of E. coli tRNA1 Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the apparent folding pathway. This assay allows the use of 32P‐labeled tRNA in integrated studies combining folding, post‐transcriptional processing and aminoacylation reactions. The assay also allows tracking folding of mitochondrial tRNAs, the native structures of many of which are poorly characterized.
Analysis of Junctions in RNA with Coaxial Stacked Helices
Authors: Fuhr Katherine Niccole , Laing Christian , Kim Namhee , Schlick Tamar
Published: June 2021

The structure of RNA is hierarchical and complex. The secondary and tertiary structure is related to the function of RNA. In order to help predict the structure of RNA, analysis of motifs (commonly occurring interactions and sequences) must be done. In coaxial stacked helices of 3–10 way junctions, we measured the length (number of nucleotides) of the single strand, Ji/(i+1), which connects the two helices, and predicted that as the length of the single strand increases, that the distance between the midpoints of the terminal base pairs of the helices will also increase.

Using the C1’ atom of each terminal base pair, we measure the respective midpoints and calculate the overall Euclidean distance. Use of Protein Data Bank data allowed us to gather the Cartesian coordinates of the atoms for use in our calculations. We analyzed junctions with degrees 3–10.

We found that 3 and 4‐way junctions are highly correlated to the length of the single strand. We also found that frequency of coaxial stacks in junctions do not signify that there will be high correlation in a linear relationship. Outliers in the data often were involved in other interactions, such as RNA‐RNA complexes, RNA‐ligand complexes, and other factors such as unusual conformations.

Further analysis of 3 and 4‐way junctions according to their respective family type gave us the normal distributions of lengths and how they correspond to the previously set parameters of the family. The distributions can act as a guide in synthesis for creating certain motifs in RNA. These results can be used in prediction software of RNA and can be further investigated as more RNA is discovered.
Characterizing the role of Puf3 in sexual development of Cryptococcus neoformans
Authors: Kaur Jan Naseer , Panepinto John C.
Published: June 2021

Mating in the human fungal pathogen Cryptococcus neoformans is one mechanism by which spores, the likely infectious propagules are produced. The multistep sexual development process involves mate recognition followed by plasmogamy, filamentation, karyogamy and meiosis ultimately resulting in the basidiospore production. The Puf (Pumilio and FBF) family of RNA binding proteins is known to bind to 3′UTRs of mRNAs and is associated with regulatory functions including translation, stabilization and localization of transcripts. Scanning of 3′UTRs of all the annotated genes of C. neoformans revealed Puf3 binding consensus sequence in the transcripts involved in pheromone signaling cascade. This led us to hypothesize that Puf3 regulates sexual differentiation of C. neoformans. To test this hypothesis we performed bilateral mating assays for wild type and puf3Δ mutants, lacking the Pumilio domain RNA binding protein Puf3. When equal number of opposite mating cells of puf3Δ mutants was cocultured on V8 agar, they were defective in filamentation as compared to the wild type cross. To determine the ability of puf3Δ mutants to produce pheromones and recognize opposite mating partner, northern blot analysis was done. The RNA obtained from mating colonies of puf3Δ mutants bilateral cross was probed for mating pheromone MFα in comparison to the wild type cross (H99α × KN99a ) and the induction of MFα was found to be normal in puf3Δ mutants mating. Our result suggests that mate recognition does occur when puf3Δ mutants are crossed. Future studies will assess the ability of puf3Δ mutants to fuse and undergo post fusion hyphal extension. Verifying the Puf3 binding to mating pheromone signaling transcripts and analyzing the transcriptional level and stability of these transcripts will lead us to define the role of Puf3 in C. neoformans.
Activity induces Arc mRNA degradation that is dependent upon translation and NMDA receptor activation
Authors: Farris Shannon L , Steward Oswald
Published: June 2021

