bluuebunny/crossref_metadata_2025_split huggingface dataset:
Perturbation of global host sumoylation by the papillomavirus E6 protein (555.3)
Authors: Wilson Van , Heaton Phillip , Deyrieux Adeline 
Published: June 2021

Human papillomaviruses (HPVs) replicate in the nucleus and modulate several host cell pathways to create a favorable environment for viral reproduction. The HPV E6 proteins utilize the host ubiquitin‐proteosome pathway to target several cellular proteins for degradation. Several HPV proteins are substrates for sumoylation, so the objective of this study was to determine if HPV E6 proteins interacted with or influenced the parallel host sumoylation system. Transient expression of E6 caused an apparent general reduction in the overall level of host sumoylation. To explore the mechanistic basis, we examined the effect of E6 proteins on Ubc9, the sole sumoylation conjugation enzyme. HPV E6 proteins reduced intracellular levels of Ubc9, without affecting transcript levels, so a direct effect on Ubc9 protein stability is likely. Consistent with typical E6‐mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. To examine the effect of E6 on sumoylated proteins in more detail, a fluorescent 2‐dimensional gel system was used to visualize sumoylated proteins. Stable expression of E6 resulted in the loss of 32 sumoylated proteins out of a matched set of over 200 protein spots. Thus, E6 exerts a significant, though selective, effect on host protein sumoylation through its action on Ubc9, which likely helps to create a cellular environment conducive to viral replication.Grant Funding Source: Supported by NIH grant CA089298 to V.G.W.
The Rpb4/7 module connects the Ccr4‐Not complex to elongating RNA polymerase II: implications for the coordination of synthesis and decay (560.10)
Authors: Reese Joseph , Babbarwal Vinod , Miller Jason , Fu Jianhua 
Published: June 2021

It is now known that the synthesis and decay of mRNAs are coordinated; however, the factors involved in this process and the mechanism of how these two distinct events are linked are not known. The Ccr4‐Not complex regulates multiple steps in mRNA metabolism, including RNA polymerase II (RNAPII) elongation and mRNA decay. Like Ccr4‐Not, the Rpb4/7 module of RNAPII has been implicated in both the synthesis and decay of mRNAs and Ccr4‐Not and RPB4 mutants display many similar phenotypes. This suggests that Ccr4‐Not and Rpb4/7 may cooperate to regulate the synthesis and decay of mRNAs. We have shown that Ccr4‐Not directly interacts with RNAPII elongation complexes and stimulates transcription elongation of arrested polymerase in vitro. Here we present new data on the structural requirements for the Ccr4‐Not‐RNAPII interaction and implicate the Rpb4/7 module of RNAPII in the process. Our biochemical data suggests that Rpb4/7 assists in the recruitment of Ccr4‐Not into elongation complexes, which is important for the coordination of transcription elongation and mRNA decay.The coordination of decay and synthesis has been demonstrated during cell stress, and the process is believed to require the remodeling of mRNP complexes and the redistribution of RNA binding factors in the cell. We have mapped the mRNAs bound to Ccr4 and other mRNA decay factors in cells during resting and stressed conditions. The results reveal a dramatic redistribution of decay factors unto mRNAs that correlate with changes in their decay and synthesis. These results provide novel insights into the mechanism of how synthesis and decay pathways are altered during cell stress.Grant Funding Source: NIH GM058672
Calcium‐mediated epigenetic regulation of alternative splicing in cardiomyocytes (560.6)
Authors: Sharma Alok , Lou Hua 
Published: June 2021

In contrast to the well‐studied regulation of tissue‐ and developmental stage‐specific alternative splicing, the regulated alternative splicing changes in cells responding to environmental cues is poorly understood. Here we report our discovery of a previously unknown calcium‐mediated epigenetic program that regulates alternative splicing in mouse cardiomyocytes. We found that elevated calcium levels in cardiomyocytes induced by KCl‐mediated depolarization or caffeine treatment led to a robust and reversible skipping of several alternative exons in a CaMKII‐ and PKD‐dependent manner. We selected two genes that showed increased skipping, NF1 and KTN1, for mechanistic studies. We demonstrated that elevated calcium levels induce a translocation of class II HDACs from the cell nucleus to the cytoplasm leading to histone hyperacetylation throughout the gene body of these two genes. Transcriptional elongation rate analysis indicated a two‐fold increase of elongation rate for these genes (3.2 to 6.8 kb/min for NF1; 2.1 to 3.9 kb/min for KTN1) but not for a gene that showed no change in splicing. The depolarizaition induced skipping can be rescued by siRNA knockdown of histone acetyltransferase CBP or p300, or camptothecin treatment, which is used to slow down transcriptional elongation. These studies have revealed a hidden network of alternative splicing that is regulated epigenetically in cardiomyocytes.Grant Funding Source: NIH
Discovering the pre‐60S ribosome biogenesis factor interactome (560.7)
Authors: McCann Kathleen , Charette J. Michael , Vincent Nicholas , Baserga Susan 
Published: June 2021

