bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S‐methylmethionine (555.2)
Authors: Patananan Alexander , Clarke Steven
Published: June 2021

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that produce secondary metabolites including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA show decreased virulence coupled with reduced secondary metabolism. Although LaeA contains the motifs characteristic of seven‐beta‐strand methyltransferases, a methyl‐accepting substrate of LaeA has not been identified. In this work, we did not find a methyl‐accepting substrate in A. nidulans with various assays including in vivo S‐adenosyl‐[methyl‐3H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self‐methylate. Amino acid hydrolysis of radioactively‐labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine‐207. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S‐adenosylmethionine, but also provide insights into the physiological function of LaeA.

Grant Funding Source: Supported by Ruth L. Kirschstein National Research Service Award GM007185
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.
Regulation of Snail stability by HIPK2‐mediated phosphorylation (555.7)
Authors: Na Wooju , Choi Dong Wook , Choi Cheol Yong
Published: June 2021

Local invasion appears to bean initial and essential step in the malignancy of carcinoma and it leads to the generation of fatal distant metastasis. During tumor invasion, changes in cell adhesion and migration are reminiscent of an important developmental process termed epithelial‐mesenchymal transition(EMT). Snail is a critical regulator of multiple signaling pathways that lead to EMT and its expression is tightly regulated. In this study, we show that HIPK2, a novel negative regulator of Snail, targets the Snail for phosphorylationleading to proteasomal degradation under normal culture condition. HIPK2 phosphorylates at multiple serine residues which partially overlaps with the GSK3β‐mediated hosphorylation sites. In hypoxic condition, however, HIPK2 is destabilized in a Siah‐2‐dependent manner and Snail is stabilized.These results demonstrate that HIPK2 is a novel regulator of Snail at the protein level and participate in EMT through regulation of Snail stability.
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
Examination of growth phenotypes and protein stability in a Saccharomyces cerevisiae model for TPI deficiency (578.2)
Authors: Barnes Carol , Koziara Kortnee , Lellock Amber , Fisher Isaac , Hrizo Stacy
Published: June 2021

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing various TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. In this study, we examined the cell growth rates of multiple mutant TPI strains by growth curve analysis and spot plate analysis. In addition, we examined the stability of the TPI proteins by cycloheximide chase at high temperature. Interestingly, not all of the mutations resulted in significant changes in growth rates. However, the mutation M80T was associated with the most severe growth defects at all temperatures and the M80T protein exhibited the highest degree of instability at high temperature. Furthermore, the mutation E104D exhibited growth defect and protein instability only at high temperature, suggesting that it is a temperature sensitive allele. Other TPI mutant proteins exhibited varying levels of instability, but no effect on growth. This suggests that protein instability is not the only mechanism of pathogenesis in TPI deficiency.
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
Suramin mediated SIRT5 oligomerization (582.6)
Authors: Yu Junru , Leedahl Travis , Singh Raushan , Mueller Dustin , Nyren Erin , Mallik Sanku , Srivastava D.K.
Published: June 2021

Silent information regulator2 (sir2) proteins, or sirtuins, are nicotinamide adenine dinucleotide dependent lysine deacetylase. There are 7 human sirtuins, which have linked to a broad range of biological functions including aging, metabolism, cell cycle and apoptosis, and are considered as therapeutic targets. SIRT5 is the only enzyme that also possesses NAD+ dependent desuccinylase activity, which is important for modulating the urea cycle. Crystallization data shows that the binding of a sirtuin inhibitor, suramin, with SIRT5 leads to protein dimerization. Here we describe the detailed binding and inhibition mechanism of suramin to SIRT5. We also did comparative study with half suramin, which binds to SIRT5 with lower affinity. The data suggest that suramin bridging leads to dimerization of SIRT5 and improves the binding affinity. SIRT5 oligomerization state varies accordingly to the change of suramin/SIRT5 ratio. Our kinetic data also shows that suramin competes with both the succinylated lysine and NAD+, which explains the discrepancy between the binding affinity (2.5µM) and inhibition constant (40µM). The overall study may provide insight for the rational design of more potent inhibitors against sirtuins.
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
Expression and purification of a potential antifolate target, dihydrofolate reductase from B. malayi (584.7)
Authors: Sanchez Karla , Gubler Ulrich , Siekierka John , Goodey Nina
Published: June 2021

