DOCSLIB.ORG

Rab6b, a Cell-Type Specific Rab6 Isoform

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Rab6b, a Cell-Type Specific Rab6 Isoform Journal of Cell Science 113, 2725-2735 (2000) 2725 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1362 The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus Frank J. M. Opdam1, Arnaud Echard2, Huib J. E. Croes1, José A. J. M. van den Hurk3, Rinske A. van de Vorstenbosch1, Leo A. Ginsel1, Bruno Goud2 and Jack A. M. Fransen1,* 1Department of Cell Biology, Institute of Cellular Signalling, University of Nijmegen, PO Box 9101, 6500 HB, Nijmegen, The Netherlands 2UMR CNRS 144, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France 3Department of Human Genetics, University Hospital Nijmegen, Nijmegen, The Netherlands *Author for correspondence (e-mail: [email protected]) Accepted 23 May; published on WWW 10 July 2000 SUMMARY Members of the Rab subfamily of small GTPases play an cell line SK-N-SH. In brain, Rab6B was found to be important role in the regulation of intracellular transport specifically expressed in microglia, pericytes and Purkinje routes. Rab6A has been shown to be a regulator of cells. Endogenous Rab6B localises to the Golgi apparatus membrane traffic from the Golgi apparatus towards the and to ERGIC-53-positive vesicles. Comparable studies endoplasmic reticulum (ER). Here, we report on the between Rab6A and Rab6B revealed distinct biochemical identification of a Rab6 isoform, termed Rab6B. The and cellular properties. Rab6B displayed lower GTP- corresponding full-length cDNA was isolated from a Caco- binding activities and in overexpression studies, the protein 2 cell library. The deduced amino acid sequence showed is distributed over Golgi and ER membranes, whereas 91% identity with the Rab6A protein and revealed that Rab6A is more restricted to the Golgi apparatus. Since the sequence divergence is dispersed over a large region of GTP-bound form of Rab6B (Rab6B Q72L) does interact the COOH-terminal domain. Rab6B is encoded by an with all known Rab6A effectors, including Rabkinesin-6, independent gene which is located on chromosome 3 region the results suggest a cell-type specific role for Rab6B in q21-q23. In contrast to Rab6A whose expression is retrograde membrane traffic at the level of the Golgi ubiquitous, northern blot analysis, immunohistochemistry, complex. and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma Key words: Golgi apparatus, Rab, Rabkinesin-6, Transport carrier INTRODUCTION Rab33 (Armstrong et al., 1996; Barbosa et al., 1995; Chen et al., 1996, 1997b; Tisdale et al., 1992). Some Rab isoforms, Transfer of cargo between intracellular membrane organelles such as Rab1A and Rab1B (Nuoffer et al., 1994) may be takes place via carrier vesicles. The correct delivery of these functionally redundant. However, distinct expression patterns vesicles to the appropriate membranes is regulated by Rab of others suggest possible differences in function. This appears proteins. This subfamily of small GTPases triggers the to be the case, for instance, for the four Rab3 isoforms (Rab3A- recruitment of specific docking complexes (Christoforidis et D). Although they are all expressed in cells with regulated al., 1999; McBride et al., 1999), and thereby might facilitate secretory pathways, Rab3A and Rab3C are preferentially the formation of SNARE complexes leading to membrane expressed in neurons and neuroendocrine cells, whereas fusion between the donor and acceptor membrane (Aridor and Rab3D is found on granules of gastric chief cells and of Balch, 1996; Chavrier and Goud, 1999; Rothman and Sollner, peritoneal mast cells (Fischer von Mollard et al., 1990, 1994; 1997; Schimmoller et al., 1998). Recently, Rab proteins have Roa et al., 1997; Tang et al., 1996). Rab3D is also the most also been found to be involved in the movement of transport abundant Rab3 isoform in adipocytes (Baldini et al., 1998). carriers along actin or microtubule cytoskeleton (Echard et al., Rab3B is highly expressed in epithelial cells (Weber et al., 1998; Nielsen et al., 1999; Walch-Solimena et al., 1997). 