The mRNA for the immediate early gene Arc is induced by synaptic activity or learning experiences. Arc is unique among neuronal mRNAs in that its newly synthesized transcripts are transported into dendrites and accumulate at activated synapses. Localization of Arc mRNA at active synapses is accompanied by Arc mRNA depletion in inactive dendritic domains. Here we assess whether this is due to activity‐dependent degradation. We used an in vivo paradigm where Arc transcription and transport into dendrites is induced by electroconvulsive seizure and subsequent targeting to active synapses is achieved by high frequency stimulation of the medial perforant path. Local blockade of translation with cycloheximide (CHX) led to an overall increase in Arc mRNA, suggesting that Arc mRNA degradation is translation‐dependent. Increases in Arc mRNA following CHX treatment were greatest in the zone of activated synapses, suggesting that degradation is occurring primarily where Arc mRNA is most actively translated. Targeting to active synapses and depletion in inactive dendritic domains was blocked by NMDA receptor antagonist MK801, indicating that NMDA receptors play a role in localization and translation‐dependent mechanisms regulating Arc mRNA. These data indicate that Arc mRNAs are subject to degradation mechanisms that are enhanced by activity and dependent upon active translation and NMDAR activation.[NS12333 to OS]
Mechanism of processive and cap‐stimulated mRNA poly(A) tail degradation
Authors: Virtanen Anders , Henriksson Niklas , Nilsson Per , Lindell Magnus
Published: June 2021

Poly(A)‐specific ribonuclease (PARN) is an oligomeric, processive and cap‐interacting exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN is a key regulator of AU‐rich element (ARE) containing mRNAs encoding proto‐oncogenes and cytokines. Here, we propose a mechanistic framework for PARN action. The model has been experimentally tested and is supported by structural evidence. In the model the RNA recognition motif (RRM) of PARN pushes the poly(A) tail into the active site, which simultaneously pulls the substrate into the hydrolytic site where the movement of a catalytically essential histidine residue is coordinated with the pushing of the RRM. The model provides a mechanistic explanation for how the cap‐structure of the mRNA can stimulate poly(A) tail hydrolysis. We have also investigated the functional significance of divalent metal ions in the active site and revealed that divalent metal ions are required for both hydrolysis and substrate translocation in the active site. Our data imply that three divalent metal ions are required for proper action. Two ions participate in hydrolysis while the third plays a key role during translocation. The generality of this proposal in relationship to other processive enzymes participating in cleavage or formation of phosphodiester bonds will be discussed.

Supported by the Swedish Research Council and VR Linnaeus Support.
Discrete LIN28 binding sites in mature messenger RNA sequences reveals regulation of a network of splicing factors and downstream alternative splicing patterns
Authors: Wilbert Melissa L , Huelga Stephanie C , Vu Anthony Q , Stark Thomas J , Massirer Katlin B , Chen Stella x , Liang Tiffany Y , Yeo Gene W
Published: June 2021

LIN28 is a conserved RNA binding protein important for pluripotency, reprogramming and oncogenesis. Studies thus far have focused primarily on the roles of LIN28 through its regulation of the microRNA (miRNA) let‐7. Here, our goal was to identify and characterize the network of mature messenger RNA targets of LIN28 in order to explain its let‐7 independent regulatory mechanisms. Using UV cross‐linking and immunoprecipitation of the LIN28 protein followed by high‐throughput sequencing (CLIP‐seq) we have identified direct LIN28 binding regions on target RNAs in human embryonic stem cells and somatic cells expressing exogenous LIN28. We found that in addition to hundreds of miRNAs, LIN28 directly binds to thousands of human mRNA genes, including LIN28 itself and dozens of splicing regulators. Furthermore, we confirm in a set of targets that this binding results in an increase in translation. Finally, splicing‐sensitive junction arrays demonstrated that LIN28 over‐expression causes widespread downstream alternative splicing differences. These findings show that LIN28 contributes to cellular homeostasis via direct mRNA interactions and downstream AS events, alterations of which may result in cancer and differentiation.
Regulation of exclusively testis‐expressed microRNA‐469 by Gonadotropin‐Regulated Testicular RNA Helicase controls male germ cells development
Authors: Dai L-S , Tsai-Morris C. H. , Sato H. , Villar J. , Zhang J-B , Dufau M. L.
Published: June 2021

Gonadotropin‐regulated testicular RNA helicase (GRTH/DDX25), a testis‐specific member of the DEAD‐box family, is an essential post‐transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNAs expression was observed in GRTH−/− mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis‐specific miR‐469, which is increased in the GRTH−/− mice. Further analysis demonstrated that miR‐469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary‐miRNAs and the expression levels of Drosha and DGCR8 (mRNA and protein) were increased significantly in the GRTH−/− mice. miR‐469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA biogenesis and consequently their function during spermatogenesis. (Supported by the IRP, NICHD)
The ER Lumenal Chaperone, Lhs1/GRP170, Plays a Unique Role in the Degradation of the Epithelial Sodium Channel
Authors: Buck Teresa M , Plavchak Lindsay , Kashlan Ossama , Kleyman Thomas , Brodsky Jeffrey
Published: June 2021

