bluuebunny/crossref_metadata_2025 huggingface dataset:
Subtelomere recombination is frequent in tumors lacking telomerase
Authors: Morrish Tammy , Behera Vivek , Dria Stephen , Wheelan Sarah J. , Greider Carol W.
Published: June 2021

The unlimited growth of tumors requires telomere maintenance. Telomerase typically maintains telomeres. Yet some tumors lack telomerase and instead use recombination‐based mechanisms for telomere maintenance. Yeast deleted for telomerase use multiple mechanisms of break‐induced replication (BIR) that can occur within the telomeres or subtelomeres. To study BIR in mammalian cells, we are using a mouse model, myc+mTR−/− that generates B‐cell lymphomas lacking telomerase. Using this model we detected subtelomere recombination in tumors lacking telomerase. To examine and quantify subtelomere recombination, we are using comparative genomic hybridization (aCGH) to quantify changes in subtelomere copy numbers. Using these arrays we find frequent copy number changes within the subtelomeres of some tumors lacking telomerase, consistent with a mechanism of BIR. To examine the mechanism of subtelomere recombination we are testing whether Rad50 and Rad51a contribute to subtelomere recombination in tumors lacking telomerase. These studies address the role of different recombination genes during telomere maintenance and subtelomere recombination in tumors lacking telomerase. NIH/NCI K99/R00
Extracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression of MCF‐7 cells
Authors: Bilbao Paola Gabriela Scodelaro , Boland Ricardo Leopoldo
Published: June 2021

Forkhead box‐O (FoxO) transcription factors play an important role in tumour suppression by regulating the expression of genes involved in DNA damage repair, apoptosis and cell cycle arrest. We studied the role of extracellular ATP in the modulation of FoxO transcription factors and of cell cycle progression in MCF‐7 breast cancer cells. Western blot analysis showed that 5 μM ATP induced the phosphorylation of FoxO1/3a at threonine 24/32, and also reduced the expression of FoxO1. The use of the phosphatidilinositol 3 kinase (PI3K) inhibitor Ly294002 and of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. Immunocytochemistry and subcellular fractionation studies showed that ATP induces the translocation of FoxO3a from the nucleus to the cytoplasm. Flow cytometry analysis revealed that ATP increases the number of cells in the S phase of cell cycle. In addition, analysis by Western blot showed that ATP increases the expression of the cyclins D1 and D3 and diminishes the inhibitory proteins p21Cip1 and p27Kip1. However, these effects were independent of the PI3K/Akt pathway. Taken together, our results suggest that ATP induces the inactivation of FoxO transcription factors in a PI3K dependent manner and also induces changes in the expression of proteins involved in cell cycle regulation in MCF‐7 cells. (PIP‐CONICET Argentina)
Functional redundancy between Cdc20 ubiquitination and p31comet
Authors: Jia Luying , Li Bing , Warrington Ross , Hao Xing , Wang Shixuan , Yu Hongtao
Published: June 2021

The spindle checkpoint senses unattached kinetochores in a mitotic cell, and inhibits the function of Cdc20, the mitotic activator of the anaphase‐promoting complex or cyclosome (APC/C), thus delaying anaphase onset. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins and produce diffusible APC/C‐inhibitory signals. The mitotic checkpoint complex (MCC) consisting of BubR1 (Mad3 in yeast), Bub3, Mad2, and Cdc20 is a critical checkpoint inhibitor of APC/C. When all kinetochores achieve proper microtubule attachment and are under tension, the spindle checkpoint turns off and MCC disassembles to allow APC/C activation. Several mechanisms have been implicated in MCC disassembly, including p31comet‐and UbcH10‐dependent Cdc20 autoubiquitination and proteasomal degradation. However, the role of Cdc20 autoubiquitination in checkpoint inactivation has remained controversial, because preventing Cdc20 ubiquitination by mutating all lysines in Cdc20 to arginines does not prevent MCC disassembly.

In this study, we have further investigated the roles and coordination of p31comet, proteasomal degradation, and Cdc20 ubiquitination in MCC disassembly. We show that both the proteasome and p31comet are required for timely MCC disassembly in HeLa cells by treatment of proteasome inhibitor MG132 or RNA interference (RNAi). Depletion of p31comet further delays MCC disassembly in cells treated with MG132, suggesting that p31comet has a proteasome‐independent role. Consistently, p31comet depletion delays Mad2–Cdc20 dissociation and mitotic exit in cells expressing Cdc20 mutants deficient for autoubiquitination. Taken together, our results reconcile earlier conflicting data, and suggest p31comet and Cdc20 ubiquitination act in somewhat redundant pathways to promote MCC disassembly.
Replication Protein A and Sex: What's Phosphorylation Got To Do With It?
Authors: Wilson Timothy M. , Piya Gunjan , Haring Stuart J.
Published: June 2021

Meiosis is an essential part of sexual reproduction that is required to create viable haploid gametes from diploid parental cells. A unique feature of meiosis is the programmed exchange of genetic information through homologous recombination (HR), which is also essential for the proper segregation of chromosomes during the reductional division. Replication Protein A (RPA) is a heterotrimeric protein complex conserved amongst all eukaryotes that binds single‐stranded DNA (ssDNA) and participates in DNA replication, repair, cell cycle regulation, and HR. Interestingly, the N‐terminus of the Rfa2 subunit is hyper‐phosphorylated in response to DNA damage. Rfa2 is also phosphorylated during meiosis by a meiosis‐specific kinase (Ime2) and by a damage‐inducible checkpoint kinase (Mec1). Our goal is to understand the role of Rfa2 N‐terminal phosphorylation in meiosis. To do this, we have generated phospho‐mimetic and non‐phosphorylatable Rfa2 mutants. These mutants show DNA damage‐dependent sensitivities in mitotic cells, and the importance of the phosphorylation state of Rfa2 in meiotic cells is being examined. This work is supported through funds provided by Chemistry and Biochemistry (NDSU) to SJH.
Posttranscriptional regulation of telomerase reverse transcriptase in Human T cells
Authors: Streater Courtney , An Jie , Weng Nan Ping
Published: June 2021

