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tisdag 24 april 2018

TRIM9 (Kr.14q22.1), RNF81, SPRING ,(C_I, fnl, pryspry)

TRIM9 (Kr. 14q22.1), SPRING, RNF91 (C_I, FNL2, PRYSPRY)

Tällä geenillä on muita nimiä SPRING ja RNF91.
Sen rakenne on RBCC (RING, Bbox1, Bbox2, Coiled-coil) ja C-terminaalin osalta alaluokkaa C_I (FNL, PRY-SPRY).

Geeni koodaa kahta isoformia. Toisessa, pidemmässä, on 710 aminohappoa (isoformi1). Domeenien sijainnit ovat : RING( 9..38), Bbox (224..266), Bbox ( 273..398). FN3 domeenit sijoittuvat jaksoon 453..532. SPRY..PRY jakso on 529..700 ja se on samankaltainen myös TRIM67:ssä, joka myöskuuluu tähän alaluokaan C-I . FN3- domeeni tarkoittaa fibronektiini tyyppi3:n kaltaista domeenia.
TRIM9 proteiini lokalisoituu sytoplasmisiin kappaleisiin. Sen funktiota ei ollut tunnistettu vielä 2008 mennessä, mutta paljon artikkelia on kertynyt sen jälkeen. Geeniä ilmenee eniten aivoissa, sappirakossa ja vielä hieman yhdessä kudoksessa. Alla on PubMed Gene lähteestä-tiedot 1-isoformista ja muutaman artikkelin suomennos.

  • https://www.ncbi.nlm.nih.gov/gene/114088
  • RNF91; SPRING Summary.The protein encoded by this gene is a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. The protein localizes to cytoplasmic bodies. Its function has not been identified. Alternate splicing of this gene generates two transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008] Expression Biased expression in brain (RPKM 15.3), gall bladder (RPKM 1.4) and 1 other tissue See more

Mielenkiintoisia artikkeleita TRIM9 funktioista netriini-1 säätelyjärjestelmässä


Avainsanoja:
DCC tässä yhteydessä tarkoittaa ”deleted in colorectal cancer”, ”puuttuu kolorektaalisyövässä”. DCC on netriini-1 reseptori.
SNARE= soluble N-ethylmaleimide attachment protein receptor https://en.wikipedia.org/wiki/SNARE_(protein)
FAK= Focal Adhesion Kinase, reseptori proteiinityrosiinikinaasi PRK2 (https://sv.wikipedia.org/wiki/PTK2)
src- family, ei-reseptori-proteiinimtyrosiinikinaasi Non-receptor tyrosine kinases, https://en.wikipedia.org/wiki/Src_family_kinase

KUVA:

(Suomennosta):
TRIM9:stä riippuva netriini1-reseptorin (DDC) ubikitinaatio pitää aisoissa kinaasisignalointia, exosytoosia ja aksonien haaroittumista. Extrasellulaarinen netriini-1 ja sen reseptori DCC edistävät aivokuoren kehittyvässä neuronissa neuroninjatkeen (aksonin) haaroittumista. Netriinistä riippuvaa morfogeneesiä edeltää netriinireseptorin (DCC) multimerisoituminen, Kinaasin FAK-aktivaatio js Src-perheen kinaasien aktivoituminen – ja se lisää exosyyttisten rakkuloiden fuusioitumista- mutta näiden tapahtumien keskinäinen linkkiytyminen on ollut tuntematon seikka.
Tässä työssä tutkijat osoittavat, että TRIM9:n tekemä netriinireseptorin (DCC) ubikitinaatio estää FAK- interaktion ja fosforyloitumisen. Mutta kun netriini-1 stimuloi tätä reseptoriaan DCC, niin TRIM9 edistää reseptorin multimerisaatiota, mutta samalla vähenee DCC:n ubikitinoitumisen aste, mistä taas fokaaliadheesiokinaasin (FAK) interaktio pääsee edistymään ja samoin sen aktivaatio.
Tutkijat havaitsivat, että FAK-aktiivisuuden inhibitio ( estäminen) blokeerasi exosytoosin kohonneet frekvenssit koeputkessa ( in vitro) ja aksonihaaroittumisen lisääntymisen in vitro ja in vivo.
Vaikka fokaaliadheesiokinaasin vähensi SNARE- välitteistä exosytoosia, SNARE-kompleksien ja rakkuloiden kokoontuminen plasmakalvon läheisyyteen lisääntyi , mikä viittaisi siihen, että FAK omaa jonkin uuden roolin SNARE-kompleksien kokoontumisen progressiossa ja rakkuloiden fuusioitumisessa koe-eläimen kehittyvässä neuronissa.
  • TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching. Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown.
  • Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation.
  • We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.

