bluuebunny/crossref_metadata_2025 huggingface dataset:
Estradiol Regulates Neuropeptidase Activity in Hypothalamic Regions Involved in Regulating Gonadotropin‐Releasing Hormone
Authors: Qiao Jana W. , Cyr Nicole E. , Bruce Lisa A. , Tetel Marc J. , Wolfson Adele J.
Published: June 2021

Thimet oligopeptidase (TOP) and prolyl endopeptidase (PEP) are neuropeptidases involved in the degradation of gonadotropin‐releasing hormone (GnRH), an important regulator of the ovarian steroid hormone estradiol (E) via the hypothalamic‐pituitary‐gonadal axis. PEP's action to remove the N‐terminal glycinamide of GnRH occurs prior to hydrolysis of the Try5‐Gly6 bond, the rate‐determining step catalyzed by TOP. E levels in turn regulate GnRH by exertion of positive and negative feedback control. TOP and PEP may potentiate GnRH effects and therefore may be regulated by E. We have previously shown that TOP and the estrogen receptor co‐localize within individual hypothalamic cells, and that levels of TOP in specific regions are down‐regulated by E. In order to determine whether E regulates TOP or PEP enzymatic activity in brain regions associated with reproduction, homogenized hypothalamic brain samples of the medial preoptic area (MPA), periaqueductal gray (PAG), and ventral medial nucleus of the hypothalamus (VMN) were collected from ovariectomized mice treated with E or oil control. Fluorometric kinetic assays were conducted to analyze differences in levels of enzymatic activity between treatments. Kinetic assay data showed that E alters TOP and PEP activity in both the VMN and mPOA, but not in the PAG. These results suggests that, in female mice, TOP and PEP are regulated by E in the hypothalamic regions involved in reproductive physiology.
Deciphering the High Mobility Group Proteins (HMGs) Code
Authors: Beltran Andrea , Garcia Benjamin
Published: June 2021

HMGs are the second most abundant proteins found in the chromatin and, like histones, have been associated with nuclear processes. HMGA is subdivided into three types of proteins: HMGA1a, HMGA1b and HMGA2. The current study investigates the effect of post‐translational modifications (PTMs) of HMGA1a and their implications on different cell processes. It is believed that there might be a PTMs code or pattern that determines cellular processes similar to the histone code, yet this remains elusive. To determine the existence of the suggested code, we isolated HMGA1a by acid extraction and purified it utilizing HPLC. Our objectives include mapping the array of modifications on HMGA1a using mass spectrometry based proteomics. To date, we have currently mapped twenty eight PTMs occurring on nineteen amino acid residues. Novel PTMs were found and also residues with more than one modification suggesting that the same residue with different PTMs might act as a switch for different biological functions, similar to histones i.e. H3K9me3 versus H3K9ac. Future work includes experiments to determine the functional characterization of the novel PTMs on HMGA1a in order to correlate them to distinct biological processes.
Enzymology of DHHC‐mediated protein palmitoylation
Authors: Jennings Benjamin Carey , Linder Maurine E
Published: June 2021

Protein S‐palmitoylation is a post‐translational modification in which a 16‐carbon fatty acid is thioester linked to a cysteine residue. It is mediated by a family of enzymes with a conserved Asp‐His‐His‐Cys sequence (DHHC). Incubation of DHHC proteins with palmitoyl‐Coenzyme A (palm‐CoA) both in the presence and absence of a protein substrate results in the DHHC protein itself being acylated. Enzyme autoacylation is a prerequisite for substrate palmitoylation. Using two approaches, we are testing the hypothesis that DHHC enzymes use a two‐step transfer mechanism where the autoacylated species represents an acyl‐enzyme intermediate.

Mutation of the DHHC cysteine residue results in loss of both enzyme and substrate acylation. Enzyme acylation is hydroxylamine‐sensitive, suggesting palmitate is attached via a thioester linkage to cysteine. In our first approach to directly monitor transfer a DHHC protein is reacted with [3H]palm‐CoA and then purified from any free [3H]palm‐CoA. The charged [3H]palm‐DHHC is then incubated with a protein substrate to determine if the radioactive acyl group is transferred. Our second approach is to use steady‐state kinetics to determine if DHHC proteins use a Bi‐Bi (ping‐pong) mechanism. A fluorescent peptide, HPLC‐based protein acyltransferase assay has been developed to achieve the higher substrate concentrations needed for kinetic analysis. Funding was provided by a pre‐doctoral fellowship from the American Heart Association (BCJ) and NIGMS (ML).
Sumoylation impacts the transcriptional activity of Nrf2 and is necessary for its localization to promyelocytic leukemia nuclear bodies
Authors: Theodore Melanie , McIntosh Deneshia , Kawai Yumiko , Arinze Ifeanyi J.
Published: June 2021

The transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2) induces transcription of a variety of genes by binding to the antioxidant response element(s) (ARE) in target gene promoters. Covalent modification(s) that may impact the biology of Nrf2 after its separation from its inhibitor protein Keap1 are not well‐delineated. Using reporter gene assays and fluorescence imaging in HepG2 cells, we have explored the role of sumoylation in the biological action of Nrf2. In the presence of the SUMO‐specific protease SENP‐1, Nrf2‐induced transcription from ARE‐driven reporter gene constructs was greatly enhanced, suggesting that sumoylation inhibits Nrf2‐induced gene transcription. Treatment with the proteasome inhibitor MG‐132 directed green fluorescent protein‐Nrf2 (GFP‐Nrf2) to promyelocytic leukemia nuclear bodies (PML‐NBs); this treatment also induced co‐localization of GFP‐Nrf2 with red fluorescent protein‐SUMO‐1 (RFP‐SUMO‐1). This localization was abrogated by co‐transfection with an expression plasmid for SENP‐1. We interpret these results to mean that Nrf2 traffics to PML‐NBs in a SUMO‐dependent manner. The results also implicate sumoylation in the transcriptional action of Nrf2.