1994). Rab3A and Rab3B appear to have distinct functional To date, the Rab family consists of approximately 50 family properties in PC12 cells, although the four isoforms are present members. Several members have been shown to be part of on secretory granules in PC12 cells and each of them inhibits subgroups based on structural relationships and high sequence secretion (Iezzi et al., 1999; Chung et al., 1999). identities (80%-95%). Such isoforms have been described for In addition to Rab6A, involved in retrograde membrane Rab1, Rab3, Rab4, Rab5, Rab8, Rab11, Rab22, Rab27 and traffic between the Golgi complex and the endoplasmic 2726 F. J. M. Opdam and others reticulum (ER) (Martinez et al., 1994, 1997; White et al., RNA isolation and northern blotting 1999), two additional Rab6 subfamily (Rab6A′ and Rab6B) HT-29 cells were grown with (+) or without D-glucose (−) to compare members have now been identified. Rab6A′ was recently found nondifferentiated versus differentiated cells (Darmoul et al., 1992). to be generated by alternative splicing of a duplicated exon Total RNAs from cultured cell lines and various human tissues within the Rab6A gene. Rab6A and Rab6A′ are ubiquitously (rectum, jejunum, stomach, oesophagus) were prepared following the expressed and differ in only three amino acid residues (F. J. M. guanidium isothiocyate-phenol-chloroform extraction method µ Opdam et al., unpublished). Interestingly, Rab6A′ does not (Chirgwin et al., 1979). Samples of total RNA (15 g) were separated interact with the Rab6A effector Rabkinesin-6, a kinesin-like by electrophoresis on a 1% formamide agarose gel and transferred to nylon membrane according to standard procedures (Sambrook et al., protein associated with the Golgi apparatus (Echard et al., ′ ′ 1989). Blots were probed with a 0.7 kb 3 PstI/EcoRI fragment of 1998). In addition, the GTPase-deficient mutant Rab6A Q72L Rab6B (Fig. 1A, 530-1270), and a 1.35 kb glyceraldehyde-3- does not induce the redistribution of Golgi resident proteins phosphate dehydrogenase (GAPDH) cDNA probe was used to enable into the ER, as Rab6A Q72L does (Martinez et al., 1997; F. J. comparison of RNA loading. The human tissue blot with 2 µg M. Opdam, unpublished). Here, we isolated the full length poly(A)+ RNA samples was purchased from Clontech. cDNA encoding Rab6B, whose partial sequence has previously been reported (Chen et al., 1997a). Rab6B displays 91% Expression plasmid construction identity with Rab6A and is encoded by a different gene. Like Constructs for the expression of GST fusion and epitope-tagged Rab6A/A′, the bulk of Rab6B localises to the Golgi apparatus. versions of Rab6B were generated. Using the complete cDNA clone However, Rab6B shows a cell-type specific expression pattern as template the open reading frame of Rab6B including the initiator AUG codon and the stop codon for proper termination of translation in brain and GTP-binding properties distinct from that of ′ was PCR amplified with specific primers. One set of primers (forward; Rab6A/A . Despite these differences, Rab6B interacts with the 5′-GGAATTCCGGATGTCCGCAGGGGGAGA-3′ introducing an same Rab6A effector molecules, including Rabkinesin-6. Our EcoRI site in front of the start codon, and reverse; 5′- results suggest a cell type-specific role for Rab6B. CCGCTCGAGCGGTTAGCAGGA-3′ introducing an XhoI site downstream of the stop codon) was used to subclone Rab6B in-frame into an EcoRI/XhoI digested pMyc vector. pMyc is a pCDNA3- ′ MATERIALS AND METHODS derived vector in which at the 5 end of the pCDNA3 (Invitrogen) multiple cloning site a synthetic DNA fragment was introduced that entails an initiator AUG codon followed by the cMyc-epitope tag and RT-PCR and library screening an EcoRI site. An other forward primer (5′-CGGGATCCATGTCC- Total RNA (1.5 µg) from polarised Caco-2 cells was reverse GCAGGGGGAGA-3′, containing a BamHI site) was used to subclone transcribed using random hexamers (2 µg, Pharmacia) and Rab6B in-frame into a BamHI/XhoI digested eukaryotic expression Superscript reverse transcriptase (100 U, Gibco/BRL). One-sixth of vector pSG5 (Green et al., 1988), which was modified to generate an the cDNA was subsequently analysed by PCR using degenerate ‘Rab- N-terminal VSV epitope tag, or into the multiple cloning site of the specific’ primers (forward; 5′-GGCGGCGGCTCGAGGGI(A0.2/G0.8) prokaryotic