Toinen artikkeli ja kuva netriini-1 signaloinnista ja TRIM9 osuudesta


Tunnettuja TRIM9- interaktioita.
Sanastoa: F-actin, on filamenttimuotoinen aktiini.
VASP = prosessiivinen aktiinipolymeraasi https://www.ncbi.nlm.nih.gov/pubmed/21041447


Kuva A. Netriini-1:n poissaollessa TRIM9 inhiboi SNARE kompleksin jäseniä (kuten SNAP25) ligaasifunktiostaan riippumattomalla tavalla estäen rakkuloiden exosytoosia. TRIM9 toimii filopodia rakenteille ”OFF” vaihteen tavoin ubikitinoimalla VASP-polymeraasin. Ubikitinoitu prosessiivinen aktiinipolymeraasi VASP irtoaa aktiinifilamenttien päistä jolloin aktiinipolymerisaatio vähenee.
Kuva B. Kun netriini-1 on läsnä, TRIM9 tekee interaktion netriini-1-reseptorin (DCC) kanssa ligaasista riippuvalla tavalla. TRIM9:n interaktio SNAP25:n kanssa estyy, kun netriini-1 tulee reseptoriinsa. Ja nyt pääsee tapahtumaan exosytoosivälitteistä kalvon laajenemista ja filopodia ”ON” vaihde, sillä TRIM9:n tekemä VASP-ubikitinaatio estyy ja VASP ( aktiinipolymeraasi ) pääsee assosioitumaan aktiinifilamentteihin ja aktiinifilamentit alkavat polymerisoitua ja filopodiamuodostus edistyy.
  • Figure 1. Known TRIM9 interactions. A, In the absence of Netrin-1, TRIM9 inhibits SNAP25 in a ligase-independent manner to inhibit exocytosis. TRIM9 also acts as a filopodia " off " switch by ubiquitinating actin polymerase VASP. Ubiquitinated VASP is dissociated from the ends of actin filaments, leading to decreased actin polymerization. B, When Netrin-1 is present, TRIM9 interacts with Netrin-1 receptor DCC in a ligase-dependent manner. TRIM9's interaction with SNAP25 is inhibited upon Netrin-1 introduction, leading to exocytosis-mediated membrane expansion. TRIM9 ubiquitination of VASP is also inhibited, leading to VASP association with actin filaments, increased actin filament polymerization, and promotion of filopodia formation. F-actin, filamentous actin; Ub, ubiquitination.  
    Cell Res. 2016 May;26(5):613-28. doi: 10.1038/cr.2016.27. Epub 2016 Feb 26.

Lyhempi TRIM9 isoformi omaa kaksoisvaikutuksia antivirusimmuunivasteessa.


  • TRIM9 short isoform preferentially promotes DNA and RNA virus-induced production of type I interferon by recruiting GSK3β to TBK1. Qin Y1, Liu Q1, Tian S1, Xie W2, Cui J1,3, Wang RF4,5,6.
(Suomennosta) . Avainsanoja:
MAVS =Mitochondrial Anti-Viral Signaling-protein
STING=double-strand (ds) DNA receptors activate the adaptor molecule STimulator of IFN Genes (STING) at the endoplasmic reticulum (ER)
Kuva tässä linkissä osoittaa MAVS ja STING proteiinien sijainnin. MAVS on mitokondriaan assosioitunut ja STING endoplasmiseen retikulumiin. Niitten avulla solu lajittelee virusvihollisiaan.