Supported by NIH grants # SC1CA143985 and T32HL007737
Protein Phosphatase 1 controls function of Ikaros transcriptional factor and regulates chromatin remodeling during lymphocyte differentiation
Authors: Dovat Sinisa , Song Chunhua , Gurel Zafer , Payne Kimberly J , Popescu Marcela
Published: June 2021

Ikaros encodes a zinc finger protein that is essential for lymphoid development. Ikaros protein binds to the upstream regulatory elements (URE) of target genes and regulates their transcription via chromatin remodeling. We have reported that the dephosphorylation of Ikaros by protein phosphatase 1 (PP1) regulates Ikaros’ subcellular localization and protein stability. (Popescu et al. J Biol Chem 2009 284:13869). We studied the effect of PP1 on Ikaros function in primary thymocytes. Inhibition of PP1 activity abolishes the repression of Ikaros target genes during thymocyte differentiation. Co‐immunoprecipitation experiments show that Ikaros phosphomimetic mutants and Ikaros protein with a point mutation at the PP1‐interaction site: 1) do not associate with Sin3a, a component of the NuRD histone deacetylase complex; 2) have decreased DNA‐binding affinity to the URE of Ikaros target genes, and 3) are unable to repress transcription of target genes in a luciferase reporter assay. The introduction of phosphoresistant Ikaros mutations restored the association of Ikaros with Sin3a, the ability Ikaros to bind DNA, and the transcriptional repression to the level observed in wild‐type Ikaros. These results show that PP1 regulates thymocyte differentiation by controlling Ikaros’ association with chromatin remodeling complexes and its ability to repress the transcription of developmentally regulated genes.
Effects of Site‐specific Phosphorylation on the Structure and Functions of the Intrinsically Disordered N‐terminal Activation Function (AF1) Domain of the Glucocorticoid Receptor
Authors: Kumar Raj , Garza Anna S , Khan Shagufta H
Published: June 2021

In eukaryotic cells, phosphorylation regulates protein structure and functions. The glucocorticoid receptor (GR) is a phosphoprotein with all the conserved sites located within its AF1 activation domain. Structure of AF1 has been difficult to determine because it exists as an intrinsically disordered (ID) protein, frequently found in transcription factors. ID regions are disproportionately higher in cell signaling proteins and are predicted to have much higher frequency of phosphorylation sites than ordered regions, suggesting an important role in their regulatory capacity. It has been suggested that phosphorylation plays an important role in the regulation of GR activity. In this study, we found that ID AF1 adopts a functionally folded conformation due to phosphorylation of S211 by p38 MAPK. Further, this induced secondary/tertiary structure in AF1 specifically facilitates its interactions with critical coregulatory proteins, and subsequent AF1‐mediated transcriptional activity of the GR. Together, we propose that under physiological conditions, site‐specific phosphorylation plays a crucial role in allowing AF1 to adopt functionally active conformation(s), which facilitates AF1’s interactions with specific coregulatory proteins, and possibly additional modulations that form the basis for the multi‐protein assemblies involved in GR‐mediated regulation of transcription.
Consequences of activation loop phosphorylation on protein kinase A
Authors: Steichen Jon M , Iyer Ganesh H , Li Sheng , Woods Virgil L , Taylor Susan S
Published: June 2021

Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP‐dependent protein kinase (PKA) autophosphorylates Thr197 in the activation loop when expressed in E. coli. While mutation of Arg194 to Ala prevents autophosphorylation, phosphorylation of Thr197 can still be achieved by phosphoinositide‐dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of PKA by comparing the wild‐type C‐subunit to the Arg194Ala mutant either in the presence or absence of activation loop phosphorylation. Phosphorylation of Thr197 increased the catalytic efficiency, and increased the thermodynamic stability of the enzyme. Additionally, deuterium exchange coupled to mass spectrometry (H/DMS) was used to compare the structure and dynamics of these proteins. All regions of the C‐subunit analyzed were more solvent exposed in the unphosphorylated state. The largest changes occurred at the C‐terminus of the activation segment in the P+1 loop/APE regions and the alphaH‐alphaI loop motifs. Mutations in either of these regions blocks phosphorylation of PKA by PDK1. Supported by NIH grant GM19301.
Cholecystokinin‐Induced PKC α Activation Mediates the Translocation of RhoA in Mouse Pancreatic Acini
Authors: SABBATINI MARIA EUGENIA , WILLIAMS JOHN A
Published: June 2021

Introduction

RhoA is a small G protein belonging to the Rho family that cycle between an inactive GDP‐bound and an active GTP‐bound forms. In previous work we showed that cholecystokinin (CCK) induces RhoA activation and translocation from the cytosol to the membrane.

Aim

To study which intracellular signals are involved in CCK‐induced RhoA translocation in mouse pancreatic acini.