Telomerase, a reverse transcriptase consisting of a catalytic subunit (TERT) and a RNA template (TERC), plays an essential role in maintaining the length of telomeres and cellular replicative lifespan. TERT mRNA is present in resting human T cells but with undetectable level of telomerase activity. It is unclear whether the lack of telomerase activity is a result of non‐functional Alternate Splicing Products (ASP) of TERT mRNA and/or missing proper post‐translational modifications of TERT protein. It was hypothesized that missing proper post‐translational modifications of TERT protein lead to the lack of telomerase activity. We analyzed two common TERT splicing forms in resting and activated human T cell subsets using single cell RT‐PCR method. We found that 1) 39% of the resting T cells expressed TERT full‐length alone, 34% expressed both full‐length and ASP and 27% expressed only ASP; 2) activation decreased expression of full‐length TERT and increased expression of ASP; and 3) all live and dead T cells after activation had ASP. These findings show that resting T cells express mostly full‐length TERT whereas activated T cells express mostly ASP. Further studies will be needed to elucidate this seemingly paradoxical finding. The findings of this study may lead to pharmaceutical interventions to slow or stop the aging process.
Accelerated aging during chronic oxidative stress: a role for PARP‐1?
Authors: Boesten Danielle M , Houben Joyce M , Timmermans Leen , Bast Aalt , Hageman Geja J
Published: June 2021

Oxidative stress plays a major role in the pathophysiology of chronic inflammatory diseases, but it has also been linked to accelerated telomere shortening. Telomeres are specialized structures at the ends of linear chromosomes that protect these ends from degradation and fusion. Loss of telomeric DNA will result in telomere shortening and eventually cellular senescence. Research has shown that poly(ADP‐ribose)‐polymerase‐1 (PARP‐1) plays a role in telomere length regulation. We hypothesized that PARP‐1 plays a role in accelerated aging in chronic inflammatory diseases. In cultured human fibroblasts, effects of mild and strong inhibition of PARP‐1 under conditions of chronic oxidative stress, induced by tert‐butyl hydroperoxide (t‐BHP), were investigated. Fisetin (Fis), a flavonoid, was used as a mild inhibitor and Minocycline (Mino), an antibiotic, as a strong inhibitor of PARP‐1. Results indicated that t‐BHP and Mino alone induced a delay in cell growth and accelerated telomere shortening. However, when Mino was given in combination with t‐BHP, cells did not show a decrease in telomere length. Additionally, both Fis and Mino lowered the transcription of inflammation‐related genes in cells treated with t‐BHP. We conclude that PARP‐1 inhibition appears to be beneficial in conditions of chronic oxidative stress, but may be detrimental under relatively normal conditions.
Integrating undergraduate research/ research training into the curriculum at primarily undergraduate institutions
Authors: Provost Joseph J
Published: June 2021

Research based laboratory courses engage students in a real and meaningful manner immersing students in a research experience. Because this style of teaching laboratories is growing in popularity there is a need to review the current state of research based laboratories and how they are integrated into specific laboratories and throughout an undergraduate curriculum. This presentation will review the benefits and costs of undergraduate research based courses for both students and faculty and address best practice approaches to consider when creating a research based laboratory and undergraduate curriculum. Several case studies from a diverse set of institutions will also be presented as examples of overall curriculum and individual teaching laboratories. Finally, assessment of research based courses and curriculum will be presented.
Integrating undergraduate research/research training into the curriculum at a minority‐serving institution
Authors: Andraos-Selim Cecile
Published: June 2021

In an effort to integrate undergraduate research experience in the curriculum at Hampton University (HU), a Historically Black University, two successful cancer biology and epidemiology curricula were developed in collaboration with the University of Pittsburgh Cancer Institute (UPCI). These curricula included the development of a total of four research‐intense undergraduate courses in cancer biology and epidemiology: a cancer biology and bioinformatics lecture course; a cancer epidemiology lecture course; a cancer biotechnique laboratory course; and a cancer seminar/workshop course. These courses were open to any student who had at least one year of general biology. In addition, a subset of students were recruited not only to participate in these classes but to also spend one summer at either UPCI or the State University of New York ‐ Downstate School of Public Health engaging in cancer and/or epidemiology research. Students enrolled in these curricula gained knowledge in cancer biology, various forms of epidemiological study designs, data analyses, hypothesis generation, results interpretation, manuscript writing, and hands‐on experience in cancer research. This collaboration resulted so far in 22 poster presentations and 7 publications. Five of the 7 publications are co‐authored by students. About 90% of the students involved in the summer research matriculated in either graduate or professional school after graduation from HU. In conclusion, the inclusion of undergraduate research experience in our curricula has enhanced the ability of our students to matriculate in and graduate from graduate and professional schools.