The Epithelial Sodium Channel (ENaC) is composed of three homologous subunits – α, β, and γ – that assemble in the endoplasmic reticulum (ER) to form the mature ENaC channel. However, channel maturation is inefficient, thus targeting the channel subunits for Endoplasmic Reticulum Associated Degradation (ERAD). To characterize the ENaC degradation pathway, a yeast ENaC expression system was developed. Using this system, we found that Jem1 and Scj1, two ER lumenal Hsp40 chaperones, play a role in ENaC degradation. Jem1 and Scj1 act as cochaperones for the ER lumenal Hsp70, BiP, but, surprisingly BiP does not play a role in ENaC degradation. Therefore, we hypothesized that another ER chaperone with an Hsp70‐like domain, Lhs1, may be involved. We found that Lhs1 facilitates αENaC subunit degradation, but that Lhs1 ATPase activity is dispensable for ENaC degradation. This result further supports our conclusion that the BiP cochaperones are acting in a BiP independent fashion. Our data also indicate the human Lhs1 homolog, GRP170, affects ENaC biogenesis in an oocyte system. In conclusion, we have identified a new ER chaperone as a novel effector of αENaC biogenesis. Funded in part by K01: DK090195.
The Role of the Vacuolar H+‐ATPase Subunit a Isoforms in Secretion and Activation of Cathepsin L in Human Breast Cancer Cells
Authors: Folkers Kelly McBride , Hinton Ayana Makeda
Published: June 2021

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231, and may contribute to the metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating local tumor invasion. We are investigating the role of subunit a isoforms in secretion and activation of cathepsin L. The 4 subunit a isoforms are responsible for cellular localization of the V‐ATPase. siRNAs were used to reduce mRNA expression of each isoform. RT PCR showed that each isoform was successfully and specifically knocked down. Immunoblotting and activity assays are being used to analyze cathepsin L expression and activity in conditioned media of MB231 cells. Knockdown of the a4 isoform decreased the level of processed cathepsin L in conditioned media, but not the unprocessed form. Activity assays will be used to confirm that reduction of a4 affects the activity of secreted cathepsin L. Our previous findings indicate a4 targets V‐ATPases to the cell surface, thus our data support their role in activation of secreted cathepsin L in the tumor microenvironment of MB231 cells. Funded by DU Anderson Fellowship.
Identification and Function of Leash Forming Residues in the FoF1 ATP Synthase Molecular Motor Using Single Molecule Measurements
Authors: Frasch Wayne D , Martin James L.. , Hudson Jennifer
Published: June 2021

In single molecule rotation measurements, the transient dwells in the proteolipid ring rotation of E. coli FoF1‐ATP synthase were eliminated by mutation of either aE196L or cR50L to subunit a and subunit c, respectively, of the Fo motor. These data indicate the involvement of these residues in the formation of the Fo motor leash that allows rotary motion of the c‐ring to a limit of ~36° while engaged. The rate of oxidative phosphorylation dependent growth observed with these mutations was about one half of that observed with wild type. The ability of ATPase dependent or NADH‐dependent proton gradient formation across inverted membrane vesicles from E. coli was measured by ACMA quenching. Mutations to either residue aE196 or cR50 that altered the ability to form a salt bridge between these subunits altered the rate and extent of proton gradient formation induced by either ATP or NADH. However, none of these mutations decreased the rate of ATP hydrolysis activity of the FoF1 in the inverted membrane vesicles significantly. These results are consistent with the hypothesis that the ability to form the leash between Fo subunits a and c biases rotation of the c‐ring in the ATP synthesis direction. This work was supported by NIH grant 5R01GM097510 to WDF.
Investigation of 2‐Fold Disorder of Inhibitors and Relative Potency by Crystallizations of HIV‐1 Protease in Ritonavir and Saquinavir Mixtures
Authors: Olajuyigbe Folasade M. , Demitri Nicola , Geremia Silvano
Published: June 2021