Maturation of the 60S ribosomal subunit is a highly coordinated and dynamic process that begins in the nucleolus and ends in the cytoplasm. Over 80 different proteins, including several helicases, are found in the 60S pre‐ribosome and play a role in ribosome assembly. However, little is known about whether subcomplexes exist and how these subcomplexes function and regulate pre‐60S ribosome assembly. To begin to elucidate how the 60S pre‐ribosome is assembled, we performed three iterations of a high‐throughput yeast two‐hybrid analysis. We identified 234 high‐confidence interactions among 61 nucleolar proteins, representing a 7.5‐fold increase from current knowledge. Independent validation experiments confirm interaction in 98% of those tested. The 60s pre‐ribosome interactome has revealed several novel likely subcomplexes. One, formed from RNA helicases essential for growth, suggests the presence of a “helicasosome”. Glycerol gradient sedimentation analysis revealed that these helicases do, in fact, co‐peak as part of a 2 MDa complex. Furthermore, Dbp10, a component of the putative helicasosome, is found in smaller fractions on glycerol gradients suggesting that it is part of a smaller subcomplex as well. Since most of the yeast pre‐60S biogenesis factors are conserved to humans, many of the interactions and potential regulatory mechanisms that we will uncover are likely to be conserved as well.Grant Funding Source: NIHGM52581
rRNA processing intermediates may not contribute to ribosome biogenesis (560.9)
Authors: Nazar Ross , Van Loggerenberg Warren , Nellimarla Srinivas 
Published: June 2021

Much of the ribosomal RNA in a eukaryotic ribosome is derived from a common nucleolar transcript via a seemingly complex cleavage pathway which can vary greatly from organism to organism. In S, cerevisiae, for example, two steps have been reported for the five 5’ ETS but in S. pombe as many as six intermediates have been observed. The significance of these steps in for ribosome biogenesis remains unclear including the differences which are observed between organisms. In this study, the processing of the 5’ ETS in S. pombe cells has been reexamine using RT‐PCR, ligation‐mediated RT‐PCR and targeted mutagenesis. The results suggest that some intermediate steps are very minor or may be artifacts of the mapping techniques. More important, the complete removal of a cleavage site can leave levels of mature rRNA essentially unchanged. Taken together, such observations raise the possibility that at least some intermediates are unnecessary, are rare events or even experimental artifacts which do not contribute to rRNA maturation or ribosome biogenesis.Grant Funding Source: Supported by the Natural Sciences and Engineering Research Council of Canada
Elongation factor 1A binds the 3’ translation element of the barley yellow dwarf virus (566.4)
Authors: Gustilo Estella , Goss Dixie 
Published: June 2021

The Barley Yellow Dwarf Virus (BYDV) is especially devastating due to its global widespread distribution and its infection of staple cereal crops. Our research aims to understand mechanisms of BYDV mRNA translation to gain insight into combating the disease and its dire economic implications. Previous studies have shown that BYDV initiates translation in a way that is unique to most eukaryotic messages. Almost all eukaryotic mRNA consist of a 5’ cap and initiates translation at the 5’ untranslated region (UTR). BYDV mRNA, however, consists of a cap‐independent translation element at the 3’ UTR (BTE). More interestingly, eukaryotic initiation factor 4F (eI4F) binds the 3’ end of BYDB mRNA, at the BTE, and translation occurs when the 3’ UTR interacts with the 5’ UTR. Pull‐down experiments using the BTE RNA as bait have revealed not only eIF4F binding but also a small array of other proteins not present when a nonfunctional BTE (BTEBF) or the 5’ UTR is used as bait. One prominent protein that appears to bind BTE but not the control BTEBF or 5’ UTR RNA is an unknown 50 kDa protein. Using Mass Spectrometry, we identify this protein to be elongation factor 1A (EF1A). EF1A has previously been shown to bind the 3’ UTR of Tobacco Etch Virus (TEV) mRNA and may have a role in in regulation and replication of the viral genes.Grant Funding Source: NSF MCB 1157632
Investigation of Hsp90 regulation by a conserved N‐terminal motif in the Aha1/Hch1 co‐chaperone family (567.6)
Authors: Mercier Rebecca , Mann Udaybir , Wolmarans Annemarie , Horvat Natalie , LaPointe Paul 
Published: June 2021