This work was supported in part by two National Cancer Institute P20 grants: 5P20CA101445 and 5P20CA132532.
Specific domains in yeast eukaryotic Initiation Factor (eIF) 4G bias the RNA unwinding specificity of eIF4F towards duplexes with a 5′‐overhang
Authors: Rajagopal Vaishnavi , Park Eun-Hee , Hinnebusch Alan G , Lorsch Jon R
Published: June 2021

Translation initiation factor 4A (eIF4A) is an essential protein in yeast Saccharomyces cerevisiae, and plays a crucial role in the recruitment of mRNA to the 43S preinitiation complex. While the human ortholog of the protein has been extensively characterized, much remains to be elucidated regarding the biochemical properties of the yeast protein. Based on its conserved helicase motifs, eIF4A is believed to unwind secondary structures in the 5′‐untranslated region (UTR) of the mRNA to permit efficient translation. In our present study, we have sought to characterize the RNA binding and unwinding activities of yeast eIF4A by itself and in the context of the eIF4F complex, comprised of eIF4A, eIF4E, and eIF4G. Our results show that the ATPase activity of eIF4A is stimulated >250‐fold when it binds to eIF4E•eIF4G. Unlike its human cousin, the yeast eIF4F complex preferentially unwinds duplexes with a 5′‐overhang. Experiments with deletion mutants of eIF4G indicate that RNA‐binding domains of eIF4G co‐operate to impart this 5′‐dependence to the eIF4F complex.
The mechanism and machinery of spliced leader RNA silencing (SLS), a stress induced mechanism leading to programmed cell death in Trypanosoma brucei
Authors: Michaeli Shulamit
Published: June 2021

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, trypanosomes possess a stress response mechanism, the spliced leader RNA (SL RNA) silencing (SLS) pathway. SLS is induced under prolonged ER stress or when translocation to the ER is perturbed upon RNAi silencing of factors involved in protein translocation across the ER membrane such as (SRP receptor, and the ER translocation factors SEC63 or SEC61). SLS leads to shutoff of SL RNA transcription by avoiding the binding of the SL RNA‐specific transcription factor tSNAP42 to its cognate promoter (Lustig al., EMBO Rep. 2007). Since SL RNA donates the SL exon to all mRNAs via trans‐splicing, SLS induction inhibits mRNA production. Induction of SLS leads to programmed cell death (PCD), evident by exposure of phosphatidyl serine and DNA laddering (Goldshmidt et al., PLoS Pathogens, 2010). We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase‐mediated PCD observed in higher eukaryotes. To investigate the mechanism of SL RNA transcription shut‐off during SLS, the SL RNA transcription complex was purified under SLS and mass‐spectrometry analysis indicated unique phosphorylation of the TATA‐binding protein TBP. A PERK‐like kinase was shown to be involved in SLS signaling, and its silencing abolished SLS induction. Using iTRAQ analysis we identified proteins that their level was elevated during SLS and focused on two proteases; rhomboid and calpain‐like proteases. Silencing of calpain‐like protease abolished SLS. I will discuss our approaches to explore the sequential events and the interplay between the different cascades leading to SLS and PCD.
The intracellular parasite Toxoplasma injects polymorphic proteins into the host cell that subvert host defenses including recruitment of host mitochondria and membrane attack by p47 GTPases
Authors: Pernas Lena , Boyle Jon P. , Fleckenstein Martin A. , Reese Michael L. , Konen-Waisman Stephanie , Howard Jonathan C. , Steinfeldt Tobias , Boothroyd John C.
Published: June 2021

The eukaryotic parasite, Toxoplasma gondii, is unusual in being able to infect almost any cell in almost any warm‐blooded animal. During invasion into the host cell, the parasite injects proteins that commandeer and/or subvert host functions. In this talk, I will describe the molecules Toxoplasma uses to recruit host mitochondria to the parasitophorous vacuolar membrane (PVM) within which the parasites divide. This recruitment has important consequences for how the host senses the parasite as well as for how the recruited mitochondria function. In the second part, I will describe how injected Toxoplasma kinases phosphorylate key host defense proteins, including a class of p47 GTPases encoded by the interferon‐gamma response genes (IRGs) that attack the PVM. Interestingly, this inactivation/phosphorylation is completely dependent on a family of injected pseudokinases that bind the IRGs and “present” them for phosphorylation by the active kinase. Our results on the structural features of these interactions will be discussed. Surprisingly, all of the parasite “effectors” involved in these processes differ between Toxoplasma strains resulting in major differences in their virulence. Speculative but plausible evolutionary reasons for these differences will be discussed.
Ribosomal Protein Transcripts as an endogenous source of nucleotide precursors in Cryptococcus neoformans
Authors: Banerjee Dithi , Panepinto John
Published: June 2021

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised individuals. The spores of C. neoformans establish primary infection in the lungs, a nutrient deprived environment. The ability to adapt and thrive in such an adverse condition is a significant trait in the pathogenesis of Cryptococcus neoformans. Previous studies have shown that ccr4Δ mutant, defective in mRNA degradation, is sensitive to replicative stress. The ccr4Δ mutant stabilizes ribosomal protein (RP) transcripts during temperature stress, which led us to investigate the effects of starvation stress on these transcripts. We hypothesize that when de novo synthesis of nucleotides is inhibited by nutrition deficiency or by the use of mycophenolic acid, the RP transcripts degrade providing nucleotide precursors for the salvage pathway. Northern blot stability assays demonstrated that under conditions of carbon and nitrogen starvation there was rapid degradation of RP transcripts in the wild type whereas they remain stabilized in ccr4Δ mutant. Steady state experiments showed that under similar conditions, there was reduced expression of RP‐transcripts in the wild type in comparison to ccr4Δ mutant. Northern blots for the expression of different de novo and salvage enzymes during carbon starvation showed a higher expression in ccr4Δ compared to H99 which most likely suggests nucleotide deficiency. Kill curve experiments with mycophenolic acid, an inhibitor of de novo synthesis of guanine demonstrated a drastic reduction in the viability of ccr4Δ mutants when compared to H99. This suggests that in the absence of de novo synthesis, internal sources of nucleotide precursors may aid in survival. These data also suggest that RP transcripts may play a significant role in stress adaptation response by providing a potential source of endogenous nucleotides during stress.
Interplay of microRNAs and RNA binding proteins
Authors: Port J. David , Polk Jeremy
Published: June 2021