HIV‐1 protease (PR) was cocrystallized in competitive mixtures of saquinavir (SQV) and ritonavir (RTV) in an attempt to compare the relative potencies of inhibitors using a crystallographic approach. The mixture ratio of RTV/SQV was in the range of 1:1 to 50:1. The crystal form obtained with 1:1 and 5:1 ratios of RTV/SQV was monoclinic, while ratios 15:1 and 50:1 gave orthorhombic crystal form. The four crystal structures of PR/RTV/SQV were solved at 1.03, 1.12, 1.25, and 1.72 Å resolutions. The X‐ray crystal structures reveal that the crystal forms are dependent on the occupancy of either SQV or RTV in the active site of PR. At low RTV/SQV concentrations, PR/SQV complex is dominant, and at higher ratios, PR/RTV is found. The absence of a crystal structure having both inhibitors statistically disordered in the catalytic site of PR suggests that the two protein complexes are sufficiently different in properties to be discriminated in crystal growth process. The X‐ray structures of the dimeric enzyme with C2 pseudosymmetry show a 2‐fold‐disorder phenomenon for the SQV, while the RTV inhibitor is detected in a single orientation. The dominancy of PR/SQV crystal form at an equimolar mixture of inhibitor and the presence/absence of the 2‐fold disorder of inhibitors have given new insight into the relative potency of these drugs and both suggest a higher potency of SQV with respect to RTV.

Research supported by Schlumberger FFTF.
An equation to model fluorescent probe displacement by non‐fluorescent competitive ligands
Authors: Beckmann Joe D. , Martin Emily D. , Gaffney Kristen
Published: June 2021

Measurement of ligand binding to a receptor to evaluate Kd values is fundamental. If binding is spectrally silent, then a probe with dynamic optical properties can be employed. Such is the case in our studies of nucleotides binding to phenol sulfotransferase, SULT1A1, one such enzyme that requires 3’‐phosphoadenosine‐ 5’‐phosphosulfate (PAPS). Whereas binding of PAP(S) and other nucleotides is spectrally invisible, 3’‐trinitrophenylated‐adenylates (e.g. TNP‐AMP) form a fluorescent SULT:TNP‐AXP complex. These TNP probes have seen considerable applications for kinases and other ATP‐binding proteins. However, many published analyses of probe displacement by biological nucleotides are confounded by assumptions and questionable analyses. We have derived an equation that predicts binding constants by adequately modeling competition fluorimetric titrations, and have applied it to both published works and to our SULT analyses. The procedure requires titration with the probe and determination of its Kd value, followed by competitive titration with another ligand. Parameters required or derived are: total receptor, probe, and ligand concentrations; Kd (probe), Kd (ligand), Fmax, and observed F values. We see agreement between Kd values obtained by this method and by kinetic and isothermal titration methods, for the binding of PAP to recombinant bovine SULT1A1.
A kinetic approach to distinguish between catalytic mechanisms of protein kinase autophosphorylation
Authors: Wang Jue , Wu Jia-Wei , Wang Zhi-Xin
Published: June 2021

Protein kinase autoactivation, via phosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation‐dependent signaling cascades. Their molecular mechanisms can be simply categorized to intra‐molecular(in cis) or inter‐molecular(in trans). Specifically, the trans‐autophosphorylation kinases vary in their initiation modes. In this study, we give a full analysis for the four initiation modes in trans‐autophosphorylation and develop a kinetic approach, which is based on the kinetic theory of substrate reaction during modification of enzyme activity, to quantitively distinguish them. Applying our kinetic method to p21‐activated kinase 2 kinase domain(PAK2‐KD), we identified the molecular mechanism of PAK2‐KD trans‐autophosphorylation and determined all microscopic kinetic constants for its autoactivation process, coincident with our structural studies on PAK1 kinase domain. The unphosphorylated PAK2‐KD, which is lack of structural constraints in context, could dynamically adopt both the active and inactive conformations, thus initiates the autoactivation process differently from the full‐length PAK2. Our kinetic method gives a general solution to describe distinct catalytic mechanisms of protein kinase autophosphorylation and is particular useful during the fast modification reaction.
Inhibiton of CYP 3A4 with novel synthetic coumarin and flavanone analogs
Authors: Swift David Aaron , Hopkins Nancy
Published: June 2021