expression vector PGEX (Pharmacia) to produce GST- (G0.2/A0.8)II(A0.2/G0.2/C0.6)IIII(A0.2/T0.2/G0.6)(G0.2/C0.2/T0.6)(A0.2/ Rab6B fusion protein in Escherichia coli following manufacturer’s T0.8)GGIAA(A0.5/G0.5)(A0.5/T0.5)C-3′ containing a XhoI restriction instructions (Pharmacia). Similar constructs were prepared for Rab6A site, and reverse; 5′-GGCGGCGGATCCTTC(C0.5/T0.5)TGICC(A0.5/ following the exact cloning procedures of Rab6B. All PCR constructs T0.5) GCIGT(A0.5/G0.5)TCCCA-3′ containing a BamHI restriction were checked for absence of mutations by DNA sequencing. site] matching conserved domains PM1 and PM3 of GTP-binding; see Fig. 1B). Purified BamHI/XhoI-digested PCR products were ligated Antibodies into the pBluescript vector KS+. Library cloning and subsequent Purified GST-Rab6B fusion protein was used to immunise a rabbit. preparations and screening of replica filters were carried out following Affinity-purified polyclonal antibodies were obtained by applying standard procedures (Sambrook et al., 1989). whole serum to Affigel-10-immobilised (Bio-Rad, Richmond, CA) The partial Rab6B sequence was radiolabeled by random GST-Rab6B fusion protein and eluting bound antibodies. For oligonucleotide priming (Feinberg and Vogelstein, 1983) and used as elimination of cross-reactivity on western blot, affinity-purified a probe to screen a human Caco-2 λgt11 cDNA library consisting of antibodies were purified three times over a GST-Rab6A bound approximately 4×106 independent clones (Lacey et al., 1989).
Load more

Recommended publications
  • Uwaterloo Latex Thesis Template
    New Algorithms for Predicting Conformational Polymorphism and Inferring Direct Couplings for Side Chains of Proteins by Laleh Soltan Ghoraie A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Computer Science Waterloo, Ontario, Canada, 2015 c Laleh Soltan Ghoraie 2015 I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Protein crystals populate diverse conformational ensembles. Despite much evidence that there is widespread conformational polymorphism in protein side chains, most of the x- ray crystallography data are modelled by single conformations in the Protein Data Bank. The ability to extract or to predict these conformational polymorphisms is of crucial im- portance, as it facilitates deeper understanding of protein dynamics and functionality. This dissertation describes a computational strategy capable of predicting side-chain poly- morphisms. The applied approach extends a particular class of algorithms for side-chain prediction by modelling the side-chain dihedral angles more appropriately as continuous rather than discrete variables. Employing a new inferential technique known as particle belief propagation (PBP), we predict residue-specific distributions that encode informa- tion about side-chain polymorphisms. The predicted polymorphisms are in relatively close agreement with results from a state-of-the-art approach based on x-ray crystallography data. This approach characterizes the conformational polymorphisms of side chains us- ing electron density information, and has successfully discovered previously unmodelled conformations.
    [Show full text]
  • High-Throughput Discovery of Novel Developmental Phenotypes
    High-throughput discovery of novel developmental phenotypes The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Dickinson, M. E., A. M. Flenniken, X. Ji, L. Teboul, M. D. Wong, J. K. White, T. F. Meehan, et al. 2016. “High-throughput discovery of novel developmental phenotypes.” Nature 537 (7621): 508-514. doi:10.1038/nature19356. http://dx.doi.org/10.1038/nature19356. Published Version doi:10.1038/nature19356 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:32071918 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Nature. Manuscript Author Author manuscript; Manuscript Author available in PMC 2017 March 14. Published in final edited form as: Nature. 2016 September 22; 537(7621): 508–514. doi:10.1038/nature19356. High-throughput discovery of novel developmental phenotypes A full list of authors and affiliations appears at the end of the article. Abstract Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms #Corresponding author: [email protected].