Results

The PKC stimulator phorbol 12‐myristate 13‐acetate increased the amount of RhoA in the membrane fraction and the PKC inhibitor GF‐203109X blocked CCK‐induced RhoA translocation while agents affecting calcium or cAMP had no effect. In order to test which isoform of PKC mediated the effect of CCK, we infected acini with adenoviruses expressing dominant negative (DN) isoforms of PKC α, δ and ε. Only in DN PKC α‐expressing acini was the effect of CCK on RhoA translocation reduced.

Conclusion

PKC α, but not calcium or cAMP, is required for CCK‐induced RhoA translocation.
Elucidating the Role of the DNA Helicase Pfh1 in DNA Repair in Fission Yeast
Authors: McMurdie Karen Elaine , Little Jamie , Sielge Jasmin , Dunaway Stephen
Published: June 2021

Helicases are crucial enzymes that utilize energy from ATP hydrolysis to unwind double‐stranded DNA and RNA molecules. DNA strand unwinding is essential for cellular processes such as DNA replication, transcription, repair and recombination, and as such they are considered vital for maintaining genomic integrity. The PIF1 family of 5′ to 3′ DNA helicases play important roles in the maintenance of telomeric, ribosomal, and mitochondrial DNA. This gene family is highly conserved and can be found in a wide range of eukaryotes including humans. In the fission yeast, S. pombe, only one Pif1 family member exists, pfh1+, and is the only member of the PIF1 family that is vital for cell viability. While the essential function of Pfh1 is still unknown, several studies have suggested important roles for this helicase in DNA replication. One possible role for Pfh1 is in Okazaki fragment maturation, an essential step in completing replication. Another possibility is that Pfh1 plays a role in helping replication forks proceed once they have stalled. Additionally, Pfh1 is believed to play a role in the repair of DNA. To further investigate this role, a cold sensitive allele of Pfh1 was challenged with various DNA damaging agents and assayed for viability. The cold sensitive allele was found to be sensitive to camptothecin, cisplatin, and nitrogen mustard. The loss in viability observed following treatments was not due to a defective DNA damage checkpoint response suggesting that Pfh1 may have a role in global DNA repair in fission yeast.
Defining the interaction between the Escherichia coli RecA and DinI proteins
Authors: Bane Lukas B. , Britt Rachel L. , Cox Michael M. , Egelman Edward H.
Published: June 2021

The RecA protein of Escherichia coli participates directly in recombinational repair of stalled replication forks and also indirectly increases expression of other DNA repair genes via regulation of the SOS response. The RecX and DinI proteins are competing modulators of RecA filament dynamics. RecX blocks extension of RecA filaments along the bound DNA, while DinI stabilizes the filaments. Both proteins bind to RecA filaments within the major filament groove. Electron microscopy reconstructions yielded a structure of DinI protein bound to a RecA filament. This structure revealed that DinI interacts with RecA similarly to RecX. However, DinI also has a second, unique mode of interaction with RecA. RecX and DinI share a structurally homologous alpha‐helical motif that both proteins may use to interact with RecA. It is possible that the β‐sheet DinI possesses, and RecX lacks, could provide the second RecA interaction site on DinI. To test this, four residues which are absolutely conserved in the DinI β‐sheet (Glu4, Arg43, Ala45 and Ser51) were mutated. The four mutant proteins have been purified and their interactions with RecA biochemically analyzed. An E4A substitution did not affect the RecA‐DinI interaction. The effects of other substitutions will be described.
Developing Reporter Systems to Monitor the Structural Dynamics of MutS
Authors: Doucette Christopher William , Biro F. Noah
Published: June 2021

DNA mismatch repair (MMR) is an evolutionarily conserved process that locates and repairs post‐replicative errors in nascent DNA and is critical for maintaining the genome integrity. The first step in the MMR pathway is recognition of the base‐base mismatch/insertion deletion loop (IDL) by MutS in prokaryotes and the MutS homologues, Msh2‐Msh6 and Msh2‐Msh3, in eukaryotes. While there is a significant body of literature concerning DNA‐binding and ATPase activities of MutS, information on MutS conformational changes involved in recognizing an array of non‐Watson‐Crick DNA structures is lacking. Here we present two fluorescence reporter assays developed to examine T. aquaticus MutS conformational changes and better understand the relationship between its structure/dynamics and function. Two mutant proteins were prepared: 1) F39, which contacts DNA, was replaced with tryptophan to monitor intrinsic protein fluorescence during the reaction; 2) M88 in the DNA binding domain was replaced by cysteine and labeled with a thiol reactive fluorophore that is sensitive to protein conformation. We are currently measuring changes in T. aquaticus MutS conformation on mismatch/IDL binding in equilibrium and in real time by transient kinetic analysis.
PCNA Phosphorylation at Tyrosine211 Inhibits Mismatch Repair at the Initiation Step
Authors: Ortega Janice , Zhang Tianyi , Huang Jian , Li Guo‐Min , Gu Liya
Published: June 2021