We have demonstrated previously that β‐adrenergic receptor (AR) mRNAs are targets for RNA‐binding proteins (BPs) that bind to A+U‐rich elements (AREs) within their 3′untranslated regions (UTRs). Our hypothesis is that ARE‐BPs and miRNAs interact affecting each other's ability to modulate gene expression. Herein, we focus on miR let‐7 and the ARE‐BP, HuR.

Results

The human β1‐AR 3′UTR was placed down‐stream of a dual luciferase reporter and cells were treated with miR mimics/inhibitors. Compared to control (1.00), let‐7 mimic down‐regulated LUC/β1‐AR expression (0.61 ± 0.04). Conversely, let‐7 inhibitor increased β1‐AR expression (1.80 ± 0.21). Mutagenesis of the let‐7 site, (−)let‐7, completely abrogated the effects of let‐7 mimic/inhibitor. HuR knockdown (~75%) also resulted in decreased LUC/β1‐AR expression (0.62 ± 0.03). In cells expressing LUC/β1‐AR(−)let‐7, β‐AR down‐regulation 2o to HuR knockdown was attenuated (0.85 ± 0.06). The effects of let‐7 mimic/inhibitor were also examined in cells expressing LUC/β1‐AR(−)ARE. Absence of the ARE completely abrogated the effects of both let‐7 mimic/inhibitor.

Conclusion

The current data supports the conclusion that modulation of β‐AR expression by HuR is let‐7 dependent. Reciprocally, let‐7 function appears dependent on the presence of an ARE and on the proteins that bind to it.
The ER chaperones BiP and Jem1/Scj1 are genetic modifiers of TorsinA, the AAA+ ATPase associated with the neurological disease primary torsion dystonia
Authors: Zacchi Lucia Florencia , Wu Hui-Chuan , Niemeyer Katie , Bell Samantha , Zolkiewski Michal , Brodsky Jeffrey
Published: June 2021

Dystonia is a disease characterized by involuntary and sustained muscle contractions that lead to paralysis and abnormal postures. The most severe type of dystonia, early‐onset Torsion dystonia, is associated with a deletion of a glutamate (ΔE) near the C‐terminus in TorsinA. TorsinA is an endoplasmic reticulum (ER) AAA+ ATPase of ill‐defined function, and the reasons that the ΔE variant is associated with the disease are unclear. Further, the penetrance of the disease is highly variable, suggesting that genetic modifiers affect disease severity. In order to identify modifiers of TorsinA stability and folding, we developed a TorsinA expression system in Saccharomyces cerevisiae. Similar to TorsinA in mammalian cells, TorsinA and TorsinAΔE in yeast associate with the luminal side of the ER membrane and are glycosylated. We found that the stability and glycosylation of TorsinA and TorsinAΔE depend on the yeast ER Hsp70 chaperone BiP and Hsp40 co‐chaperones Jem1 and Scj1. Mutations in TorsinA that prevent glycosylation, ATP binding, or ATP hydrolysis, or that eliminate the hydrophobic domain led to BiP‐dependent degradation, and to differences in protease sensitivity and membrane association. Therefore, we have identified conserved ER chaperones that are critical for TorsinA conformation and stability. We are testing these findings in mammalian systems. Supported by the Dystonia Medical Research Foundation.
Protein Disulfide Isomerases promote the Endoplasmic Reticulum Associated Degradation of diverse substrates using different mechanisms
Authors: Grubb Sarah , Guo Liang , Fisher Edward A. , Brodsky Jeffrey L.
Published: June 2021

Endoplasmic Reticulum Associated Degradation (ERAD) clears misfolded or incorrectly processed proteins from the ER. One family of ER resident proteins that are involved in ERAD and exhibit disulfide redox, isomerization, and chaperone activity is the Protein Disulfide Isomerase (PDI) family. In humans there are twenty PDI homologs, whereas in Saccharomyces cerevisiase there are five. To address substrate specificity among the PDI family members and their mechanisms of action during ERAD, we investigated the contributions of distinct yeast PDIs on the ERAD of model substrates that either contain disulfide bonds or lack cysteines. Through the use of a yeast expression system for Apolipoprotein B (ApoB), which is disulfide‐rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY*, an ERAD substrate containing five disulfide bonds. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to ERAD through different mechanisms.
Characterization of the interaction between Hsp70s and lipids: evolutionary and functional implications
Authors: Nikolaidis Nikolas , McCallister Chelsea , Donnelly Gabrielle , Le Dat
Published: June 2021