    [Show full text]
  • RAB6B Polyclonal Antibody
    For Research Use Only RAB6B Polyclonal antibody Catalog Number:10340-1-AP 2 Publications www.ptgcn.com Catalog Number: GenBank Accession Number: Recommended Dilutions: Basic Information 10340-1-AP BC002510 WB 1:500-1:2000 Size: GeneID (NCBI): IP 0.5-4.0 ug for IP and 1:500-1:2000 450 μg/ml 51560 for WB IHC 1:20-1:200 Source: Full Name: IF 1:10-1:100 Rabbit RAB6B, member RAS oncogene family Isotype: Calculated MW: IgG 23 kDa Purification Method: Observed MW: Antigen affinity purification 24 kDa Immunogen Catalog Number: AG0322 Applications Tested Applications: Positive Controls: IF, IHC, IP, WB,ELISA WB : mouse brain tissue; C6 cells, rat brain tissue Cited Applications: IP : mouse brain tissue; IF, WB IHC : human gliomas tissue; Species Specificity: human, mouse, rat IF : C6 cells; Cited Species: mouse, rat Note-IHC: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0 The human RAB genes share structural and biochemical properties with the Ras gene superfamily. Accumulating Background Information data suggests an important role for RAB proteins either in endocytosis or in biosynthetic protein transport. The transport of newly synthesized proteins from endoplasmic reticulum to the Golgi complex and to secretory vesicles involves the movement of carrier vesicles, a process that appears to involve RAB protein function. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23.
    [Show full text]
  • 1 Supplementary Materials and Methods Gene Set Enrichment Analysis for Gene Expression Data We Compared the Gene Expression Leve
    Supplementary Materials and Methods Gene set enrichment analysis for gene expression data We compared the gene expression levels from 2 different phenotypes (i.e., drug sensitive versus resistant) and picked up the genes which had significant different expression for Gene set enrichment analysis (GSEA) by using Molecular Signatures Database(V3.0). Gene set enrichment analysis was carried out by computing overlaps with canonical pathways(CP) and gene ontology (GO) gene sets(C5), obtained from the Broad Institute [1]. Genes in Gene Set (K), Genes in Overlap (k), k/K and P value were used to rank the pathways enriched in each phenotype. We used with 10363 genes in 2334 pathways as the gene set in this study. Supplementary References 1.Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102:15545– 50. 1 Supplementary Results Other drug response-genotype associations in this study Cells with wild genotype were more sensitive to irinotecan than those with TGFβR2 mutation and deletion together (P=0.03) (Supplementary Table S6). PC cells with wild genotype in ADAM gene family were more sensitive to gemcitabine and docetaxel than those with mutation in this family (p<0.05) (Supplementary Table S6). PC cells with wild genotype were more sensitive to docetaxel than those with DEPDC or TTN mutation (p<0.05) (Supplementary Table S6). PC cells with BAI3 mutation were more sensitive to cisplatin than those with wild genotype (p=0.02) (Supplementary Table S6).