” Antivirusimmuunivasteessa on tyypin 1 interferoni(IFN) signaalinvälittäjäna ja sen sanomana on, että solun virussensorijärjestelmä (DNA-sensori cGAS ja RNA-sensorit RIG-1 ja MDA5) ovat tunnistaneet DNA ja RNA virusta. Näiden DNA ja RNA-sensoreiden aktivoituminen johtaa STING- ja vastaavasti MAVS- proteiinien rekrytoitumiseen- ja nämä herätteet konvergoituvat TBK1- signalointitiehen ja siitä seuraa IRF3- fosforyloituminen ( ja aktivoituminen) ja 1 tyypin IFN:n tuotto ( katso kuva)

  • Type I interferon (IFN) is an important component of antiviral innate immune signaling mediated by viral DNA and RNA recognition by the DNA sensor cGAS and RNA sensors RIG-I and MDA5. Activation of these DNA and RNA sensors leads to the recruitment of STING and MAVS, respectively, and converges on TANK-binding kinase 1 (TBK1) signaling for subsequent phosphorylation of IFN regulatory factor 3 (IRF3).
(TANK= TRAF family member associated NfkB activator)
(TRAF = TNR receptor associated factor)
(TNR = tumor necrosis factor, proinflamamtorinen sytokiini)
(GSK3beta =glykogeenisyntaasikinaasi , 3 beta https://en.wikipedia.org/wiki/GSK-3, kiinnsotava entsyymi ja ilmenee monessa patologiassa)


”TBK1 kinaasin aktivaatio ei ole täysin yksityiskohtaisesti selvitetty. Tässä työssä on tunnistettu TRIM9 geenin koodaama lyhempi isoformi 1-tyypin interferonisignaloinnin positiiviseksi säätelijäksi. Virusinfektion aikana TRIM9 autopolyubikitinoituu K63 asemaan ja toimii alustana, jolla GSK3beta liittyy TBK1 entsyymiin ja tätä tietä pääsee IRF3-siganlointi aktivoitumaan. Tutkijat havaitsivat, että TRIM9 pystyy selektiivisesti inhiboimaan proinflammatoristen sytokiinien tuotantoa, mutta lisää 1- tyypin interferonin ja interferonilla stimuloituvien geenien ilmenemistä. Tutkijoitten löytönä on TRIM9:n kaksoisvaikutukset antivirusimmuniteetissä ja ne tasapainottavat proinflammatorisia vasteita ja 1- tyypin interferonien tuotantoa”.

  • However, the mechanisms that control TBK1 activation are still poorly defined. Here, we identify tripartite motif 9 short isoform (TRIM9s) as a positive regulator in type I IFN signaling. Upon viral infection, TRIM9s undergoes Lys-63-linked auto-polyubiquitination and serves as a platform to bridge GSK3β to TBK1, leading to the activation of IRF3 signaling. Interestingly, we found that TRIM9s selectively inhibits the production of pro-inflammatory cytokines, but enhances the expression of type I IFNs as well as IFN-stimulated genes, in response to viral infection. Our findings reveal novel dual functions of TRIM9s in antiviral immunity, which serve to balance pro-inflammatory response and production of type I IFNs.
  • TRIM9 pitempi isoformi 16-393X/5/3/23/htm

TRIM9 on aivospesifinen NF-kB:n negatiivinen säätelijä.

TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

E3 ubiquitin-protein ligase TRIM9 isoform 1 [Homo sapiens]

NCBI Reference Sequence: NP_055978.4
Identical Proteins FASTA Graphics
LOCUS       NP_055978                710 aa            linear   PRI 08-APR-2018
DEFINITION  E3 ubiquitin-protein ligase TRIM9 isoform 1 [Homo sapiens].
ACCESSION   NP_055978
VERSION     NP_055978.4
DBSOURCE    REFSEQ: accession NM_015163.5
KEYWORDS    RefSeq.
SOURCE      Homo sapiens (human)
  ORGANISM  Homo sapiens
            Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
            Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;
            Catarrhini; Hominidae; Homo.
REFERENCE   1  (residues 1 to 710)
  AUTHORS   Plooster M, Menon S, Winkle CC, Urbina FL, Monkiewicz C, Phend KD,
            Weinberg RJ and Gupton SL.
  TITLE     TRIM9-dependent ubiquitination of DCC constrains kinase signaling,
            exocytosis, and axon branching Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons.