Human Cytochrome P450 3A4 (CYP 3A4) is an oxidative enzyme found in hepatocytes and enterocytes and is a key agent in the metabolism of pharmaceutical drugs. CYP 3A4 is very important in the understanding of drug interaction and drug efficiency with implications in the fields of pharmacokinetics and chemotherapy. Novel synthetic analogs of a variety of Flavanones and Coumarins will be screened to test their ability to inhibit CYP 3A4 in vitro. These compounds are analogs of natural dietary products that have shown efficacy in inhibiting 3A4 or of compounds that have proven methods of inhibition in other non‐hepatic and enteroceullar p450's. The IC50 of these compounds will be tested using a rapid assay on a known flourocoumarin substrate. Then a 96 well fluorescence plate reader and computer analysis will be used to test the degree of inhibition each substance has on the substrate. By altering CYP 3A4 activity we can manipulate the amount of time drugs stay in tissue. This is particularly important with certain chemotherapy drugs that have high metabolic rates. In inhibiting CYP 3A4 we can increase the amount of time that these drugs work in tissue.
Thioredoxin1 as a novel serum marker for human ovarian cancer
Authors: Cha Mee-Kyung , Park Byung-Joon , Kim Il-Han
Published: June 2021

Thioredoxin1(Trx1), a constituent of cellular antioxidant defense systems, controls the redox state in estrogen‐dependent cells. Trx1 is suggested to play an important role in malignant promotion and progression of estrogen‐associated cancer such as ovarian and breast cancers. We have recently reported Trx1 as a potential biomarker for human breast cancer. In this study, we investigated whether serum Trx1 levels are elevated in patients with ovarian carcinoma. To clarify whether serum levels of Trx1 could be a serum marker for ovarian carcinoma, we measured the serum levels in patients with ovarian carcinoma using an ELISA, and investigated its associations with the tumor staging from I to IV. The serum Trx1 levels were significantly higher than both levels of normal control and benign disease subjects and were correlated with the cancer progress. At the cut‐off point 0.5527 at A540nm on the receiver operating characteristic curve, Trx1 could well discriminate ovarian carcinoma from normal controls with a sensitivity of 85.7%, specificity 91.8%, and area under the curve (AUC) 0.93+/−0.0203. Elevated serum levels of the Trx1 were associated with the increased cancer stage and grade. Trx1 may therefore be a helpful new biomarker for diagnosing, monitoring, and risk assessing ovarian cancer patients. *This work was financially supported by project B0009735) funded by the Ministry of Knowledge Economy of Korea.
A Statistical Analysis and Approach to Protein Surface Modeling
Authors: Doucette Luticha , Craig Paul A. , Halavin James
Published: June 2021

In studying protein‐protein interactions it is important to accurately describe the surface of the proteins, where the interactions occur. Surface can be represented in two ways in RasMol ‐ as Lee‐ Richards surfaces which are generated by rolling a solvent molecule on the surface of the protein, or as an approximation to the Lee‐Richards surface by adding pseudo‐Gaussian electron densities. The second approach is a reasonable approximation to a Lee‐Richards surface with less computational burden. Either approach allows the user to identify atoms that are on the surface of the protein. A new algorithm for surface modeling was developed to improve speed and accuracy. In this approach protein surfaces are defined by their individual atoms as data points. The algorithm was implemented first in Minitab, then converted to Python code to increase speed. The surface atoms are identified by both Minitab and Python, but there are discrepancies between the output of the two programs. Further work will be done to resolve these discrepancies, the code will be converted to GPU processing to further increase the speed, and continued analysis on the trends seen with how many atoms are found on the outside and their biological significance. Thanks to the NSF LSAMP at RIT for funding this research.
Proteomic analyses of genes regulated by heterogeneous nuclear ribonucleoproteins A/Bs in Jurkat cells
Authors: Yeh Yuan-ming , Huang Pei-Rong , Wu Chih-Ching , Wang Tzu-Chien Vant
Published: June 2021