The molecular chaperone Hsp90 functions in a complex cycle for the maturation and activation of a diverse, but specific, group of client proteins. These clients range from transcription factors to kinases and many are key players in cancer. Consequently, understanding the cycle through which Hsp90 acts is imperative. Hsp90 has a large cohort of co‐chaperone proteins which regulate cycle progression and facilitate client maturation. The co‐chaperone Aha1p is of particular importance as it is the only known co‐chaperone which is a potent stimulator of Hsp90 ATPase activity. The co‐chaperone Hch1p, found in some fungi, is a homologue of Aha1p but regulates Hsp90 function differently. The N‐terminal motif, NxxNWHW, is conserved across the Aha1p/Hch1p family. We show that this motif is necessary for Hch1p function in vivo but is dispensable for the in vitro activity of Aha1p. Functional assays developed in both the model organism Saccharomyces cerevisiae and in mammalian cells reveal the importance of this N‐terminal motif. Additionally, isolation of Hsp90/co‐chaperone complexes provides clues regarding the Hsp90 functional cycle. We are able to provide further support for the dual role of the Aha1p/Hch1p co‐chaperone family in the regulation of Hsp90 activity, both early and late in the Hsp90 cycle.Grant Funding Source: Supported by CIHR and NSERC
The timing of EF‐G:A‐site tRNA distance changes during translocation (569.4)
Authors: Shen Rong , Chen Chunlai , Goldman Yale , Cooperman Barry 
Published: June 2021

During the polypeptide elongation cycle, prokaryotic elongation factor G (EF‐G) catalyzes the coupled translocations on the ribosome of mRNA and A‐ and P‐site bound tRNAs with high precision. However, the detailed dynamics of this important step have not been fully resolved. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Cy3 fluorescent probe attached within domain IV of EF‐G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A‐site tRNA. Two different sites on L11 and two A‐site tRNAs, Phe‐tRNAPhe or Val‐tRNAVal, were labeled with Cy5. During EF‐G occupancy on the ribosome the EF‐G:L11 FRET efficiency is unchanged, whereas for a significant fraction of complexes the EF‐G:tRNA FRET proceeds from high (0.50) to intermediate (0.29) to low (0.20) FRET values, with dwell times of 0.09 s, 1.0 s, and 0.10 s, respectively. This indicates that EF‐G:A‐site tRNA distance increases progressively during translocation, from EF‐G initially bound to a pretranslocation complex, to a translocation intermediate, and finally to a postranslocation complex just prior to EF‐G dissociation. Each change in the FRET value corresponds to a relative distance change of about 5 to 8 Å, so in total, we capture a 13 Å relative movement of A‐site tRNA away from EF‐G during translocation. The dwell times for some EF‐G:A‐site tRNA FRET states are sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state and destabilizing the intermediate FRET state, while leaving the dwell time for the low FRET state unchanged. Each of the translocation inhibitors viomycin and spectinomycin stabilize the high FRET state and destabilize the intermediate FRET state, while blocking formation of the low FRET state. Viomycin leaves the FRET values of the high and intermediate states unchanged, whereas spectinomycin decreases both values.Grant Funding Source: NIH grant R01GM080376
Excess Ca2+ and succinate‐induced mitochondrial H+ permeability (572.2)
Authors: Aldakkak Mohammed , Stowe David , Camara Amadou 
Published: June 2021