    [Show full text]
  • Prognostic Significance of Autophagy-Relevant Gene Markers in Colorectal Cancer
    ORIGINAL RESEARCH published: 15 April 2021 doi: 10.3389/fonc.2021.566539 Prognostic Significance of Autophagy-Relevant Gene Markers in Colorectal Cancer Qinglian He 1, Ziqi Li 1, Jinbao Yin 1, Yuling Li 2, Yuting Yin 1, Xue Lei 1 and Wei Zhu 1* 1 Department of Pathology, Guangdong Medical University, Dongguan, China, 2 Department of Pathology, Dongguan People’s Hospital, Southern Medical University, Dongguan, China Background: Colorectal cancer (CRC) is a common malignant solid tumor with an extremely low survival rate after relapse. Previous investigations have shown that autophagy possesses a crucial function in tumors. However, there is no consensus on the value of autophagy-associated genes in predicting the prognosis of CRC patients. Edited by: This work screens autophagy-related markers and signaling pathways that may Fenglin Liu, Fudan University, China participate in the development of CRC, and establishes a prognostic model of CRC Reviewed by: based on autophagy-associated genes. Brian M. Olson, Emory University, United States Methods: Gene transcripts from the TCGA database and autophagy-associated gene Zhengzhi Zou, data from the GeneCards database were used to obtain expression levels of autophagy- South China Normal University, China associated genes, followed by Wilcox tests to screen for autophagy-related differentially Faqing Tian, Longgang District People's expressed genes. Then, 11 key autophagy-associated genes were identified through Hospital of Shenzhen, China univariate and multivariate Cox proportional hazard regression analysis and used to Yibing Chen, Zhengzhou University, China establish prognostic models. Additionally, immunohistochemical and CRC cell line data Jian Tu, were used to evaluate the results of our three autophagy-associated genes EPHB2, University of South China, China NOL3, and SNAI1 in TCGA.
    [Show full text]
  • Inhibition of Mitochondrial Complex II in Neuronal Cells Triggers Unique
    www.nature.com/scientificreports OPEN Inhibition of mitochondrial complex II in neuronal cells triggers unique pathways culminating in autophagy with implications for neurodegeneration Sathyanarayanan Ranganayaki1, Neema Jamshidi2, Mohamad Aiyaz3, Santhosh‑Kumar Rashmi4, Narayanappa Gayathri4, Pulleri Kandi Harsha5, Balasundaram Padmanabhan6 & Muchukunte Mukunda Srinivas Bharath7* Mitochondrial dysfunction and neurodegeneration underlie movement disorders such as Parkinson’s disease, Huntington’s disease and Manganism among others. As a corollary, inhibition of mitochondrial complex I (CI) and complex II (CII) by toxins 1‑methyl‑4‑phenylpyridinium (MPP+) and 3‑nitropropionic acid (3‑NPA) respectively, induced degenerative changes noted in such neurodegenerative diseases. We aimed to unravel the down‑stream pathways associated with CII inhibition and compared with CI inhibition and the Manganese (Mn) neurotoxicity. Genome‑wide transcriptomics of N27 neuronal cells exposed to 3‑NPA, compared with MPP+ and Mn revealed varied transcriptomic profle. Along with mitochondrial and synaptic pathways, Autophagy was the predominant pathway diferentially regulated in the 3‑NPA model with implications for neuronal survival. This pathway was unique to 3‑NPA, as substantiated by in silico modelling of the three toxins. Morphological and biochemical validation of autophagy markers in the cell model of 3‑NPA revealed incomplete autophagy mediated by mechanistic Target of Rapamycin Complex 2 (mTORC2) pathway. Interestingly, Brain Derived Neurotrophic Factor
    [Show full text]
  • Viewed and Published Immediately Upon Acceptance Cited in Pubmed and Archived on Pubmed Central Yours — You Keep the Copyright
    BMC Genomics BioMed Central Research article Open Access Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice Claudia Prinzen1, Dietrich Trümbach2, Wolfgang Wurst2, Kristina Endres1, Rolf Postina1 and Falk Fahrenholz*1 Address: 1Johannes Gutenberg-University, Institute of Biochemistry, Mainz, Johann-Joachim-Becherweg 30, 55128 Mainz, Germany and 2Helmholtz Zentrum München – German Research Center for Environmental Health, Institute for Developmental Genetics, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany Email: Claudia Prinzen - [email protected]; Dietrich Trümbach - [email protected]; Wolfgang Wurst - [email protected]; Kristina Endres - [email protected]; Rolf Postina - [email protected]; Falk Fahrenholz* - [email protected] * Corresponding author Published: 5 February 2009 Received: 19 June 2008 Accepted: 5 February 2009 BMC Genomics 2009, 10:66 doi:10.1186/1471-2164-10-66 This article is available from: http://www.biomedcentral.com/1471-2164/10/66 © 2009 Prinzen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results: To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn) of this enzyme.