Brugia malayi is one of the three causative parasites of lymphatic filariasis, a neglected parasitic disease. There is current literature suggesting that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Conditions for the expression and purification of B. malayi DHFR have not yet been investigated. As such, this study aims to establish ideal conditions to express and purify B. malayi DHFR and to determine Michaelis‐Menten kinetic values for the enzyme (kcat, Km, Vmax). The gene for B. malayi DHFR was designed, incorporating an N‐terminal His6‐tag, synthesized, and subcloned into pET15b, an E.coli expression plasmid. Expression conditions for BL21 DE3 cells were optimized at 30°C. Purification of the enzyme was completed using IMAC chromatography, eluting with 250 mM imidazole in PBS. Further work consists of optimizing protein yield, determination of kcat, Km, and Vmax and determination of Ki and IC50 values for a set of existing DHFR inhibitors such as trimethoprim and pyrimethamine, to validate B. malayi DHFR as a priority drug target.
Evaluation of new cathepsin B inhibitors (585.2)
Authors: Forgas David , Wen Lisa , Huang Jenq‐Kuen , McConnell Rose
Published: June 2021

Cathepsin B, a cysteine protease that naturally occurs in human cells, is primarily responsible in protein degradation and bone resorption. However, Cathepsin B has been observed in excessive concentrations in patients suffering from certain cancers, as well as Alzheimer's Disease. Though the direct involvement of Cathepsin B within these cells is not currently known, the potential medical benefits of reducing Cathepsin B activities on these cells are promising. The purpose of this research was to evaluate the activity of 5 new synthetic inhibitors of Cathepsin B and to observe the effectiveness of each inhibitor by determining their half maximal inhibitory concentration (IC50). Effectiveness of each inhibitor was determined by the enzyme's ability to cleave the chromogenic substrate, N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride. The activity of the enzyme was observed at 405 nm over a span of 3 minutes. A dose‐response curve was then constructed to interpret the results. The benefits of this research could possibly pave the way for future anti‐cancer treatments, as well as other diseases characterized by over‐expression of cathepsin B.

Grant Funding Source: National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1) and WIU
The calcineurin signaling network evolves via conserved kinase‐phosphatase modules that transcend substrate identity (586.3)
Authors: Cyert Martha , Goldman Aaron , Roy Jagoree , Bodenmiller Bernd , Wanka Stefanie , Landry Christian , Aebersold Ruedi
Published: June 2021

To define the first functional network for calcineurin, the conserved Ca2+/calmodulin‐regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by co‐purification and dephosphorylation assays. This ‘calcineurinome’ establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the calcineurinome by acquiring a calcineurin‐recognition motif. Calcineurin substrates in yeast and mammals are distinct, due to network rewiring, but, surprisingly, are phosphorylated by similar kinases. We postulate that co‐recognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin‐kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase‐phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop and establish common regulatory motifs in signaling networks.

Grant Funding Source: Supported by NIH 5R01GM048729
A CtBP‐miRNA network links metabolism with the acquisition of stem cell traits and drug resistance (59.4)
Authors: Byun Jung , Gardner Kevin
Published: June 2021

One of the most common causes of mortality in breast cancer is recurrence and/or the development of resistance to chemotherapy. In particular the acquisition of multiple drug resistance (MDR) has posed one of the greatest challenges in treating breast cancer. Recent findings indicate that some forms of drug resistance can be regulated by microRNAs (miRNAs), a relatively newly described class of non‐coding genes that can dramatically influence gene expression by controlling mRNA degradation and/or translation inhibition. This has led to numerous pre‐clinical therapeutic strategies to modulate or reverse drug resistance by miRNA targeting. The C‐terminal binding protein (CTBP) is an epigenetic regulator that is activated in the presence of NADH to recruit and target a variety of histone modifying complexes and transcriptional regulators to chromatin, thus providing a defined mechanism through which carbohydrate metabolism can drive epigenetic regulatory events. We have recently shown that CtBP drives epithelial mesenchymal transition (EMT), genome instability, and the acquisition of stem cell tumor‐initiating cell‐like pathways in breast cancer. Interestingly we find that CTBP also directly targets and inhibits miR‐Let7i expression, a central regulator of cellular pluripotency that downregulates major drivers of pluripotency including Lin28, and HMG2a. miR‐Let7i also represses expression of the IGF2BP1,2,3 proteins that stabilize the mRNA transcripts of HRAS, MYC and MDR1. Moreover, it has also been recently shown that miR‐137 inhibits CtBP expression. This surprising confluence of pathways suggest a regulatory CtBP‐miR network in which CtBP and miR‐ Let7i act as major hubs that provide means through which metabolic imbalance can influence both the acquisition of drug resistance and tumor‐initiating cell like properties.
Differentiation of equine mesenchymal stromal cells into neural cells: potential for clinical applications (596.4)
Authors: Cruz Villagran Claudia , Amelse Lisa , Neilsen Nancy , Dhar Madhu
Published: June 2021