Hsp70s are molecular chaperones indispensable for cellular homeostasis under both normal and pathological conditions. Apart from their associations with protein substrates, Hsp70s also associate with membrane lipids. This association has been related to their membrane localization and anchorage, secretion and endocytosis, as well as their functions in stabilizing membranes and cell signaling. Despite the essential role of membrane‐associated Hsp70s in cell survival, their interactions with membranes and lipids, as well as the effect that lipid‐binding has on their chaperone activities remain unknown. To this end, we determined that several eukaryotic and prokaryotic Hsp70s bind to phospholipids with different affinities suggesting that Hsp70s are functionally differentiated with respect to their lipid‐binding properties. We next established that the nucleotide binding domain of Hsp70s contains multiple lipid‐binding sites, a result that was also supported by mutagenesis experiments, which further identified critical residues for different lipids. Furthermore, we determined that lipid‐binding significantly reduces the refolding activity of Hsp70s, suggesting a new regulatory mechanism of chaperone function. Collectively, these results provide fundamental knowledge towards understanding the full spectrum of biologically significant activities mediated by Hsp70‐lipid interactions.

This project was supported by funds from CSUPERB and CSUF to NN.
Analysis of substrate specificity of 6‐hydroxynicotinate‐3‐ monooxygenase (NicC) from Bordetella bronchiseptica
Authors: Bauerle Matthew R. , Ammons William , Shvets Karina , Snider Mark J.
Published: June 2021

Bordetella species are known to catabolize nicotinate to fumarate. In an effort to better understand the mechanism of decarboxylation and subsequent hydroxylation of 6‐hydroxynicotinate (6‐HNA) to 2,5‐dihydroxypyridine (2,5‐DHP), the second step in the conversion of nicotinate to fumarate, the gene coding the putative enzyme (NicC) has been characterized. Analysis of the products of the enzymatic reaction by HPLC‐UV and LCMS are consistent with 2,5‐DHP as the expected product. Steady‐state kinetic analysis of the enzyme activity by isothermal titration calorimetry provides a kcat = 29 +/− 1 s−1, a KM (6‐HNA) = 447 +/− 52 μM and a KM (NADH) = 11 +/− 1 μM. Analysis of the binding of 6‐HNA by following the quenching of the protein's inherent fluorescence by titration yields a KD = 6 +/− 1 μM. The substrate analogue, 6‐ chloronicotinate, known to modulate B. pertussis virulence, is shown to be a non‐competitive inhibitor of NicC. Kinetic activity and thermodynamic binding data will be reported for substrate analogues of NicC in an effort to better understand the structural and electronic determinants of substrate specificity.
Dynamics of induced‐fit in HIV reverse transcriptase specificity and resistance
Authors: Johnson Kenneth A , Kirmizialtin Serdal , Nguyen Virginia , Li An , Elber Ron
Published: June 2021

Structural studies show that nearly every enzyme undergoes a significant change in structure after binding it's substrate; however, the role of substrate‐induced conformational changes in enzyme specificity has been controversial. Recent studies on DNA polymerases show that nucleotide‐induced changes in enzyme structure are key determinants of specificity by showing that a large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. Here we present new experimental and theoretical analysis of the role played by changes in enzyme structure to select a correct substrate during DNA polymerization by HIV reverse transcriptase. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile commensurate with experimental data and provide molecular details defining the large motions responsible for enzyme specificity. The inability of a mismatched nucleotide to lock onto the protein structure shortens its waiting time at the binding site, and hence lowers its reaction probability. In contrast, the initial weak interactions with a correct substrate rapidly grow in strength and number as the enzyme proceeds toward the final closed state leading to alignment of active resides to promote catalysis.
Efficacy and security of 3 analogues of L‐carnitine in vitro and in vivo models
Authors: Rodríguez-Fragoso Lourdes , Gómez-Solis Antonieta , De la Cruz Cordero Ricardo , Esparza Jorge Reyes , Avalos-Soriano Anaguiven , Duarte Miguel Angel
Published: June 2021

The metabolic syndrome (MS) is the common denominator of the relationship between Diabetes Mellitus and cardiovascular risk. The L‐carnitine has proven to be effective in treating a variety of diseases, among them Diabetes Mellitus and hyperlipidemia. The aim was to evaluate the efficacy and security of 3 analogues of L‐carnitine. In vitro model we developed a tolerance to glucose (g) in liver cells, the cells were treated with L‐carnitine and 3 analogues, it was determinate the intracellular values of glucose, cholesterol (c) triglycerides (t) and glycogen (gy). In vivo model we developed a MS model in rats, were treated only with the analogue 3 and evaluated the serum levels of g, c and t, as well as the gy levels in liver and muscle. In vitro and in vivo assay demonstrated that analogue 3 was safe in cells and rats. The results showed a change in the intracellular levels of g, c and t in the liver cells treated with the analogue 2 and 3 and decrease in the serum levels of g and t in rats. The security evaluation did not show evidences of alterations on cell viability and proliferation in human cell lines treated with the analogues and only was observed an increase in the lymphocyte levels in rats. In conclusion, the analog 3 was effective and safe in this study. Research support CONACYT 131321.
Neuronal dCbl downregulates the EGFR‐ERK pathway and controls the production of Insulin‐like Peptides in Drosophila
Authors: Yu Yue , Sun Ying , He Shengqi , Li Wenjun , Liu Yong
Published: June 2021