The proliferating cell nuclear antigen (PCNA) plays an important role in DNA replication and repair. Recent in vivo studies have shown that PCNA functions can be modulated by tyrosine211 (Tyr211) phosphorylation, leading to increase in cell proliferation and poor survival in some cancer patients. Since PCNA is required for DNA mismatch at the initiation and DNA re‐synthesis steps of DNA mismatch repair (MMR), we examined the effect of Tyr211‐phosphorylated PCNA on MMR. Tyr211‐phosphorylation and non‐phosphorylation mimic PCNAs were obtained and assayed for MMR in vitro. We demonstrate here that Tyr211‐phosphorylated PCNA blocks the in vitro MMR reaction at the initiation stage in both HeLa nuclear extract and the reconstituted MMR system, as little incision/excision product was detected in these reactions in the presence of Tyr211‐phosphorylated PCNA. However, in vitro gap‐filling and primer extension experiments suggest that the phosphorylated PCNA does not block DNA synthesis catalyzed by HeLa nuclear extract or pol δ. These observations suggest that Tyr211 phosphorylation regulates PCNA functions, possibly by promoting DNA replication but inhibiting DNA repair.
Characterization of the Functional Domains of TnsC
Authors: Spencer Jeanelle M. , Craig Nancy L.
Published: June 2021

Tn7 is a transposable element isolated from E. coli that encodes five transposon‐specific proteins, Tns A, B, C, D, and E. TnsC is essential in the formation and activity of a Tn7 nucleoprotein complex comprised of TnsA,B,C, and D proteins with donor DNA as well as target DNA. The goal of this work is to identify and characterize the functional domains of TnsC. The Tn7 reaction mechanism suggests an interaction between TnsB and C. Crosslinking experiments were performed with a TnsB peptide and TnsC to stabilize and capture the complex. The data show that TnsB and TnsC participate in a direct physical interaction. Mass spectrometry of the complex will show which residues of TnsC interact with the peptide, and thereby reveal the TnsBC domain. In order to determine the remaining functional domains, Tn7 was used as a mutagen to generate a TnsC mutant library. The mutants were screened for transposition activity in a promoter capture assay. The mutable regions of TnsC were mapped and dominant negative mutants that, presumably, retain one or more essential activities of TnsC were identified. Future experiments will reveal which activity and, correspondingly, functional domain is disrupted. Experiments that identify the functional domains of TnsC will provide deeper insights into how Tn7 and other nucleoprotein complexes are controlled. This work was supported by Howard Hughes Medical Institute.
Increased DNA Damage and Decreased DNA Repair Following Poly(ADP‐ribose) Glycohydrolase Inhibition and Chemotherapy
Authors: KOH DAVID W , Zhou Yiran , Feng Xiaoxing , Garcia Katherine D.
Published: June 2021

The synthesis of poly(ADP‐ribose) (PAR) is an immediate response to DNA damage. We reported previously that the failure to degrade PAR by inhibiting PAR glycohydrolase (PARG) leads to hypersensitivity to DNA damage.

OBJECTIVE

To investigate the ability of PARG inhibition to increase the effectiveness of DNA‐damaging agents.

METHODS

Utilizing PARG null trophoblast stem cells, chromatin structure was analyzed by the detection of PAR‐modified nuclear proteins, DNA damage and repair were analyzed by Comet assay after treatment with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), and cell death was measured by FACS.

RESULTS

PARG null cells displayed high levels of PAR‐modified histones, suggesting chromatin structural change. Low‐dose MNNG led to high levels of DNA damage, which suggests greater DNA access due to PARG inhibition. Further, this DNA damage was not repaired after 12 hr, which suggests unrepairable levels of DNA damage or decreased DNA repair. Finally, treatment of PARG null cells with current chemotherapeutic agents led to increased cell death.

CONCLUSION

The results show that PARG inhibition enhances the efficacy of DNA‐damaging agents by inducing greater amounts of DNA damage and decreasing DNA repair. Therefore, targeting PARG is expected to enhance the effectiveness of DNA‐modifying anti‐cancer agents. Support was provided by ASPET, ACS, and the PhRMA Foundation.
A Role for Np95 in Genome Integrity Maintenance.
Authors: Mistry Helena Sumantrai
Published: June 2021

Np95 (also known as Uhrf1) is a proliferation‐associated E3 ubiquitin ligase that participates in maintenance of DNA methylation and histone H3 lysine 9 trimethylation trimeH3K9) associated with heterochromatin. Although cells deficient in Np95 have previously been shown to exhibit greater sensitivity to DNA damaging agents, the role of this protein in the cellular response to DNA damage is unknown. Here we report that Np95 is critical for the maintenance of genome integrity. In the absence or presence of DNA damaging agents, Np95 deficiency leads to an increase in the number of cells with chromosomal instability and elevated levels of γH2AX, a cellular marker that signifies the presence of DNA double strand breaks. Although H2AX status has previously been shown to impact checkpoint activation, Np95 deficient cells exhibit intact checkpoint activation compared to wild‐type cells. Furthermore, DNA damage signalling pathways that activate Chk2 and FancD2 are intact in Np95 deficient cells. Loss of Np95 results in a redistribution of focal trimeH3K9 staining to the nuclear periphery. We observed that (ii) focal staining of heterochromatin protein HP1β but not HP1α dissipates following DNA damage and (ii) this loss of focal HP1β staining is accentuated in Np95 deficient cells. Our findings indicate that maintenance of heterochromatin by Np95 is crucial for genome integrity and protection from DNA damage.
Apex1 haploinsufficiency leads to age‐dependent increases in mtDNA lesions in liver
Authors: Torres‐Ramos Carlos A , Torres‐Ortíz Ceidy , Walter Christi , Ayala‐Torres Sylvette
Published: June 2021

