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Mayo, FL, United States

Caulfield T.R.,Mayo Clinic Jacksonville | Fiesel F.C.,Mayo Clinic Jacksonville | Springer W.,Mayo Graduate School
Biochemical Society Transactions | Year: 2015

The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependentmitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Parkinson's disease (PD) [Springer and Kahle (2011) Autophagy 7, 266-278]. Upon mitochondrial stress, PINK1 activates the E3 ubiquitin (Ub) ligase Parkin through phosphorylation of the Ub-like (UBL) domain of Parkin and of the small modifier Ub itself at a conserved residue [Sauvé and Gehring (2014) Cell Res. 24, 1025-1026]. Recently resolved partial crystal structures of Parkin showed a 'closed', auto-inhibited conformation, consistent with its notoriously weak enzymatic activity at steady state [Wauer and Komander (2013) EMBO J. 32, 2099-2112; Riley et al. (2013) Nat. Commun. 4, 1982; Trempe et al. (2013) Science 340, 1451-1455; Spratt et al. (2013) Nat. Commun. 4, 1983]. It has thus become clear that Parkin must undergo major structural rearrangements in order to unleash its catalytic functions. Recent published findings derived from X-ray structures and molecular modelling present a complete structural model of human Parkin at an all-atom resolution [Caulfield et al. (2014) PLoS Comput. Biol. 10, e1003935]. The results of the combined in silico simulations-based and experimental assay-based study indicates that PINK1-dependent Ser65 phosphorylation of Parkin is required for its activation and triggering of 'opening' conformations. Indeed, the obtained structures showed a sequential release of Parkin's intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cellbased screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin's activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular pathogenic dysfunctions of Parkin mis-sense mutations have been recently disseminated (Fabienne C. Fiesel, Thomas R. Caulfield, Elisabeth L. Moussaud-Lamodiere, Daniel F.A.R. Dourado, Kotaro Ogaki, Owen A. Ross, Samuel C. Flores, and Wolfdieter Springer, submitted). Such a structure-function approach provides the basis for the dissection of Parkin's regulation and a targeted drug design to identify small-molecule activators of this neuroprotective E3 Ub ligase. © The Authors Journal compilation © 2015 Biochemical Society.

Wang X.,Mayo Medical School | Bledsoe K.L.,Mayo Graduate School | Graham R.P.,Mayo Medical School | Viswanatha D.S.,Mayo Medical School | And 6 more authors.
Nature Genetics | Year: 2014

Biphenotypic sinonasal sarcoma (SNS) is a newly described tumor of the nasal and paranasal areas. Here we report a recurrent chromosomal translocation in SNS, t(2;4)(q35;q31.1), resulting in a PAX3-MAML3 fusion protein that is a potent transcriptional activator of PAX3 response elements. The SNS phenotype is characterized by aberrant expression of genes involved in neuroectodermal and myogenic differentiation, closely simulating the developmental roles of PAX3.© 2014 Nature America, Inc. All rights reserved.

Kurklinsky S.,Mayo Graduate School | Chen J.,Center for Basic Research in Digestive Diseases | McNiven M.A.,Center for Basic Research in Digestive Diseases
Journal of Neurochemistry | Year: 2011

Neuronal growth cone (GC) migration and targeting are essential processes for the formation of a neural network during embryonic development. Currently, the mechanisms that support directed motility of GCs are not fully defined. The large GTPase dynamin and an interacting actin-binding protein, cortactin, have been localized to GCs, although the function performed by this complex is unclear. We have found that cortactin and the ubiquitous form of dynamin (Dyn) 2 exhibit a striking co-localization at the base of the transition zone of advancing GCs of embryonic hippocampal neurons. Confocal and total internal reflection fluorescence microscopies demonstrate that this basal localization represents point contacts. Exogenous expression of wild-type Dyn2 and cortactin leads to large, exceptionally flat, and static GCs, whereas disrupting this complex has no such effect. We find that excessive GC spreading is induced by Dyn2 and cortactin over-expression and substantial recruitment of the point contact-associated, actin-binding protein α-actinin1 to the ventral GC membrane. The distributions of other point contact proteins such as vinculin or paxillin appear unchanged. Immunoprecipitation experiments show that both Dyn2 and cortactin reside in a complex with a-actinin1. These findings provide new insights into the role of Dyn2 and the actin cytoskeleton in GC adhesion and motility. © 2011 Mayo Clinic Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Sebo Z.L.,Mayo Medical School | Sebo Z.L.,University of Missouri - Kansas City | Lee H.B.,Mayo Graduate School | Peng Y.,Mayo Medical School | Guo Y.,Mayo Medical School
Fly | Year: 2014

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility. © 2014 Landes Bioscience.

Ramirez-Giraldo J.C.,Rochester College | Thompson S.M.,Rochester College | Krishnamurthi G.,Rochester College | Knudsen B.E.,Mayo Graduate School | And 3 more authors.
American Journal of Roentgenology | Year: 2013

OBJECTIVE. The purpose of this study is to evaluate radiation dose reduction strategies in perfusion CT by using a biologic phantom. MATERIALS AND METHODS. A formalin-preserved porcine liver was submerged in a 32-cm-wide acrylic phantom filled with water. The portal vein was connected to a continuous flow pump. The phantom was scanned with a perfusion CT protocol using 80 kVp and 400 mAs, every 1 second, for 50 seconds. This was repeated using 100 and 20 mAs. It was also repeated again using 400 mAs to assess reproducibility. A sparser scan frequency was simulated retrospectively. Blood flow was determined for each dataset using the maximum slope and deconvolution methods. RESULTS. Measurements of the mean blood flow values in identical regions of interest had a percent difference of 7% for repeated perfusion CT protocols using the same settings regardless of perfusion model used. The 100 mAs scans agreed with 400 mAs scans, with percent differences of 21% and 31% for the maximum slope and deconvolution methods, respectively. At a simulated frequency of one scan every 4 seconds, blood flow values differed up to 17% and 60% from the reference scan for the maximum slope and deconvolution methods, respectively. At 20 mAs and one scan every 1 second, or 400 mAs and a simulated frequency of one scan every 8 seconds, both computation methods failed to provide accurate blood flow estimates. CONCLUSION. The biologic phantom showed reproducible measurements that can help in optimizing perfusion CT protocols by determining both the acquisition parameters that affect radiation dose and the accuracy of estimates from different perfusion models. © American Roentgen Ray Society.

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