The hnRNP A/Bs (A1, A2/B1, and A3) are the most abundant proteins in the nuclear extracts that bind specifically to G‐strand telomeric DNA. While the telomeric DNA‐binding activities of hnRNP A/Bs in vitro have been characterized in detail, the biological functions of hnRNP A/Bs in vivo remains poorly understood. In this study, we have employed RNAi knock‐down and proteomic approach to investigate the biological functions of hnRNP A/Bs. Knock‐down of hnRNP A2, but not A1 or A3, produced a profound effect on cellular proliferation in Jurkat cells. A slight increase of apoptotic cells was observed in the A2‐knock‐down cells, but not in the A1 or A3 knock‐down cells. Analysis of the proteomes in the cells singly knock‐down for hnRNP A1, A2 or A3 has identified a total of 1604 quantifiable proteins. Among the down‐regulated proteins, one was commonly regulated by A1 and A3, three were commonly regulated by A1 and A2, and three were commonly regulated by A2 and A3. A total of 19 proteins were specifically altered only in the cells knock‐down for A2. Studies are underway to investigate biological functions of A2‐regulated genes. This study was supported by research grant CMRPD1A0421.
Structural Adaptation of Microvessels in Disease States
Authors: Threlkeld Elizabeth Fay , Little Larissa , Park Jea Young , Varin Patrick , Geddes John , Sarang-Sieminski Alisha
Published: June 2021

Hypertension, diabetes, and obesity are often associated with impaired microvascular function and structural adaptation. A.R. Pries and T.W. Secomb have developed a mathematical model of structural adaptation based on known physiological responses to shear stress, circumferential stress, and metabolic demand under healthy conditions. While this model captures key aspects of microvascular remodeling, it does not explicitly incorporate signaling pathways. As altered signaling pathways are a prominent feature of many disease states, in its current state this model cannot be used to predict the effects of diseases on vessel remodeling. Using the Pries and Secomb model as a framework, we have developed a model that incorporates relevant signaling pathways. In our model, diameter changes with nitric oxide, a vasodilator, and endothelin‐1, a vasoconstrictor, which are both functions of shear stress. Wall area changes with circumferential stress as well as the growth and death of vascular smooth muscle cells. Currently our model reflects the steady state trends in vessel geometry under normal conditions. Work is ongoing to further validate the model and examine vascular remodeling in disease states. Our long term goal is to improve the understanding of vascular adaptation in disease states and to create modeling tools which provide biologically testable hypotheses for experimentalists and clinicians.
Development of a Parkinsonˈs disease model in medaka fish
Authors: Ferng Shiana J. , Gonzalez David E. , Falk Torsten , Rilo Horacio L. , Sherman Scott J.
Published: June 2021

Parkinson's disease (PD) is the 2nd most common neurodegenerative disease. Dopaminergic (DA) neurons in the substantia nigra of the brain are the most vulnerable to PD, and PD's primary symptoms only become evident when 60–70% of DA neurons have already died. Current PD treatments only provide symptomatic relief. 6‐hydroxydopamine (6‐OHDA) is a neurotoxin structurally similar to dopamine that is transported inside the brain and destroys DA neurons. The resulting PD symptoms can be identified through behavioral analysis and verification of DA neuron loss through immunohistochemical analysis in medaka fish (Oryzias latipes). Medaka are well‐suited as a PD model because they are transparent during most stages of development, produce numerous offspring, and readily absorb toxicants through their skin. To establish a PD medaka model to time and cost‐efficiently screen drugs that may slow, stop, or reverse the progression of PD, medaka were exposed to varying concentrations of 6‐OHDA through the water they swam in. The acquired PD symptoms were evidenced by a decrease in total distance swam and velocity, and an increase in angular velocity and turn angle in the 6‐OHDA‐treated medaka; these results are promising for the verification of a PD medaka model. This research was supported in part by a grant to the University of Arizona (UA) from Howard Hughes Medical Institute (#52006942) and the UA Department of Neurology.
Protein Phosphatase 1α is involved in the regulation of the thyrotropin‐releasing hormone receptor
Authors: Gehret Austin U. , Hinkle Patricia M.
Published: June 2021

G protein‐coupled receptors (GPCRs) are widely expressed transmembrane proteins involved in signal transduction of neurotransmitters, hormones and various other ligands. Their signaling is desensitized through phosphorylation and dissociation from their cognate G proteins. They are resensitized by mechanisms involving dephosphorylation, but details about the phosphatases responsible are generally lacking. We describe here the use of an siRNA‐based knock down screening approach to identify phosphatase(s) acting on a GPCR, the thyrotropin‐releasing hormone (TRH) receptor, in conjuction with phosphatase inhibitor studies. Protein phosphatase PP1α was specifically identified as critical in TRH receptor regulation. Inhibition of PP1α synthesis with an siRNA directed against the PP1α catalytic subunit and overexpression of a dominant negative mutant preserved receptor phosphorylation under conditions favoring dephosphorylation whereas overexpression of PP1α accelerated dephosphorylation. Parallel studies with PP1β and PP1γ showed no significant change on dephosphorylation. We conclude that the PP1 class of phosphatases is directly responsible for dephosphorylating the TRH receptor and likely to be involved in regulation of other GPCRs.
Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit
Authors: Smetana Juliana H C , Migueleti Deivid L , Nunes Hugo F , Kobarg Jörg , Zanchin Nilson I
Published: June 2021