The base excision repair pathway repairs DNA lesions induced by reactive oxygen species (ROS). A central component of this pathway is the apurinic/apyrimidinic endonuclease 1 (APEX1). Knockout of both copies of Apex1 results in murine embryo lethality, whereas heterozygous mice (Apex1+/−) are viable and can live to old age. Apex1 haploinsufficiency does lead to a phenotype characterized by increased mutation frequencies in germ cells. Because of the prominent role of the liver in detoxification reactions, we hypothesize that APEX1 plays a prominent role in protecting this organ from the deleterious effects of ROS. Our hypothesis predicts that Apex1 haploinsufficiency will lead to increased DNA lesions, particularly in the mitochondrial genome (mtDNA). For this purpose we determined the amount of mtDNA lesions in liver tissue obtained from 6, 16, and 28‐month old wild type (WT) and Apex1+/− mice using a gene specific assay based on PCR. This assay can detect a variety of DNA lesions such as abasic sites, strand breaks and oxidized bases, among others. The results of these experiments show a significant increase in mtDNA lesions in both WT and Apex1+/− 28‐month‐old mice as compared to 6 month‐old animals. Moreover, the 28‐month‐old Apex1+/− mice show a 2.5 fold increase in mtDNA lesions over aged‐matched WT animals suggesting that Apex1 haploinsufficiency leads to an age‐dependent increase in mtDNA lesions. These results underscore the role of BER in maintaining mtDNA stability in liver. Supported by 5SC3GM084759, 5R25GM061838 and G12RR03051.
The N‐terminal domain of BLM/Sgs1 displays a strand‐annealing and strand‐exchange activity that is required for in vivo function
Authors: Chen Chi‐Fu , Brill Steven
Published: June 2021

Mutations in BLM result in Bloom's syndrome, a rare autosomal disease characterized by a variety of symptoms including a predisposition to cancer. BLM is conserved in the budding yeast Saccharomyces cerevisiae as Sgs1. BLM/Sgs1 is a 3′‐5′ DNA helicase with a large N‐terminal domain of unknown function. To determine the role of the N‐terminus, we assayed purified recombinant sub‐domains using biochemical methods. We found that an internal domain of the Sgs1 N‐terminus (amino acids #103–322, named the SA domain) can bind single‐stranded (ss) DNA using gel‐shift assay. Moreover, this ssDNA‐binding activity is conserved in the N‐termini of human and Drosophila BLM. Further studies demonstrate that the SA domain exhibits strand‐annealing and strand‐exchange activities in vitro. To determine the biological function of this domain, we tested the sgs1Δ103–322 allele for a variety of sgs1Δ phenotypes including MMS sensitivity, top3Δ slow‐growth and sgs1Δ slxΔ synthetic‐lethality. The results show that sgs1Δ103–322 complements MMS sensitivity but is defective for top3Δ slow‐growth promotion and sgs1Δslx4Δ and sgs1Δslx5Δ synthetic lethality. While further defining its modular structure, these results identify an essential pro‐recombinogentic function for BLM/Sgs1 and provide a framework to analyze the coordinated activities of Top3, Rmi1 and strand annealing.

This work is supported by NIH‐R01 GM071268
Toward the isolation of a novel RNA‐RNA interaction based on an activity of a trans‐acting ribozyme
Authors: Ishikawa Junya , Furuta Hiroyuki , Ikawa Yoshiya
Published: June 2021

The DSL ribozyme, a class of artificial ligase ribozymes, has a highly modular architecture, which catalyzes template‐directed RNA ligation on a helical substrate that can be either covalently connected (cis‐DSL) or physically separated (trans‐DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively.

In the field of organic chemistry, modular functional groups are attached into parent compounds to generate new or improved properties. This strategy is well established and a variety of the mudular groups renders the number of compounds infinite. Along with the fact, an abundance of RNA motifs could expand possibilities of functional RNAs. Recent works demonstrated artificially designed RNAs composing naturally occurring structural motifs could be a scaffold to isolate a small RNA unit.

In the present study, we are attempting to isolate an artificial loop receptor motif based on the activity of trans‐DSL. For the selection, we have constructed a new derivative of the trans‐DSL by replacing one of its clamp modules with a target loop and a random sequence.

This research was supported by Grant‐in‐Aids on Innovative Areas “Emergence in Chemistry” [No. 21111518 to Y.I.] and for the Global COE program, “Science for Future Molecular Systems” [to H.F.] from The MEXT of Japan.
In Vivo Selection of mRNA Repair Ribozymes from Randomized Pools
Authors: Müller Ulrich F , Olson Karen E.
Published: June 2021

Group I introns are catalytic RNAs (ribozymes) that self‐splice from pre‐mRNA. These cis‐splicing ribozymes can be converted for gene therapeutic purposes into trans‐splicing ribozymes, which replace the (mutated) 3′‐terminus of an mRNA by the 3′‐exon of the ribozyme, thereby repairing the mRNA. The major limiting step for this strategy is a low in vivo mRNA repair efficiency. We report here an in vivo selection that improves the in vivo mRNA repair efficiency, from millions of ribozyme variants. The improved portions on the ribozymes are the extended guide sequences (EGSs). The predicted secondary structures of the resulting EGSs form several classes of secondary structure motifs, some of which have been found before. The newly discovered motifs could become important for evading cellular responses in therapeutic applications. The absence of a correlation between in vitro and in vivo activities indicates that in vivo methods are most promising for future, stepwise optimizations of mRNA repair ribozymes. This work was supported by the NIH [T32DK007233 to K.E.O].
Mechanisms involved in efficient removal of multiple introns
Authors: Crabb Tara L. , Lam Bianca J , Hertel Klemens J
Published: June 2021