  JOURNAL   Mol. Biol. Cell 28 (18), 2374-2385 (2017)
   PUBMED   28701345
  REMARK    GeneRIF: Authors demonstrate that tripartite motif protein 9
            (TRIM9)-dependent ubiquitination of DCC blocks the interaction with
            and phosphorylation of FAK.
REFERENCE   2  (residues 1 to 710)
  AUTHORS   Qin Y, Liu Q, Tian S, Xie W, Cui J and Wang RF.
  TITLE     TRIM9 short isoform preferentially promotes DNA and RNA
            virus-induced production of type I interferon by recruiting
            GSK3beta to TBK1
  JOURNAL   Cell Res. 26 (5), 613-628 (2016)
   PUBMED   26915459
  REMARK    GeneRIF: TRIM9s undergoes Lys-63-linked auto-polyubiquitination and
            serves as a platform to bridge GSK3beta to TBK1, leading to the
            activation of IRF3 signaling.
REFERENCE   3  (residues 1 to 710)
  AUTHORS   Yang X, Coulombe-Huntington J, Kang S, Sheynkman GM, Hao T,
            Richardson A, Sun S, Yang F, Shen YA, Murray RR, Spirohn K, Begg
            BE, Duran-Frigola M, MacWilliams A, Pevzner SJ, Zhong Q, Trigg SA,
            Tam S, Ghamsari L, Sahni N, Yi S, Rodriguez MD, Balcha D, Tan G,
            Costanzo M, Andrews B, Boone C, Zhou XJ, Salehi-Ashtiani K,
            Charloteaux B, Chen AA, Calderwood MA, Aloy P, Roth FP, Hill DE,
            Iakoucheva LM, Xia Y and Vidal M.
  TITLE     Widespread Expansion of Protein Interaction Capabilities by
            Alternative Splicing
  JOURNAL   Cell 164 (4), 805-817 (2016)
   PUBMED   26871637
REFERENCE   4  (residues 1 to 710)
  AUTHORS   Shi M, Cho H, Inn KS, Yang A, Zhao Z, Liang Q, Versteeg GA,
            Amini-Bavil-Olyaee S, Wong LY, Zlokovic BV, Park HS, Garcia-Sastre
            A and Jung JU.
  TITLE     Negative regulation of NF-kappaB activity by brain-specific
            TRIpartite Motif protein 9
  JOURNAL   Nat Commun 5, 4820 (2014)
   PUBMED   25190485
  REMARK    GeneRIF: TRIM9 is a brain-specific negative regulator of the
            NF-kappaB pro-inflammatory signalling pathway.
            Publication Status: Online-Only
REFERENCE   5  (residues 1 to 710)
  AUTHORS   Kanazawa T, Ikeda M, Glatt SJ, Tsutsumi A, Kikuyama H, Kawamura Y,
            Nishida N, Miyagawa T, Hashimoto R, Takeda M, Sasaki T, Tokunaga K,
            Koh J, Iwata N and Yoneda H.
  TITLE     Genome-wide association study of atypical psychosis
  JOURNAL   Am. J. Med. Genet. B Neuropsychiatr. Genet. 162B (7), 679-686
            (2013)
   PUBMED   24132900
REFERENCE   6  (residues 1 to 710)
  AUTHORS   Tanji K, Kamitani T, Mori F, Kakita A, Takahashi H and Wakabayashi
            K.
  TITLE     TRIM9, a novel brain-specific E3 ubiquitin ligase, is repressed in
            the brain of Parkinson's disease and dementia with Lewy bodies
  JOURNAL   Neurobiol. Dis. 38 (2), 210-218 (2010)
   PUBMED   20085810
  REMARK    GeneRIF: These results suggest that TRIM9 plays an important role
            in the regulation of neuronal functions and participates in
            pathological process of Lewy body disease through its ligase
            activity.
REFERENCE   7  (residues 1 to 710)
  AUTHORS   Li Y, Chin LS, Weigel C and Li L.
  TITLE     Spring, a novel RING finger protein that regulates synaptic vesicle
            exocytosis
  JOURNAL   J. Biol. Chem. 276 (44), 40824-40833 (2001)
   PUBMED   11524423
REFERENCE   8  (residues 1 to 710)
  AUTHORS   Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L,
            Riganelli D, Zanaria E, Messali S, Cainarca S, Guffanti A, Minucci
            S, Pelicci PG and Ballabio A.
  TITLE     The tripartite motif family identifies cell compartments
  JOURNAL   EMBO J. 20 (9), 2140-2151 (2001)
   PUBMED   11331580
REFERENCE   9  (residues 1 to 710)
  AUTHORS   Ohara O, Nagase T, Ishikawa K, Nakajima D, Ohira M, Seki N and
            Nomura N.
  TITLE     Construction and characterization of human brain cDNA libraries
            suitable for analysis of cDNA clones encoding relatively large
            proteins
  JOURNAL   DNA Res. 4 (1), 53-59 (1997)
   PUBMED   9179496
REFERENCE   10 (residues 1 to 710)
  AUTHORS   Dawson SJ and White LA.
  TITLE     Treatment of Haemophilus aphrophilus endocarditis with
            ciprofloxacin
  JOURNAL   J. Infect. 24 (3), 317-320 (1992)
   PUBMED   1602151
COMMENT     REVIEWED REFSEQ: This record has been curated by NCBI staff. The
            reference sequence was derived from BC063872.1, DA101991.1,
            DA173440.1 and D87458.2.
            On Jun 13, 2008 this sequence version replaced NP_055978.3.
            