PP2A is the main serine/threonine‐specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL, the methyl‐transferase LCMT‐1, and the methyl‐esterase PME‐1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. FLAG‐tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME‐1. Consistently, α4 outcompetes PME‐1 and LCMT‐1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit.
Isolation and amplification of a true lipase gene expressed during the rapeseed germinatif process
Authors: BELGUITH Hatem
Published: June 2021

Lipases are useful enzymes that are responsible for the hydrolysis of triacylglycerides and play an important role in plant growth. In this study, we report a rapid molecular method developed to amplify a partial sequence of the rapeseed (Brassica napus L.) lipase class 3 family (lip3) gene, which can be used to follow and analyze the expression of this gene during seed germination. Therefore, we conceived PCR homologous primers to amplify the active site encoding region of the lip3 family genes. Subsequently to sequence determination of the 582 bp fragment, we have deduced the Brassica napus lip3 specific primers for a nested RT‐PCR application. The deduced 194 amino acid sequence was submitted to the Genbank (1160264). It was found to share 85 % of identity with Arabidopsis thaliana lip3. The GxSxG consensus motif was found in the amplified sequence. Our result suggests that we have amplified the active site, which was found to contain the catalytic triad, particularly the active serine residue. The sequence alignment showed that the Brassica napus lip3 peptide presented a little homology with mammalian and microbial lipases. RT‐PCR analysis indicated that lip3 gene was only expressed during the Brassica napus seeds germinatif process.
Elucidation of the function of 16 newly identified genes of D. hansenii and their role in osmoadaptation
Authors: Rodriguez-Santiago Ronald Enrique , Maysonet Vivian D. , de la Cruz Wilanet , Arroyo Monica M. , Arroyo Nancy
Published: June 2021

Debaromyces hansenii, a halotolerant yeast, is a model system for the study of osmoadaptation, tolerating up to 4M NaCl. This osmotic stress has been monitored with microarrays, where 109 genes displayed a significant change in expression. Among them, 16 genes have not had their function annotated. The characterization of these genes or their protein products by various bioinformatics analysis tools is key for the determination of their role in the halotolerant properties and preparedness of D. hansenii as an extremophilic organism. These results will assist in the determination of the appropriate experimental conditions for the complete elucidation of these genes, and the analysis of the potential application of D. hansenii in environmental and agricultural biotechnology currently underway in our laboratory.
Evaluation of Immunoassay Interference Reduction Technologies
Authors: Jentz Tim
Published: June 2021

Problems with non‐specific binding and assay interferences can cause high background and loss of sensitivity in many IVD applications including ELISA, western blotting and microarrays. Interference issues can arise due to inadequate blocking of a membrane and ELISA plates, or inferior surface chemistries, or heterophilic antibodies such as; HAMA and RF. Here we examine interference reduction technologies versus in‐house and commercial products across multiple IVD applications. Interference reduction technologies were evaluated for four common assay formats: 1. Non‐specific binding was reduced in a functional ELISA using both protein containing and protein‐free blocker formulations developed for dried stability applications. These novel formulations were also able to stabilize the structure/function of the antibody in the dried state, leading to an increase in shelf life of immunoassay plates over common in‐house formulations. 2. In a traditional membrane immunoassay, western blotting results indicated a decrease in background with a set of new blocker formulations compared to common in‐house buffers. Sensitivity for the novel formulations was shown to be superior allowing for improved signal‐to‐noise for membrane immunoassay's. 3. In a standard microarray format, comparison of a novel surface chemistry coating to commercially available epoxysilane surfaces, showed less background in a protein array format. The superior slides display more distinct spots with consistently low background across the slide. 4. Finally, matrix interference and false positive/negative results caused by heterophilic antibodies were reduced by 43% compared to negative controls using a synthetic assay diluent. Solutions discussed here provide the tools necessary to develop and optimize immunoassays that are sensitive, reproducible, and robust while demonstrating the ability to reduce assay interference across multiple IVD applications.