    [Show full text]
  • Download Tool
    by Submitted in partial satisfaction of the requirements for degree of in in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Approved: ______________________________________________________________________________ Chair ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Committee Members Copyright 2019 by Adolfo Cuesta ii Acknowledgements For me, completing a doctoral dissertation was a huge undertaking that was only possible with the support of many people along the way. First, I would like to thank my PhD advisor, Jack Taunton. He always gave me the space to pursue my own ideas and interests, while providing thoughtful guidance. Nearly every aspect of this project required a technique that was completely new to me. He trusted that I was up to the challenge, supported me throughout, helped me find outside resources when necessary. I remain impressed with his voracious appetite for the literature, and ability to recall some of the most subtle, yet most important details in a paper. Most of all, I am thankful that Jack has always been so generous with his time, both in person, and remotely. I’ve enjoyed our many conversations and hope that they will continue. I’d also like to thank my thesis committee, Kevan Shokat and David Agard for their valuable support, insight, and encouragement throughout this project. My lab mates in the Taunton lab made this such a pleasant experience, even on the days when things weren’t working well. I worked very closely with Tangpo Yang on the mass spectrometry aspects of this project. Xiaobo Wan taught me almost everything I know about protein crystallography. Thank you as well to Geoff Smith, Jordan Carelli, Pat Sharp, Yazmin Carassco, Keely Oltion, Nicole Wenzell, Haoyuan Wang, Steve Sethofer, and Shyam Krishnan, Shawn Ouyang and Qian Zhao.
    [Show full text]
  • Presynaptic Mechanisms for Cargo Recruitment
    Presynaptic Mechanisms for Cargo Recruitment The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Nyitrai, Hajnalka. 2019. Presynaptic Mechanisms for Cargo Recruitment. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013129 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Presynaptic Mechanisms for Cargo Recruitment A dissertation presented by Hajnalka Nyitrai to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts August 2019 © 2019 Hajnalka Nyitrai All rights reserved. Dissertation Advisor: Dr. Pascal S. Kaeser Hajnalka Nyitrai Presynaptic Mechanisms for Cargo Recruitment Abstract Delivery and assembly of presynaptic material over long axonal distances is a daunting challenge for neurons. Many presynaptic proteins are thought to be delivered from the Golgi apparatus via precursor vesicles, the recruitment of which at nerve terminals is crucial for the maintenance of presynaptic function. But how do these transport vesicles know where to stop along the axon? In most cellular secretory pathways, Golgi-derived cargos are labeled with Rab GTPases that are recognized by tethering complexes at their fusion sites. Surprisingly, we know very little about what presynaptic tethers and cargo labels facilitate presynaptic cargo capture.
    [Show full text]
  • Computational Proteogenomic Identification and Functional
    bioRxiv preprint doi: https://doi.org/10.1101/168377; this version posted July 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Computational proteogenomic identification and functional interpretation of translated fusions and micro structural variations in cancer Yen Yi Lin1;y, Alexander Gawronski1;y, Faraz Hach1;3;y, Sujun Li2, Ibrahim Numanagic,´ 1, Iman Sarrafi1;3, Swati Mishra5, Andrew McPherson1, Colin Collins3;4, Milan Radovich5, Haixu Tang2, S. Cenk Sahinalp 1;2;3; ∗ 1School of Computing Science, Simon Fraser University, Burnaby, BC, Canada, 2School of Informatics and Computing, Indiana University, Bloomington, IN, USA, 3Vancouver Prostate Centre, Vancouver, BC, Canada, 4Dept. of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada 5Department of Surgery, Indiana University, School of Medicine, Indianapolis, IN, USA Motivation: Rapid advancement in high throughput genome and transcriptome sequencing (HTS) and mass spectrometry (MS) technologies has enabled the acquisition of the genomic, transcriptomic and proteomic data from the same tissue sample. In this paper we in- troduce a novel computational framework which can integratively analyze all three types of omics data to obtain a complete molecular profile of a tissue sample, in normal and disease conditions. Our framework includes MiStrVar, an algorithmic method we developed to identify micro structural variants (microSVs) on genomic HTS data. Coupled with deFuse, a popular gene fusion detection method we devel- oped earlier, MiStrVar can provide an accurate profile of structurally aberrant tran- scripts in cancer samples. Given the breakpoints obtained by MiStrVar and deFuse, our framework can then identify all relevant peptides that span the breakpoint junc- tions and match them with unique proteomic signatures in the respective proteomics data sets.