The Cbl proteins are a family of E3 ubiquitin ligases and adaptor proteins that regulates a diversity of cellular signaling and play essential roles in growth, development and survival. To explore the mechanism by which Cbl exerts its regulatory actions in vivo, here we show that in the fruit fly Drosophila melanogaster, dCbl controls multiple aspects of physiological processes through downregulating the expression of insulin‐like peptides (dilps) in the brain. We found knockdown by RNA interference of the expression of dCbl in neurons or specifically in insulin‐producing cells led to increased sensitivity to oxidative or nutrient stresses, decreased carbohydrate storage, and shortened lifespan. Moreover, neuronal dCbl deficiency caused elevations in the p‐dAkt and p‐dERK, which was accompanied by increased expression of dilps. Interestingly, genetic interaction analysis showed blocking neuronal dEGFR‐dERK signaling abolished dCbl deficiency‐mediated upregulation of dilps. In parallel, suppression of c‐Cbl in rat insulin‐producing INS‐1 cells also increased the production of insulin in an ERK‐dependent manner. Collectively, these results reveal that Cbl downregulation of the EGFR pathway is coupled to the dynamic control of insulin biosynthesis, suggesting Cbl may act through an evolutionarily conserved mechanism to participate in systemic modulation of growth, metabolism and lifespan.
The structural basis for neuropilin‐ligand interactions
Authors: Parker Matthew W. , Li Xiaobo , Guo Hou-Fu , Xu Ping , Vander Kooi Craig W.
Published: June 2021

Neuropilin is an essential cell surface receptor that functions in vascular endothelial growth factor (VEGF) dependent vascularization and semaphorin dependent axon guidance. We demonstrate that neuropilin ligands necessarily possess a C‐terminal arginine. We show that the presence of a C‐terminal arginine is critical for VEGF binding to neuropilin. This result has allowed us to explain the marked difference in the activity of different VEGF‐A splice forms. We further demonstrate that a C‐terminal arginine is critical for the binding of semaphorin to neuropilin, but that this is dependent on furin proteolysis. This modification is found to regulate not only semaphorin binding to neuropilin, but it directly modulates competitive binding between Sema3F and VEGF‐A for Nrp1. Having defined this necessary ligand feature, we are working to define additional molecular interactions that regulate ligand binding specificity. Our work also opens new avenues towards the development of selective neuropilin inhibitors.

Supported by NIH Grants T32HL072743 and R01GM094155.
Mining Open Reading Frames (ORF) of the human secretome identifies a novel candidate ligand in Esophageal Cancer Related Gene‐4 (ECRG4)
Authors: Dang Xitong , Podvin Sonia , Larocca David , Muchinyi Stephen , Coimbra Raul , Eliceiri Brian , Baird Andrew
Published: June 2021

Objective

These studies aimed to use phage display to identify hidden ligands and ligand‐domains in orphan ORF of genes of the human secretome.

Methods

Libraries containing 100–200 base pair fragments were generated by random fragmentation of either the 4.5 Kb human epidermal growth factor (EGF) or the 0.5Kb human ECRG4 ORF using DNase I. DNA fragments were then displayed on filamentous phage and the libraries were biopanned for phage capable of internalizing into PC3 epithelial cells. After PCR recovery of internalized phages, candidate ligands or ligand‐domains were sequenced and nucleotides that were in‐frame with both geneIII and the ORF were identified by comparative analyses.

Results

After one round biopanning, 8 colonies contained a core 53‐aminoacid human EGF ligand, in‐frame with pIII and all derived from the C‐terminus of the EGF pre‐pro‐precursor. When the method was applied to the hormone‐like precursor Ecrg4, a potential ligand, Ecrg4(37–62), was in‐frame to both geneIII and the ORF was identified. The authenticity of this ligand‐domain was confirmed by immunostaining intracellular phages in PC3 and HEK cells and by quantifying recovery using real time PCR of treated PC3 cells and normal human dermal fibroblasts.

Conclusion

The data establish that phage display can be used for mining ORFs of the human secretome and that a ligand‐like domain in the Ecrg4 ORF that can target epithelial cells.
An Unconventional Rab GTPase is Involved in the Regulation of Unique Secretory Organelles in Toxoplasma gondii
Authors: Heng Joanne , Kremer Katrin , Meissner Markus
Published: June 2021

The apicomplexan parasite, Toxoplasma gondii, has a highly polarised secretory system and relies on the sequential secretion of virulence factors from unique organelles (micronemes, rhoptries and dense granules) for parasite invasion and propagation. The timely delivery of proteins to these organelles involves correct protein sorting and transport and thus highly organised trafficking pathways. Rab GTPases (of which apicomplexan parasites have a reduced set) are small G‐proteins that act as molecular switches during the transport of a vesicle from a donor to an acceptor membrane. In T. gondii, the role of the conserved endocytic Rab5 proteins (Rab5A and C) has been modulated to traffick proteins to distinct micronemal subsets. Rab5B is alveolate‐specific and its role in protein trafficking in T. gondii has been investigated using an inducible protein expression system. Over‐expression of Rab5B has a severe effect on parasite growth as a result of disrupted host‐cell egress. The elucidation of Rab5B‐dependent protein trafficking has also highlighted a role in micronemal secretion and may thus indicate a role in signalling pathways important during the parasite lifecycle.

Supported by Wellcome Trust.