Peripheral nerve injuries cause poor performance in horses. They are difficult to manage and clinicians mostly rely upon physical therapy and anti‐inflammatories. The long term effects are time and personnel consuming. The aim of this research is to test the ability of equine mesenchymal stromal cells (MSCs) to differentiate into neuronal lineage, and trans‐differentiate into Schwann cells for nerve regeneration. Bone marrow‐derived MSCs were obtained from 3 young and 4 adult horses. Firstly, their stemness was demonstrated. Low passaged cells were neuronally induced in 24 well plates and assessed by microscopy at 3, 6, 12, 24 and 48 h. Results showed that the plate PRIMARIA™ supported proliferation and differentiation. Survival was improved by high number of cells. Morphological changes were observed as early as 3 hours; at 12 h approximately 60% of the cells were neuron‐like. Morphology was similar in all horses, but proliferation was variable. Protein concentration was influenced by cell number and method of protein extraction. Expression of neuronal progenitor proteins, vimentin, β3 tubulin and GFAP, was detected by immunoblot analysis; nestin was not evident. Results demonstrate that equine MSCs can differentiate into neuronal lineage. Future work is aimed to commit these cells into Schwann cells. Expression of additional neural‐specific markers, S100B, Krox20 and CD104 will be tested.
Erythrocyte membrane proteins in sickle cell disease (598.1)
Authors: Craik James , Al‐Shammali Hadeel , Adekile Adekunle , Khan Islam
Published: June 2021

Sickle cell disease (SCD) is a common inherited hematological disorder where mutation causes substitution of valine for glutamate in β‐globin, giving hemoglobin S (HbS). HbS polymerization and cell dehydration cause cell distortion (sickling) at low oxygen tension, often leading to microvascular occlusion. Membrane changes are found in SCD, and increased Na+/H+ exchange activity mediated by NHE‐1 transporter has been reported. This study assayed levels of key nutrient (GLUT‐1 and hENT‐1) and ion (AE‐1 and NHE‐1) transporters to better understand pathophysiological changes in SCD.

Membrane protein levels were examined by SDS‐PAGE and quantitative immunoblot analysis (against a common standard arbitrarily set as 100%). Sodium pump (Na/K ATPase) activity was followed by surrogate assay of p‐nitrophenylphosphatase (PNPase) activity.

HPLC analysis confirmed HbSS and control phenotypes. SCD showed decreased expression of hENT‐1 (75.33 ± 3.75 n=11 vs. 98.09 ± 7.67 n=11; p < 0.05), elevation of NHE‐1 (108.92 ± 5.01 vs. 93.19 ± 4.90; p < 0.05) and GLUT‐1 (116.92 ± 5.45 vs. 90.94 ± 2.55; p < 0.05) but no significant change in AE‐1 (96.89 ± 2.99 vs. 95.55 ± 2.48) between normal and SCD samples. PNPase activity was increased in SCD (1.303 ± 0. 211 vs. 0.729 ± 0.137 µmole/mg/ml, n= 7; p < 0.05).

NHE‐1 increase is consistent with reported Na‐H exchange and decreased hENT‐1 may affect plasma and intracellular purine levels in SCD.