Mitochondrial permeability transition pore (mPTP) opening is a key event in ischemia and reperfusion injury. Several proteins were suggested to constitute mPTP, but the molecular nature of mPTP remains elusive. An increased probability of mPTP opening when using mitochondrial complex I substrates led to a suggestion that complex I might constitute part of the pore. In this study, we entertained the idea that complex I, under certain conditions that lead to excess reactive oxygen species generation, may increase permeability across mitochondrial membranes that is not the typical mPTP. Guinea pig heart mitochondria were suspended in experimental buffer +/‐ excess Ca2+ and supplemented with either pyruvate or succinate. Excess Ca2+ combined with succinate induced mitochondrial permeability that was mainly due to H+ leak as evidenced by matrix acidification without a change in mitochondrial volume. This was associated with impaired Ca2+ uptake. Adding rotenone restored/prevented matrix acidification (H+ leak) and improved Ca2+ uptake in a time dependent manner. We propose that under conditions similar to those that occur during and after extended ischemia i.e., excess Ca2+ with high succinate, mitochondrial complex I or another site may become permeable to H+. The mechanism for this H+ permeability requires further investigation.Grant Funding Source: Supported by grants from the National Institutes of Health (R01HL095122,A.K.S.Camara/R.K.Dash)
Regulation of pyruvate dehydrogenase phosphorylation by hypoxia (572.4)
Authors: Waters Alicia , Zhdanov Alexander , Papkovsky Dmitri 
Published: June 2021

Pyruvate dehydrogenase (PDH), a mitochondrial enzyme, converts pyruvate formed by glycolysis into acetyl‐CoA, thus feeding the Krebs cycle. PDH can be inactivated by phosphorylation via pyruvate dehydrogenase kinase (PDK1), which is activated under hypoxic conditions.The levels of PDH (total and phosphorylated protein) , PDK1 and Hypoxia Inducible Factor (HIF‐1α and HIF‐2α) were analysed in human colon cancer cells HCT116 wild type and SCO2‐/‐ (deficient in complex IV of the respiratory chain) grown under 20.9% and 3% O2.Surprisingly, under normoxia the levels of active non‐phosphorylated PDH were substantially higher in the low respiring HCT116 SCO2‐/‐ cells. Under continuous hypoxia WT cells exhibited lower intracellular O2 coupled to the significant elevation of HIF‐α and PDK1 protein levels. A proportion of the inactive phospho‐PDH increased in both cell lines. Interestingly, in SCO2‐/‐ cells the level of phospho‐PDH became higher than in WT cells.Our data on PDH phosphorylation state indicates that regulation of the enzyme activity under normoxia and hypoxia can occur in a manner independent of HIF‐signalling, mitochondrial respiration and the demand of the Krebs cycle in acetyl‐CoA.Grant Funding Source: EU FP7 Marie Curie ITN Program “Chebana”, 733 grant agreement no. 264772.
Yeast cells expressing the human mitochondrial DNA polymerase reveal correlations between polymerase fidelity and human disease progression (573.1)
Authors: Qian Yufeng , Kachroo Aashiq , Yellman Christopher , Marcotte Edward , Johnson Kenneth 
Published: June 2021

Mutations in the human mitochondrial polymerase (Pol‐γ) are associated with various mitochondrial disorders. To correlate biochemically quantifiable defects resulting from point mutations in Pol‐γ with their physiological consequences, we created ‘humanized’ yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol‐γ. In spite of differences in the replication and repair mechanism, the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and the yeast helicase. We examined the effects of four disease‐related point mutations (S305R, H932Y, Y951N and Y955C) and an exonuclease‐deficient mutant (D198A/E200A). In haploid cells, each mutant results in mtDNA depletion, increased mutation frequency leading to reductions in mtDNA content and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild‐type Pol‐γ suppresses mutation‐associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content and depolarized mitochondrial membranes. The severity of the Pol‐γ mutant phenotype in heterozygous diploid humanized yeast correlates with the age of disease onset and the severity of symptoms observed in humans.Grant Funding Source: Supported by NIH GM044613
Effect of palmitate on the metabolic fate of leucine and vice versa in primary adipocytes of a rat model of diet‐induced obsesity (575.7)
Authors: Salinas Rubio Daniela , Torres Nimbe , Tovar Armando , Noriega Lilia 
Published: June 2021