The vast majority of mammalian pre‐mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre‐mRNA splicing that can interfere with downstream splicing events. These considerations raise the question whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron‐containing pre‐mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions, or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, kinetic trap experiments show that spliceosomal components that are not involved in the initial splicing event remain associated with the pre‐mRNA to ensure efficient subsequent intron removal. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splices sites. NIH funding.
The Role of Wnt‐10b in Osteoblast Development
Authors: Chase Jennifer A , Moedder Ulrike , Monroe David , Khosla Sundeep
Published: June 2021

Wnt signaling plays a central role in the development of bone forming osteoblasts and bone remodeling. This study analyzed Wnt‐10b control of gene expression throughout osteoblast differentiation. Genes regulated by Wnt‐10b were identified by analyzing five mouse osteoblast lines (C3H10T1/2, ST‐2, bone marrow stromal cells, MC‐3T3, and calvarial osteoblasts). These cell lines were exposed to media from a Wnt‐10b producing cell line via a transwell system. mRNA levels of 32 osteoblastic, Wnt and BMP responsive genes were quantified by qPCR. Several genes were consistently up/down regulated, representing different stages of osteoblast commitment and differentiation. In the second part of the study, C3H10T1/2 cells were treated with BMP2 osteoblast differentiation media to stimulate the growth of osteoblastic cells. The control and Wnt‐10b treated cells were harvested at 0, 6, 9, 12, or 15 days to observe the effect of Wnt‐10b during differentiation. Analysis of 64 genes showed two known Wnt‐3a targets, complement component 3 (C3) and chemokine ligand 16 (Cxcl16), were highly regulated by Wnt‐10b. This study indentifies a number of important genes that are regulated by Wnt‐10b during osteoblast differentiation. This project was funded by the Mayo Graduate School and the Summer Undergraduate Research Fellowship.
Reduced Milk Triglycerides in Mice Lacking Phosphoenolpyruvate Carboxykinase in Mammary Gland Adipocytes and White Adipose Tissue Contribute to the Development of Insulin Resistance in Pups
Authors: Croniger Colleen M , Hsieh Chang‐Wen , Millward Carrie A , DeSantis David , Pisano Sorana , Machova Jana , Perales Jose C
Published: June 2021

Obesity and type 2 diabetes are growing problems worldwide in adults and children. This study focused on understanding the patterning of insulin resistance as a result of altered perinatal nutrition. We analyzed mice in which the binding site for peroxisome proliferator‐activated receptor γ (PPARγ) was deleted from the promoter of the cytosolic phosphoenolpyruvate carboxykinase gene (Pck1) (PPARE−/−). We analyzed pups from dams with the same genotype as well as fostered and cross‐fostered pups. Pck1 expression and triglyceride content in the milk were measured. The PPARE mutation reduced Pck1 expression in WAT to 2.2% of wild type (WT) and reduced Pck1 expression in whole mammary gland tissue to 1% of WT. The female PPARE−/− mice presented reduced lipid storage in mammary gland adipocytes and in WAT, resulting in a 40% reduction of milk triglycerides during lactation. Pups from PPARE−/− dams exhibited insulin resistance as early as 14 days after birth, a condition that persisted into adulthood. Wild type pups fostered by PPARE−/− dams have lower body weights and plasma insulin concentrations compared to WT pups reared by WT dams. PPARE−/− pups fostered by WT dams have improved glucose clearance compared to pups raised by PPARE−/− dams. PPARE+/− and PPARE−/− dams patterned the newborn pup for reduced growth and insulin resistance in utero as well. Thus, in utero environment and altered nutrition in the perinatal period cause epigenetic changes that last into adulthood and contribute to the development of insulin resistance. J Nutr. 139:1–9, 2009.
Retinoid metabolism and signaling in lung from COPD patients with different degree of emphysema
Authors: Roy Somdutta Sinha , Schäffer Michael W , Ong David E , Das Salil K
Published: June 2021

Chronic bronchitis and emphysema are two types of chronic obstructive pulmonary disease (COPD). Numerous findings suggest that retinoic acid (RA) may protect against emphysema. Vitamin A and its active metabolite RA are important for growth and differentiation of tissues. Considerable evidence demonstrates that RA is an essential signaling molecule for normal lung development. The objective of the present study is to establish a relationship between the severity of emphysema and levels of expression of genes involved in retinoid metabolism/signaling. Microarray analysis was done on samples from each of the 3 COPD groups using Human Whole Genome OneArrayTM. Data show differential gene expression in the expression of 11 genes of retinoid metabolism and signaling between 3 groups (7 up‐regulated and 4 down‐regulated). Data from western blot analysis indicate a relationship between the protein levels of several mediators [LRAT, CRBP‐I, CRABP‐II, CYP26A1, RAR (α, β, γ) and RXRα] and severity of emphysema. Immunohistochemical staining of lung sections from COPD patients was done for some of these proteins and compared with the normal lung sections. Taken together, our results strongly support the hypothesis that there is a strong relationship between an abnormality in retinoid metabolism/signaling and severity of emphysema. (Supported by NIH grant 1RO3 HL095419‐01A1)
Divalent Metal‐Dependent Regulation of Hepcidin Expression by MTF‐1
Authors: Balesaria Sara , Bayele Henry , Srai Kaila
Published: June 2021