            Summary: The protein encoded by this gene is a member of the
            tripartite motif (TRIM) family. The TRIM motif includes three
            zinc-binding domains, a RING, a B-box type 1 and a B-box type 2,
            and a coiled-coil region. The protein localizes to cytoplasmic
            bodies. Its function has not been identified. Alternate splicing of
            this gene generates two transcript variants encoding different
            isoforms. [provided by RefSeq, Jul 2008].
            
            Transcript Variant: This variant (1) represents the longest
            transcript and encodes the longer isoform (1).
            
            Publication Note:  This RefSeq record includes a subset of the
            publications that are available for this gene. Please see the Gene
            record to access additional publications.
            
            ##Evidence-Data-START##
            Transcript exon combination :: BC063872.1, SRR1660807.103429.1
                                           [ECO:0000332]
            RNAseq introns              :: mixed/partial sample support
                                           SAMEA1965299, SAMEA1966682
                                           [ECO:0000350]
            ##Evidence-Data-END##
FEATURES             Location/Qualifiers
     source          1..710
                     /organism="Homo sapiens"
                     /db_xref="taxon:9606"
                     /chromosome="14"
                     /map="14q22.1"
     Protein         1..710
                     /product="E3 ubiquitin-protein ligase TRIM9 isoform 1"
                     /EC_number="2.3.2.27"
                     /note="homolog of rat RING finger Spring; tripartite
                     motif-containing protein 9; E3 ubiquitin-protein ligase
                     TRIM9; RING finger protein 91; RING-type E3 ubiquitin
                     transferase TRIM9"
                     /calculated_mol_wt=79046
     Region          9..>38
                     /region_name="RING"
                     /note="RING-finger (Really Interesting New Gene) domain, a
                     specialized type of Zn-finger of 40 to 60 residues that
                     binds two atoms of zinc; defined by the 'cross-brace'
                     motif C-X2-C-X(9-39)-C-X(1-3)-
                     H-X(2-3)-(N/C/H)-X2-C-X(4-48)C-X2-C; probably involved
                     in...; cd00162"
                     /db_xref="CDD:238093"
     Site            41
                     /site_type="phosphorylation"
                     /experiment="experimental evidence, no additional details
                     recorded"
                     /note="Phosphothreonine. {ECO:0000250|UniProtKB:Q8C7M3};
                     propagated from UniProtKB/Swiss-Prot (Q9C026.1)"
     Site            44
                     /site_type="phosphorylation"
                     /experiment="experimental evidence, no additional details
                     recorded"
                     /note="Phosphoserine. {ECO:0000250|UniProtKB:Q8C7M3};
                     propagated from UniProtKB/Swiss-Prot (Q9C026.1)"
     Site            46
                     /site_type="phosphorylation"
                     /experiment="experimental evidence, no additional details
                     recorded"