Grant Funding Source : Wellcome Trust.
NMR Analysis of Actin Binding Protein Cofilin
Authors: Audette Nicholas Jack , Yan Si , kudryashov Dmitri , Polenova Tatyana
Published: June 2021

The cytoskeleton and its associated proteins are vital to numerous physiological functions including cell migration, mitosis, polarization, and organelle transport and have been connected to numerous physiological disorders. Despite their importance, the function and structure of these proteins are not fully understood because they are not completely soluble and F‐actin lacks long range order and resists crystallization. Proteins of ADF/cofilin family are vital regulators of the actin cytoskeleton in eukaryotes. Binding of cofilin results in dramatic reorganization of F‐actin structure and potentiates filament severing and its accelerated depolymerization from the pointed end. The long term goal of this project is to generate high resolution information on the interaction between cofilin and F‐actin through solid state nuclear magnetic resonance (NMR) spectroscopy. In this experiment, isotopically labeled yeast cofilin was expressed and purified in S. cerevisieae for solution NMR of the free protein. This provides detailed resonance assignments, structural information, and backbone dynamics of the protein. Approximately 60 g of 15N labeled cofilin has been produced at high purity and initial HSQC experiments have been run to assist resonance assignments of the protein backbone. Additionally 15N‐1H dipolar lineshapes have been measured in a residue‐specific way in U‐15N,13C cofilin sample to examine the backbone mobility of the protein on micro‐ to nanosecond timescales.
Cholesterol crystals of atherosclerotic lesions induce endothelial dysfunction via RhoA activation
Authors: Baumer Yvonne , Meiler Svenja , Anastasiadis Pavlos , Allen John , Boisvert William A.
Published: June 2021

A hallmark feature of atherosclerosis is that lipids accumulate beneath the activated endothelium and become chemically modified. Also, pH and temperature changes during atherogenesis can induce subendothelial cholesterol crystal (CC) formation. CC can come in contact with the luminal side of the inflammed endothelium lining the atherosclerotic blood vessel. To determine how CCs might alter the endothelium, human aortic endothelial cells (HAECs) were treated with CC and various measurements were made to assess the endothelial function. We found that 1) CCs added to either the basal or the apical side resulted in breakdown of endothelial barrier integrity, 2) CC treatment induced endothelial gap formation and 3) HAECs cultured on CC‐coated surfaces showed decreased viability. These CC effects, however, were completely abolished when intracellular cAMP levels were increased using Forskolin/Rolipram treatment. To gain mechanistic insight of CC's action we assessed Ca2+‐influx, initial cAMP decrease, ROS formation, Rho GTPase activation levels and gene regulatory mechanisms. Of these, only cAMP levels and RhoA activation affected the endothelial function. In summary, CC that can form on either side of the endothelium can have dramatic effects on the endothelial barrier function by, at least in part, regulating cAMP and RhoA activation. This project is funded by grants R01HL075677 and R01HL081863 to WAB.
PTEN binding properties for different model membrane morphologies
Authors: Gericke Arne , Ross Alonzo H , King Katrice E. , Neuman Brittany M. , Jiang Zhiping
Published: June 2021

PTEN acts as an agonist to PI 3‐kinase signaling by removing the phosphate group in the 3 position of PI(3,4,5)P3. PTEN membrane association and activation requires the interaction with PI(4,5)P2 and PS. In biological membranes, these lipids may form upon interaction with other molecular entities domains that exhibit distinct compositional and physicochemical characteristics. It is the objective of this study to elucidate the relationship between lipid bilayer morphologies and the extent of PTEN binding and PTEN membrane association dynamics.

Methods

To characterize lipid morphologies in the absence and presence of PTEN, we used fluorescence microscopy measurements of mixed lipid giant unilamellar vesicles (GUVs). PTEN binding extent is evaluated using SPR and Trp fluorescence measurements. The binding kinetics of PTEN bilayer interaction is elucidated using stopped‐flow experiments.

Results

The interaction of PTEN with mixed PC/PI(4,5)P2 model membranes results in the formation of PI(4,5)P2 enriched domains. Domain formation in the absence of PTEN is also observed when cholesterol is added when making mixed PC/PI(4,5)P2 vesicles. We find an increased PTEN binding in the presence of PI(4,5)P2 domains, which is probably due to increased local PI(4,5)P2 concentration. This work was supported by NIH R01 NS021716 and NSF CHEM 0922848 and 1058719.
MEMBRANE LIPID COMPOSITION INFLUENCES A NEURONAL CELL FATE DECISION IN C. ELEGANS
Authors: Kauv Kalyann , Bettinger Jill C. , Davies Andrew G.
Published: June 2021

We are determining if a neuronal cell fate decision in Caenorhabditis elegans can be altered by changing membrane lipid composition. We are using as a model of neuronal devleopment the activity dependent cell fate decision made by the AWC neurons. The AWCs are a pair of chemosensory neurons, that express different chemosensory receptors, which we can monitor by observing an asymmetrically expressed reporter construct (str‐2::gfp) that causes GFP to be expressed in one of the AWCs (which we call the AWCON cell.) The AWC neurons negotiate to become “ON” or “OFF” through a connected axon expressing the SLO‐1 channel. During this negotiation, the AWC with more SLO‐1 function decides to express the ON suite of receptors because it has less activity (Bargmann et al, 1999). Previous results from the lab indicate that lipids modulate the function of the slo‐1 BK potassium channel. We observed the AWC cell fate using a functional odor analysis and expression analysis of str‐2::gfp. Slo‐1 gain‐of‐function (gf) and loss‐of‐function (lf) mutants were tested relative to the wild‐type strain. The gain‐of‐function mutation allows a continual flow of potassium out of the cell membrane as its channel is abnormally kept open. This imbalance causes a depressed neuronal function, which we hypothesize can be corrected when the BK potassium channel shifts into thicker parts of the lipid bilayer, acting as physical protection from inappropriate activation. We also hypothesize that mutants can be rescued back to its wild‐type phenotype when triacylglycerol levels within the membrane are restored by deleting the gene lips‐7, a lipase that consumes triacylglycerols. Our results currently have shown that the deletion of lips‐7 in slo‐1 gain‐of‐function mutants partially rescued them back to wild‐type, suggesting the important influence lipids may have on a neuronal cell fate decision.
Dualistic role of tumor‐directed antibodies on carcinoma progression
Authors: Pearce Oliver Matthew , Soto Paula Campos , Secrest Patrick , Varki Nissi , Crocker Paul , Bui Jack , Varki Ajit
Published: June 2021