There appears to be cross‐talk between metal ions and their acquisition pathways. In particular, zinc and iron reciprocally inhibit each other's uptake. Hepcidin, a small acute phase and antimicrobial peptide, is the principle regulator of iron absorption. This peptide is induced by inflammation and infection, but is repressed by anaemia and hypoxia, suggesting a complex array of regulatory pathways underlying its expression. Here we further reveal that it also appears to be a sensor of other divalent metal ions, principally zinc and cadmium. Using 1.8kb of the human hepcidin promoter tagged with luciferase as a reporter, we show that hepcidin transcription can be significantly modulated by zinc and cadmium levels. This process is entirely dependent on functional metal response elements (MREs) that recruit the MRE‐binding transcription factor MTF‐1 to the promoter, as ascertained by site‐directed mutagenesis. Analysis of hepcidin expression both at the mRNA and protein levels in hepatoma cells suggests that its induction by zinc is rapid; paradoxically, cobalt and iron significantly decreased its mRNA levels. Taken together, these data show that hepcidin may be a pleiotropic sensor of divalent metals some of which are xenobiotic environmental toxins.
Regulation of arginase‐I expression in macrophages by selenium
Authors: Nelson Shakira M , Gandhi Ujjawal H , Kaushal Naveen , Prabhu K. Sandeep
Published: June 2021

Selenium (Se) is an essential micronutrient with anti‐inflammatory properties. It has been previously shown that Se supplementation of macrophages led to a decrease in bacterial endotoxin lipopolysaccharide (LPS)‐induced expression of inducible nitric oxide synthase (iNOS), a prototypical marker of classical (M1) macrophage activation. We believe that L‐arginine, a substrate for iNOS, is now acted upon by arginase‐1 (Arg‐1), a marker of alternate (M2) activation. Arg‐1 produces L‐ornithine, which serves as a precursor for the synthesis of collagen and polyamines that are essential components of catabasis or wound healing responses. Using murine bone marrow‐derived macrophages and RAW 264.7 cell models, we have examined Se effects on Arg‐1 transcription, protein expression, and enzyme activity. Following IL‐4 treatment, an alternative activation cytokine, the Arg‐1 mRNA, protein and enzyme activity were significantly increased in Se supplemented macrophages when compared to Se deficient counterparts. Stimulation of these cells with LPS caused a further increase in Arg‐1 expression suggesting a synergistic effect of Se and IL‐4 on Arg‐1 expression even under inflammatory conditions. These data show that optimal Se status is critical for alternative macrophage activation, leading to attenuated inflammation. National Institute of Health grant DK077152.
Promoting Dendritic Cell Migration by Airway Epithelial Cells through Interleukin‐17A‐Induced CCL20 Expression
Authors: Hung Li Yin , Huang Fei , Nguyen Tiffany T. , Wu Reen
Published: June 2021

We previously demonstrated that IL‐17A is a potent inducer of both human β‐defensin (hBD) 2 and CCL20/macrophage inflammatory protein (MIP)‐3α expression in well‐differentiated primary human airway epithelial cell (AEC) cultures. In addition to having a common anti‐microbial activity, both hBD2 and CCL20 are principal chemotactic ligands specific to CCR6‐positive dendritic cells (DC) and T lymphocytes. To further explore the chemotactic significance of IL‐17A in the mouse model, we conducted studies using isolated primary mouse AECs and Cc10‐Il17 transgenic animals. Consistent with human AECs, well‐differentiated mouse AECs also expressed elevated mCcl20 when treated with mIL‐17A. This induction was time‐ and dose‐dependent on the IL‐17A treatment, and the induction was inhibited by isohelenin, a specific inhibitor of NF‐κB activation. Chemotactic assays showed that conditioned media from mIL‐17A‐treated mouse AEC cultures stimulated the migration of CCR6‐positive mouse bone marrow‐derived dendritic cells (BMDC) in vitro; this chemotactic activity was blocked by anti‐CCL20 neutralizing antibody. We found a persistent elevation of mCcl20 message and the presence of CCR6‐positive DCs in bronchoalveolar lavage fluids, accompanied by increased mucin production in the lungs of Cc10‐Il17 transgenic mice, which overexpress IL‐17A specifically in the airway. Blocking the migration of CCR6 expressing cells in vivo lowered mucin production in the airway. These results suggest a functional role of IL‐17A/CCL20 in regulating CCR6‐positive DC migration and mucous cell metaplasia.

This research was supported in part by grants from NIH (HL077902, HL077315, and ES00628)
Role of mDia1 in RAGE signaling and smooth muscle cell migration
Authors: Toure Fatouma , Fritz Guenter , Li Quing , Peng Jun , Lu Yan , Rosario Rosa , Yan Shi Fang , Alberts Arthur , Ramasamy Ravichandran , Schmidt Ann Marie
Published: June 2021

