Browsing by Subject "protein"
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Item Open Access Expression, tandem repeat copy number variation and stability of four macrosatellite arrays in the human genome(2010) Tremblay, Deanna C; Alexander, Graham; Moseley, Shawn; Chadwick, Brian PBackground: Macrosatellites are some of the largest variable number tandem repeats in the human genome, but what role these unusual sequences perform is unknown. Their importance to human health is clearly demonstrated by the 4q35 macrosatellite D4Z4 that is associated with the onset of the muscle degenerative disease facioscapulohumeral muscular dystrophy. Nevertheless, many other macrosatellite arrays in the human genome remain poorly characterized. Results: Here we describe the organization, tandem repeat copy number variation, transmission stability and expression of four macrosatellite arrays in the human genome: the TAF11-Like array located on chromosomes 5p15.1, the SST1 arrays on 4q28.3 and 19q13.12, the PRR20 array located on chromosome 13q21.1, and the ZAV array at 9q32. All are polymorphic macrosatellite arrays that at least for TAF11-Like and SST1 show evidence of meiotic instability. With the exception of the SST1 array that is ubiquitously expressed, all are expressed at high levels in the testis and to a lesser extent in the brain. Conclusions: Our results extend the number of characterized macrosatellite arrays in the human genome and provide the foundation for formulation of hypotheses to begin assessing their functional role in the human genome.Item Open Access Mono- or Double-Site Phosphorylation Distinctly Regulates the Proapoptotic Function of Bax(2010) Wang, Qinhong; Sun, Shi-Yong; Khuri, Fadlo; Curran, Walter J; Deng, XingmingBax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)-> alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax(-/-) MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S -> A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S -> E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S -> A or S -> E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment.Item Open Access Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux(2010) Ma, Nan; Tulu, US; Ferenz, Nick P; Fagerstrom, Carey; Wilde, Andrew; Wadsworth, PatriciaTPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.Item Open Access Quantification of the Binding Properties of Cu2+ to the Amyloid Beta Peptide: Coordination Spheres for Human and Rat Peptides and Implication on Cu2+-Induced Aggregation(2010) Hong, Lian; Carducci, Tessa M; Bush, William D; Dudzik, Christopher G; Millhauser, Glenn L; Simon, John DThere is no consensus on the coordinating ligands for Cu2+ by A beta. However, the differences in peptide sequence between human and rat have been hypothesized to alter metal ion binding in a manner that alters Cu2+-induced aggregation of A beta. Herein, we employ isothermal titration calorimetry (ITC), circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy to examine the Cu2+ coordination spheres to human and rat A beta and an extensive set of A beta(16) mutants. EPR of the mutant peptides is consistent with a 3N1O binding geometry, like the native human peptide at pH 7.4. The thermodynamic data reveal an equilibrium between three coordination spheres, {NH2, O, NIm(His6), N-}, {NH2, O, N-Im(His6), N-Im(His13)}, and {NH2, O, N-Im(His6), N-Im(His14)}, for human A beta(16) but one dominant coordination for rat A beta(16), {NH2, O, N-Im(His6), N-}, at pH 7.4-6.5. ITC and CD data establish that the mutation R5G is sufficient for reproducing this difference in Cu2+ binding properties at pH 7.4. The substitution of bulky and positively charged Arg by Gly is proposed to stabilize the coordination {NH2, O-, NIm(His6), N-} that then results in one dominating coordination sphere for the case of the rat peptide. The differences in the coordination geometries for Cu2+ by the human and rat A beta are proposed to contribute to the variation in the ability of Cu2+ to induce aggregation of A beta peptides.Item Open Access The tricarboxylic acid cycle in Shewanella oneidensis is independent of Fur and RyhB control(2010) Yang, Yunfeng; McCue, Lee Ann; Parsons, Andrea B; Feng, Sheng; Zhou, JizhongBackground: It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. It is unclear in the gamma proteobacterium S. oneidensis whether TCA is also regulated by Fur and RyhB. Results: In the present study, we showed that a fur deletion mutant of S. oneidensis could utilize TCA compounds. Consistently, expression of the TCA cycle genes acnA and sdhA was not down-regulated in the mutant. To explore this observation further, we identified a ryhB gene in Shewanella species and experimentally demonstrated the gene expression. Further experiments suggested that RyhB was up-regulated in fur mutant, but that AcnA and SdhA were not controlled by RyhB. Conclusions: These cumulative results delineate an important difference of the Fur-RyhB regulatory cycle between S. oneidensis and other gamma-proteobacteria. This work represents a step forward for understanding the unique regulation in S. oneidensis.