Grant Funding Source: Supported by the College of Graduate Studies, Kuwait University, Research Sector Kuwait University R
Delineating desmoglein adhesive properties on living keratinocytes by atomic force microscopy (60.2)
Authors: Vielmuth Franziska , Waschke Jens , Spindler Volker
Published: June 2021

We used atomic force microscopy (AFM) to simultaneously visualize the topography of living human keratinocytes and the distribution of the Ca2+ dependent adhesion molecule desmoglein 3 (Dsg3) in the cell membrane. Adhesion events were detectable in clusters on the cell surface and at areas of cell‐cell contact. These events were blocked by removing Ca2+ and by addition of Dsg3‐specific autoantibodies. On the cell surface, the binding forces of Dsg3 molecules were lower compared to areas of cell‐cell contact (37±2pN vs. 45±2pN), indicating that Dsg adhesive properties are not uniform but can be modulated depending on the localization. We further studied morphologic changes induced by autoantibodies targeting Dsg derived from patients with the disease pemphigus vulgaris. Autoantibody incubation resulted in separation of plasma membranes between desmosomal adhesive complexes and caused retraction of intermediate filaments, the latter of which are detectable as bundles of fiber‐like structures attached to desmosomes in the topography image. These data are in line with well‐established morphological changes induced by pemphigus autoantibodies such as interdesmosomal widening and collapse of the keratin filament cytoskeleton. Taken together, the combination of topographical and functional AFM imaging on living cells represents a powerful tool for research of cell adhesion receptors.
Accelerated Plb1‐mediated turnover of phosphatidylcholine upon loss of Ypk1 in S. cerevisiae (600.2)
Authors: Surlow Beth , Cooley Ben , Patton Vogt Jana
Published: June 2021

Ypk1, the yeast homolog of the human serum‐ and glucocorticoid‐induced kinase (Sgk1), is a serine/threonine protein kinase that regulates several aspects of lipid metabolism, including sphingolipid homeostasis and flippase activity. Our results indicate Ypk1 impinges upon the turnover of the major phospholipid, phosphatidylcholine (PC). In pulse‐chase experiments, a ypk1Δ strain displays increased deacylation of PC and the resultant production of glycerophosphocholine (GroPCho) compared to wildtype. Loss of Plb1, a B‐type phospholipase, in the ypk1Δ strain largely abrogates the increase in PC turnover. Furthermore, Plb1 protein and message levels are elevated in a ypk1Δ strain compared to wildtype. A ypk1Δ plb1Δ mutant displays slowed growth and reduced cell wall integrity compared to either of the single deletion strains, suggesting that upregulated Plb1 activity is beneficial in a ypk1Δ backgound. We are in the process of further examining the mechanism by which Ypk1 regulates Plb1. Preliminary data indicate that the regulation of PLB1 expression by Ypk1 is linked to altered sphingolipid metabolism. Our data suggests a novel role for Ypk1 in the regulation of Plb1 mediated PC turnover.

Grant Funding Source: Supported by GM NIH R15 GM104876 to JPV
Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus‐free betaine‐lipid diacylglyceryl‐N,N,N‐trimethylhomoserine (605.22)
Authors: Riekhof Wayne , Naik Surabhi
Published: June 2021

Diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) is a phosphorus‐free betaine‐lipid analog of phosphatidylcholine (PtdCho) synthesized by many soil bacteria, algae, and non‐vascular plants. Synthesis of DGTS and other phosphorus‐free lipids in bacteria occurs in response to phosphorus (P) deprivation, and results in the replacement of phospholipids by non‐phosphorous lipids. The genes encoding DGTS biosynthetic enzymes have previously been identified and characterized in bacteria and the alga Chlamydomonas reinhardtii. We now report that many fungal genomes, including those of plant and animal pathogens, encode the enzymatic machinery for DGTS biosynthesis, and that fungi synthesize DGTS during P‐limitation. This finding demonstrates that replacement of phospholipids by non‐phosphorous lipids is a strategy used in divergent eukaryotic lineages for the conservation of P under P‐limiting conditions. Mutants of Neurospora crassa were used to show that DGTS synthase encoded by the BTA1 locus is solely responsible for DGTS biosynthesis, and is under the control of the fungal phosphorus‐deprivation regulon, mediated by the NUC‐1/Pho4p transcription factor. Furthermore, we describe the rational re‐engineering of lipid metabolism in the yeast S. cerevisiae such that PtdCho is completely replaced by DGTS, and show that membrane and organelle biogenesis are largely unaffected in the engineered strain.