Metabolic profiling studies have spotlighted altered fatty acid (FA) or branched chain amino acid (BCAA) oxidation that marks the obese and insulin‐resistant phenotype. Interestingly, mRNA expression of Carnitine palmitoyltransferase I (CPT1) ‐the rate‐limiting enzyme of FA oxidation‐ and Isovaleryl‐CoA dehydrogenase (IVDH) ‐a mitochondrial enzyme involved in BCAA oxidation‐ are inversely correlated in the livers of 41 recombinant inbred mouse strains of the BxD genetic reference population (http://www.genenetwork.org/). The inverse correlation between CPT1 and IVDH suggest that a high rate of FA oxidation could decrease BCAA oxidation and vice versa. To test this hypothesis, we have evaluated the effect of increasing concentrations of palmitate or leucine in the metabolic fate of leucine or palmitate in primary adipocytes of a rat model of diet‐induced obsesity. We incubated primary adipocytes with: 1) 0, 250, 500 or 1000 µM of palmitate and evaluated the oxidation, incorporation to lipids or proteins of [U‐14C]‐leucine; or 2) 0, 250, 500 and 1000 µM of leucine and determined the oxidation, incorporation to lipids or proteins of [1‐14C]‐palmitic acid. Interstingly, palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose‐dependent manner. Furthermore, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins in a dose‐dependent manner. Overall, our results demonstrate that palmitate affects the metabolic fate of leucine and that leucine modifies the metabolic fate of palmitate in primary adipocytes of a rat model of diet‐induced obsesity.Grant Funding Source: CONACYT 155949
Increased TCA cycle intermediates in response to diet with Deanna protocol in ALS mouse model (578.3)
Authors: Ari Csilla , Poff Angela , Held Heather , Fiorelli Tina , Goldhagen Craig , D`Agostino Dominic 
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons and is characterized by progressive muscle weakness, paralysis, and, eventually, death. There is no cure for ALS. This metabolic profiling study was conducted to determine the metabolic impacts of a multi‐component experimental nutritional regimen known as the Deanna Protocol (DP) to gain insights into possible mechanisms by which this diet is reported to help slow the progression of ALS.In this 30 sample study SOD1 G93A mice were fed for 6 weeks a standard diet (CTRL) or diets representing a low (12%) or high (22%) dose of DP. Comparison of the plasma global biochemical profiles were performed at endpoint by Metabolon that revealed key metabolic differences.The DP diet dose‐dependently increased compounds associated with glycolysis and the tricarboxylic acid (TCA) cycle. Alpha‐ketoglutarate, a key ingredient of DP, pyruvate, lactate succinate, fumarate, malate and phosphatidylcholine (PC) metabolites were elevated in the high DP group relative to the CTRL. Elevations of intermediates of fatty acid oxidation (FAO) and ready FAO substrates indicated a likely increased beta‐oxidation in response to the DP diet.Substantial build‐up of TCA cycle components combined with acetyl‐CoA provided by increased beta‐oxidation, likely led to greater mitochondrial ATP production, which might be the reason of improved motor function and slower progression of disease.Grant Funding Source: Winning the Fight Foundation
Molecular cloning and biochemical characterization of laccase from Rigidoporus vinctus (583.3)
Authors: Huang Jenq‐Kuen , Kuo Chia‐Yu , Pang Ka‐Lai , Wen Lisa , Lin Chi‐Tsai 
Published: June 2021

Laccases are multi‐copper oxidases that widely distributed in plants and fungi. The enzyme catalyzes oxidation of various compounds including phenolic and non‐phenolic, and has been used in many industrial processes such as paper industry, biobleaching and bioremediation. A full‐ length cDNA (1767 bp) encoding a putative Lac from Rigidoporus vinctus was cloned by polymerase chain reaction (PCR). The coding region of RvLac encodes 517 amino acids which contained four conserved putative copper‐binding regions. To further characterize the RvLac, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli BL21 (DE3)pLysS. The expression of the Lac was induced by IPTG and the recombinant His‐tagged Lac was purified by Ni2+ ‐nitrilotriacetic acid Sepharose superflow column. The purified enzyme was analyzed by 12 % SDS‐PAGE. Furthermore, the enzyme activity and kinetics were determined by measuring 2,2‐azino‐bis 3‐ethylbenzothiozone‐6‐sulphonic acid (ABTS) oxidation. The enzyme is active under a broad pH range and is most active at pH 6‐10.Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant NSC 100‐2313‐B‐019‐003‐MY3