Immunosurveillance and immunoediting of tumors comprise immune reactions that check and destroy cancer cells, and form the basis of cancer immunotherapy strategies. Paradoxically, immune reactions can also facilitate tumor progression, i.e. the immune response has a dualistic role. It is currently unclear what range of immune response defines the ends of this spectrum for a given tumor. We addressed this question using a humanized murine model in which antibodies against a human‐specific tumor antigen alters progression of tumors bearing the antigen. Tumor growth was stimulated or inhibited over a linear and narrow dose range defining an immune response curve (IRC), i.e., inverse hormesis. Tuning the relative activity of one or more immune reactants further demonstrated the dualistic role of inflammation in tumor progression. The results have implications for the etiology, prevention and treatment of human cancer. This work is supported by a Samuel and Ruth Engelberg/Cancer Research Institute fellowship and the NIH.
Menadione‐Induced Oxidative and Nitrosative Stress In C2C12 Myotubes
Authors: Venkatakrishnan Priya , Panda Satya , Masters Bettie Sue S , Roman Linda J
Published: June 2021

Skeletal muscle cells undergo oxidative challenges due to rapid changes in energy demand, high metabolic activity, and high levels of heme‐containing proteins. Nitric oxide synthases (NOSs) reduce redox‐cycling compounds, forming superoxide anion (O2·), which reacts with NO to form peroxynitrite, and causing oxidative injury. Here, we study the effect of the redox‐cycler, menadione (MD), on C2C12 skeletal muscle myotubes and the involvement of NOS in MD‐induced oxidative stress. Quantitative RT‐PCR showed >2–3‐ fold increase in nNOS expression with MD + NOS inhibitors over MD alone, suggesting that the cells compensate for decreased bioavailable NO. by an increase in nNOS expression. iNOS, NFkB, AKT1 and Cav3 levels were decreased by 40–50% with MD. Expression of the antioxidant protein, GPX1, decreased significantly but SOD1 expression was unaffected. NOS inhibitors moderately restored mRNA expression levels, and SOD1 levels increased >2‐fold. nNOS protein levels increased with MD when compared to the controls. Increased O2· formation and reactive oxygen species formation was also observed in MD‐treated cells as compared to MD + NOS inhibitors, indicating a protective role for NO (Support: NIH GM052419 to BSM and LJR).
Physiological implications and Haematological indices of Chicks fed on Feed based on Biodegraded Agrowastes
Authors: Babalola Musa Oladayo , Magbagbeola Abiola O , Ebuehi Osaretin A , Okochi Ihuaku Veronica , Okafor Uzoma A
Published: June 2021

The long‐term benefits of using agrowaste residues as feedstocks will include, reduced potential for water, air, and soil pollution. The study was designed to find out the response of broiler chicks to agrowastes based formulated feed.

Aboria sawdust was biodegraded using mixed Aspergillus niger and Penicillium chrysogenum culture. Poultry feed was formulated by replacing 25% (Group A), 50% (Group B), 75% (Group C ) and 100% (Group D)maize in conventional feed with degraded sawdust. The grouping of birds followed the standard procedure for animal care. Feeding was for 28 days, the body weight of chicks was monitored and blood analysis was done.

The body weight showed no statistical difference in entire Group. Also, no significant difference in the glucose, Hb, WBC, differential blood count, and PCV (p<0.05) in all the groups as compared to the control. However, there was significant difference in ESR (p<0.05) of Group B and D and no significant difference in Group A and C when compared with the control. There was statistical significant difference in the triglycerides of all the groups except Group D when compared with the control. Cholesterol showed, no statistical significant difference in Group A and B, however, there was a high statistical significant difference in Group C and D. The result indicated the prospect of biodegraded agrowastes utilization for poultry feed formulation.
RGS proteins maintain robustness of GPCR‐GIRK coupling
Authors: Chuang Huai-hu , Chuang Alexander
Published: June 2021

Objective

To elucidate the mechanism underlying sustained GPCR‐GIRK coupling under RGS actions.

Results

Regulators of G‐protein signaling (RGS) are GTPase activating proteins (GAP) that reduce response amplitudes of activated G‐protein coupled receptors (GPCRs). We discovered that, although RGS proteins drastically accelerate kinetics of GPCR‐coupled K+ currents (GIRK), they actually increased amplitudes of inhibitory neurotransmitter‐evoked GIRK currents. The RGS‐Box domain alone is sufficient for stimulation of transmitter activation of K+ currents, but its membrane association enhances the efficiency of stimulation. Moreover, RGS4 mutants with compromised GAP activity still augment GPCR‐GIRK coupling. Among the pertussis toxin sensitive G‐proteins, we found that RGS4 selectively stimulates Gαo to maintain robustness of GPCR‐GIRK coupling, by accelerating activation kinetics of Gαo.

Conclusion

Opposing actions of RGS proteins both stimulate and inhibit G‐proteins to modulate ultimate amplitudes of transmitter‐induced GIRK currents and to differentiate signal intensity coupled to various G‐protein isoforms.