Grant Funding Source: National Institutes of Health
Detecting O‐GlcNAc using in vitro sulfation (607.1)
Authors: Wu Zhengliang , Robey mathew , Tatge Timothy , Leymarie Nancy , Zou Yonglong , Zaia Joseph
Published: June 2021

O‐GlcNAc glycosylation, the covalent attachment of N‐acetylglucosamine to serine and threonine residues of proteins, is a post‐translational modification that shares many features with protein phosphorylation. O‐GlcNAc is essential for cell survival and plays important roles in many biological processes (e.g., transcription, translation, cell division) and human diseases (e.g., diabetes, Alzheimer’s disease, cancer). However, detection of O‐GlcNAc is challenging. Here, a method for O‐GlcNAc detection using in vitro sulfation with two GlcNAc‐specific sulfotransferases, CHST2 and CHST4, and the radioisotope 35S is described. Sulfation on free GlcNAc is first demonstrated, and then on O‐GlcNAc residues of peptides as well as nuclear and cytoplasmic proteins. It is also demonstrated that the sulfation on O‐GlcNAc is sensitive to OGT and OGA treatment. The labeled samples are separated on SDS‐PAGE and visualized by autoradiography. Overall, the method is sensitive, specific and convenient
Dynamic interactions of TATA‐box binding protein with promoters is regulated by GlcNAcylation (607.17)
Authors: Hardivillé Stéphan , Han Guanghui , Ma Junfeng , Hart Gerald
Published: June 2021

The GlcNAcylation is an intracellular posttranslational modification dynamically controlled by two enzymes. OGT catalyzes the transfer of a single O‐GLcNAc moiety from UDP‐GlcNAc to a Ser or Thr residue, whereas OGA removes it. Alterations of the GlcNAcylation profile appear in neurodegenerative disorders, diabetes and cancer. The GlcNAcylation rate of proteins is tightly correlated to the concentration of UDP‐GlcNAc, the end product of the hexosamine biosynthetic pathway, which varies according to environmental and nutritional factors. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAc. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex (PIC). DNA‐bound TBP can also be recognized by NC2 and TAF172. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with TAF172 forms B‐TFIID and promotes its release.

Here, we showed that DNA‐bound TBP is modified by O‐GlcNAc on its N‐terminal domain and this GlcNAcylation impairs the recruitment of TAF172 to the DNA‐bound TBP in vitro and the formation of B‐TFIID in vivo. Indeed, using STZ‐treated rat models, we showed that TBP/TAF172 association is decreased in diabetes. Owing the fact that B‐TFIID dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that B‐TFIID formation and TBP dynamics on gene promoters is regulated by O‐GlcNAc.

Grant Funding Source: Supported by NIH R01CA42486, R01DK61671, N01‐HV‐00240, P01HL107153
HTLV‐1 Tax protein induces autophagy via IKK in human astroglioma cells: a protective mechanism against death receptor‐mediated apoptosis (610.1)
Authors: Zheng Dexian , Wang Weimin , Zhou Jiansuo , Shi Juan , Liu Yanxin
Published: June 2021

The human T‐cell leukemia virus type 1 (HTLV‐1) Tax protein is hypothesized to play a central role in the process that leads to adult T‐cell leukemia/lymphoma and tropical spastic paraparesis/HTLV‐1‐associated myelopathy. A recent study showed that Tax can enhance autophagy in peripheral T cells, but the function and underlying molecular mechanism have not been elucidated. We demonstrate here that Tax increases autophagy, which plays a protective role in death receptor (DR)‐mediated apoptosis of U251 astroglioma cells. The cellular FLICE‐inhibitory protein (c‐FLIP), which is upregulated by Tax, also contributes to this apoptotic resistance. Both Tax‐induced autophagy and c‐FLIP expression require the activation of IκB kinase (IKK). Furthermore, Tax‐induced c‐FLIP expression is regulated via Tax‐IKK‐NF‐κB pathway, whereas Tax‐triggered autophagy depends upon the activation of IKK, but not the activation of NF‐κB. In addition, DR‐mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by inhibitors of autophagy. These data reveal a novel function of Tax in the regulation of autophagy. Therefore, Tax‐induced autophagy might represent a new pathogenic mechanism involved in the development of HTVL‐1 related‐diseases and, thus, a possible target for therapeutic interventions.