                     /note="Phosphoserine. {ECO:0000250|UniProtKB:Q8C7M3};
                     propagated from UniProtKB/Swiss-Prot (Q9C026.1)"
     Site            49
                     /site_type="phosphorylation"
                     /experiment="experimental evidence, no additional details
                     recorded"
                     /note="Phosphoserine. {ECO:0000250|UniProtKB:Q8C7M3};
                     propagated from UniProtKB/Swiss-Prot (Q9C026.1)"
     Region          163..211
                     /region_name="BBOX"
                     /note="B-Box-type zinc finger; smart00336"
                     /db_xref="CDD:197662"
     Region          224..266
                     /region_name="BBOX"
                     /note="B-Box-type zinc finger; smart00336"
                     /db_xref="CDD:197662"
     Region          273..399
                     /region_name="BBC"
                     /note="B-Box C-terminal domain; smart00502"
                     /db_xref="CDD:128778"
     Region          453..532
                     /region_name="FN3"
                     /note="Fibronectin type 3 domain; One of three types of
                     internal repeats found in the plasma protein fibronectin.
                     Its tenth fibronectin type III repeat contains an RGD cell
                     recognition sequence in a flexible loop between 2 strands.
                     Approximately 2% of all...; cd00063"
                     /db_xref="CDD:238020"
     Site            order(521..522,524..525)
                     /site_type="other"
                     /note="Cytokine receptor motif"
                     /db_xref="CDD:238020"
     Region          529..700
                     /region_name="SPRY_PRY_TRIM67_9"
                     /note="PRY/SPRY domain in tripartite motif-containing
                     proteins, TRIM9 and TRIM67; cd12889"
                     /db_xref="CDD:293947"
     CDS             1..710
                     /gene="TRIM9"
                     /gene_synonym="RNF91; SPRING"
                     /coded_by="NM_015163.5:766..2898"
                     /note="isoform 1 is encoded by transcript variant 1"
                     /db_xref="CCDS:CCDS9703.1"
                     /db_xref="GeneID:114088"
                     /db_xref="HGNC:HGNC:16288"
                     /db_xref="MIM:606555"
ORIGIN      
        1 meemeeelkc pvcgsfyrep iilpcshnlc qacarnilvq tpesespqsh raagsgvsdy
       61 dyldldkmsl yseadsgygs yggfasaptt pcqkspngvr vfppampppa thlspalapv
      121 prnscitcpq chrslilddr glrgfpknrv legvidryqq skaaalkcql cekapkeatv
      181 mceqcdvfyc dpcrlrchpp rgplakhrlv ppaqgrvsrr lsprkvstct dhelenhsmy
      241 cvqckmpvcy qcleegkhss hevkalgamw klhksqlsqa lnglsdrake akeflvqlrn
      301 mvqqiqensv efeaclvaqc dalidalnrr kaqllarvnk ehehklkvvr dqishctvkl
      361 rqttglmeyc levikendps gflqisdali rrvhltedqw gkgtltprmt tdfdlsldns
      421 pllqsihqld fvqvkasspv patpilqlee ccthnnsatl swkqpplstv padgyileld
      481 dgnggqfrev yvgketmctv dglhfnstyn arvkafnktg vspysktlvl qtsevawfaf
      541 dpgsahsdii lsndnltvtc ssyddrvvlg ktgfskgihy weltvdrydn hpdpafgvar
      601 mdvmkdvmlg kddkawamyv dnnrswfmhn nshtnrtegg itkgatigvl ldlnrknltf
      661 findeqqgpi afdnveglff pavslnrnvq vtlhtglpvp dfyssrasia










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