    [Show full text]
  • 39UTR Shortening Identifies High-Risk Cancers with Targeted Dysregulation
    OPEN 39UTR shortening identifies high-risk SUBJECT AREAS: cancers with targeted dysregulation of GENE REGULATORY NETWORKS the ceRNA network REGULATORY NETWORKS Li Li1*, Duolin Wang2*, Mengzhu Xue1*, Xianqiang Mi1, Yanchun Liang2 & Peng Wang1,3 Received 1 2 15 April 2014 Key Laboratory of Systems Biology, Shanghai Advanced Research Institute, Chinese Academy of Sciences, College of Computer Science and Technology, Jilin University, 3School of Life Science and Technology, ShanghaiTech University. Accepted 3 June 2014 Competing endogenous RNA (ceRNA) interactions form a multilayered network that regulates gene Published expression in various biological pathways. Recent studies have demonstrated novel roles of ceRNA 23 June 2014 interactions in tumorigenesis, but the dynamics of the ceRNA network in cancer remain unexplored. Here, we examine ceRNA network dynamics in prostate cancer from the perspective of alternative cleavage and polyadenylation (APA) and reveal the principles of such changes. Analysis of exon array data revealed that both shortened and lengthened 39UTRs are abundant. Consensus clustering with APA data stratified Correspondence and cancers into groups with differing risks of biochemical relapse and revealed that a ceRNA subnetwork requests for materials enriched with cancer genes was specifically dysregulated in high-risk cancers. The novel connection between should be addressed to 39UTR shortening and ceRNA network dysregulation was supported by the unusually high number of P.W. (wangpeng@ microRNA response elements (MREs) shared by the dysregulated ceRNA interactions and the significantly sari.ac.cn) altered 39UTRs. The dysregulation followed a fundamental principle in that ceRNA interactions connecting genes that show opposite trends in expression change are preferentially dysregulated. This targeted dysregulation is responsible for the majority of the observed expression changes in genes with significant * These authors ceRNA dysregulation and represents a novel mechanism underlying aberrant oncogenic expression.
    [Show full text]
  • A Systematic Genome-Wide Association Analysis for Inflammatory Bowel Diseases (IBD)
    A systematic genome-wide association analysis for inflammatory bowel diseases (IBD) Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Dipl.-Biol. ANDRE FRANKE Kiel, im September 2006 Referent: Prof. Dr. Dr. h.c. Thomas C.G. Bosch Koreferent: Prof. Dr. Stefan Schreiber Tag der mündlichen Prüfung: Zum Druck genehmigt: der Dekan “After great pain a formal feeling comes.” (Emily Dickinson) To my wife and family ii Table of contents Abbreviations, units, symbols, and acronyms . vi List of figures . xiii List of tables . .xv 1 Introduction . .1 1.1 Inflammatory bowel diseases, a complex disorder . 1 1.1.1 Pathogenesis and pathophysiology. .2 1.2 Genetics basis of inflammatory bowel diseases . 6 1.2.1 Genetic evidence from family and twin studies . .6 1.2.2 Single nucleotide polymorphisms (SNPs) . .7 1.2.3 Linkage studies . .8 1.2.4 Association studies . 10 1.2.5 Known susceptibility genes . 12 1.2.5.1 CARD15. .12 1.2.5.2 CARD4. .15 1.2.5.3 TNF-α . .15 1.2.5.4 5q31 haplotype . .16 1.2.5.5 DLG5 . .17 1.2.5.6 TNFSF15 . .18 1.2.5.7 HLA/MHC on chromosome 6 . .19 1.2.5.8 Other proposed IBD susceptibility genes . .20 1.2.6 Animal models. 21 1.3 Aims of this study . 23 2 Methods . .24 2.1 Laboratory information management system (LIMS) . 24 2.2 Recruitment. 25 2.3 Sample preparation. 27 2.3.1 DNA extraction from blood. 27 2.3.2 Plate design .
    [Show full text]