RAGE (Receptor for Advanced Glycation end Products) plays a pivotal role in the atherosclerotic process. The cytoplasmic binding domain of RAGE interacts with mDia‐1, a member of the formin family involved in actin and microtubule reorganization. Here we report that mDia‐1 is required for RAGE effects on vascular stress. Immunostaining of human and mice atherosclerotic tissue showed mDia‐1 expression in the atherosclerotic plaque, particularly in smooth muscle cells (SMCs). We established primary cultures of murine aortic SMCs from wild type (WT) and mDia1 null mice. Recombinant S100B was used as a prototypic RAGE ligand. We found that compared to control cells, SMCs devoid of mDia‐1 (KO or mdia1 siRNA transfected) showed significant and specific inhibition of S100B‐stimulated superoxide production, lamellopodia formation and cell migration. Interestingly, RAGE induced Rac activity and phosphorylation of AKT and GsK3beta were also greatly inhibited in these cells. In WT cells, blockade of RAGE‐induced ROS production with Diphenyiodinium or dominant negative Rac construct, significantly prevented RAGE‐induced AKT/Gsk3beta phosphorylation and SMCs migration. We concluded that in SMCs, mDia‐1 is essential for RAGE‐induced Rac1 activation and ROS production. ROS may then activate PI3Kinase and induce AKT dependent phosphorylation of GSK3beta, leading to cell migration.
Expression and Purification of Sunflower Cotyledon Acetoacetyl CoA Thiolase Mutants
Authors: Gomez Iris , Cadet Melissa , Oeljeklaus Silke , Schiedel Anke C. , Dyer James H.
Published: June 2021

Sunflower (Helianthus annuus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of Thiolase II from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and this construct was transformed into BL‐21 E. coli that were induced for production of the Thiolase II with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble Thiolase II was purified to apparent homogeneity using Ni‐NTA agarose affinity chromatography as indicated by a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.
Divergent biochemical roles for cytosolic and chloroplastic pyruvate phosphate dikinase regulatory protein
Authors: Failing Christopher Jon , Chastain Chris J
Published: June 2021

Pyruvate orthophosphate dikinase regulatory protein (PPDK‐RP) is a unique regulatory protein found in plants. Its properties are highly unusual among all regulatory enzymes. First, it is a bi‐functional enzyme catalyzing both its protein kinase and protein phosphatase on its target protein (PPDK). Second, it uses ADP rather than ATP as a phosphoryl donor. Third, its dephosphorylation mechanism is a Pi‐dependent, pyrophosphate (PPi) forming phosphoryltic cleavage. Bioinformatic analysis of its primary sequence reveals that it is unrelated to any other known class of protein kinase. We describe in this presentation, a PPDK‐RP isoform cloned from Arabidopsis thaliana localized in the cytoplasm that lacks protein phosphatase activity. Our biochemical analysis of this isoform at its native cytsolic pH reveals a phosphoryl substrate preference for GDP and GTP rather than ADP. This difference suggests that this RP is regulated in an unknown yet strikingly different manner than its bi‐functional counterpart.

This work was supported by NSF/RUI Grant no.0642190
Regulatory Dephosphorylation of Catalytic His‐P of Inactive PPDK in Maize
Authors: Manandhar Lumu , Chastain Chris J
Published: June 2021

Plant pyruvate phosphate dikinase (PPDK) is a major enzyme involved in C4 photosynthesis. It undergoes up/down (activation/inactivation) regulation in response to light by the enzyme PPDK Regulatory Protein (RP) via reversible phosphorylation. The rate of dephosphorylation of inactive p‐PPDK depends on the removal of the catalytic His‐P from the active site of PPDK. The mechanism involved in this removal is remains to be discovered. We hypothesized that an adjacent Ser residue participates in the His‐P dephosphorylation. We explored this hypothesis by producing a site directed mutation converting wild‐type (WT) Ser into Ala. Phosphorylation/inactivation of mutant (MT) PPDK by RP was unchanged. However, a preliminary experiment showed a higher rate of dephosphorylation (activation) in the MT vs. WT. Thus, this preliminary data showed the Ser residue may be involved in a yet to be discovered His‐P dephosphorylation mechanism.
Possible functional roles of chemerin, a new adipokine, in domestic animals
Authors: Roh Sang‐Gun , Song Sang‐Houn , Suzuki Yutaka , Katoh Kazuo
Published: June 2021

Chemerin was recently identified as a novel adipokine highly expressed in adipose tissues of mouse and human and suggested to play a role in the pathophysiology of obesity. However, the function roles of chemerin in metabolic and cellular processes in domestic animals still remain unknown. Full‐length cDNA and predicted amino acid sequences of bovine chemerin and chemerin receptor were found to be highly homologous to those of the human and mouse, suggesting similar functions of these genes in cattle as in other species. Bovine chemerin mRNA was highly expressed in adipose tissues and liver, and the transcripts of chemerin receptor was abundant in adipose tissues, muscle, liver and brain. Both transcripts of chemerin and chemerin receptor were up‐regulated during the adipocyte differentiation of bovine preadipocytes. TNF‐ƒ¿ treatment (10ng/ml), an inhibitor of adipocyte differentiation, in bovine differentiated adipocytes increased the mRNA expression of bovine chemerin and chemerin receptor. These results indicate that chemerin, highly expressed in adipose tissues, regulate the fatty metabolism in adipose tissue through its receptor in cattle.
Thioredoxin interacting protein (Txnip) is feedback regulator of S‐nitrosylation
Authors: Forrester Michael T , Seth Divya , Hausladen Alfred , Eyler Christine E , Foster Matthew W , Matsumoto Akio , Benhar Moran , Marshall Harvey E , Stamler Jonathan S
Published: June 2021

Nitric oxide exerts a plethora of biological effects via protein S‐nitrosylation, a redox‐based reaction that converts a protein Cys thiol to a S‐nitrosothiol. However, while the regulation of protein S‐nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus‐coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin‐interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin‐mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S‐nitrosylation.