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lördag 21 april 2018

TRIM 13 (Kr.13q14), RFP2, LEU5, RNF77, CAR ( C_XI TM), G-Quadruplx kohtia rakenteessa

TRIM13 (Kr.13q14). K29 ubikitiiniligaasi 

TRIM13 geeni koodaa tripartiittimotiivisen perheen jäsentä. TRIM-motiivissa on kolme sinkkiä sitovaa domeenia: RING, B-box 1 ja B-box2 sekä coiled -coil- helikaali jakso.   Se kuuluu alaluokaan C_XI, koska sillä on transmembraaninen domeeni (TM).
 Minimaalinen deleetio alue todetaan kroonisessa B-lymfosyytin leukemiassa (B-CLL).
 TRIM13 omaa  monia vaihtoehtoisesti luettuja transkriptiovariantteja. Geeniä ilmenee  testiksessä ja  luuytimessä, tyreoideassa ja 24 muussa kudoksessa. Promoottorissa on  tälle trimille  tyypillinen toistosekvenssi ja siitä tulee nimi RFP2. Siitä on yksi linkki alla.
Nimiä: CAR; LEU5; RFP2; DLEU5; RNF77

    Summary This gene encodes 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. This gene is located on chromosome 13 within the minimal deletion region for B-cell chronic lymphocytic leukemia. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jul 2008]Expression Broad expression in testis (RPKM 17.7), bone marrow (RPKM 8.8) and 25 other tissues

TRIM13 tekee K29polyubikitinaation TRAF6:een ja täten vahvistaa TLR2- välitteistä NF-KB aktivaatiota makrofagissa

  • Trim13 Potentiates Toll-Like Receptor 2–Mediated Nuclear Factor κB Activation via K29-Linked Polyubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6. Bin Huang and Suk-Hwan BaekMolecular Pharmacology April 2017, 91 (4) 307-316; DOI: https://doi.org/10.1124/mol.116.106716
(Tiivistelmästä suomennosta). Ubikitinaatio on monivivahteinen posttranslationaalinen modifikaatio, joka osallistuu NK-kB-aktivoitumiseen Tollin kaltaisen reseptorin (TLR) signaloinnin kautta. Tässä tutkijat osoittivat, että TRIM13 ( E3-ubikitiiniligaasi) säätyy makrofageissa ylös, kun TLR2 stimuloituu. TRIM13 poistogeenisyys heikonsi TLR2-välitteistä sytokiinien/kemokiinien tuotantoa, vaahtosolujen muodostumista ja NF-kB- aktivaatiota.
 TRIM13:n vuorovaikuttaa TNF- reseptoriin assosioituneeseen tekijään 6 (TRAF6) ja vahvistaa NF-kB aktiviteettiä ubikitinoimalla TRAF6:n .
 Inaktiivin mutantin tai TRIM13:n RING-domeenideleetiota kantavan mutantin yliesiintymä ei vahvistanut TRAF6 ubikitinaatiota eikä myöskään NF-kB-aktivaatiota.
Nämä tulokset viitaavat siihen, että TRIM13:n vaikutukset riippuvat sen E3-ligaasi aktiviteetista. TRAF6:n ubikitinoimiseen TRIM13 käytti K29-linkittyjä ubikitiiniketjuja edistääkseen NF-kB aktiivisuutta ja sillä tavalla vahvisti TLR2- välitteisiä immuunivasteita.
Tutkijoitten saama tieto vahvistaa, että TRIM13 on NF-kB-aktivaation positiivinen säätelijä ja tieto viittaa siihenkin, että TLR2-signaloinnin kontrolliin osallistuu spesifinen ubikitinaatiomalli, jossa on K29-linkkiytynytta polyubikitinaatiota.
  • Ubiquitination is a versatile post-translational modification involved in nuclear factor-κB (NF-κB) activation of Toll-like receptor (TLR) signaling. Here, we demonstrated that Trim13, an E3 ubiquitin ligase, is up-regulated in macrophages upon stimulation with TLR2 ligand. Knockdown of Trim13 attenuated TLR2-mediated production of cytokines/chemokines and formation of foam cells as well as activation of NF-κB. Trim13 interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and potentiates NF-κB activity via ubiquitination of TRAF6. Overexpression of inactive mutant (C10/13A) or really interesting new gene (RING) deletion mutant of Trim13 did not potentiate ubiquitination of TRAF6 or activation of NF-κB. These results suggest that the effects of Trim13 are dependent on its E3 ligase activity. Trim13 used K29-linked polyubiquitin chains for TRAF6 ubiquitination to promote NF-κB activity and thus potentiated activation of TLR2-mediated immune responses. Our data identify Trim13 as a positive regulator of NF-κB activation and suggest that K29-linked polyubiquitination is a specific ubiquitin-linked pattern involved in the control of TLR2 signaling.

Kr13q14 täysi tai osittainen deleetio syynä moneen plasmasolumaligniteettiin

  • TRIM13 (RFP2) downregulation decreases tumour cell growth in multiple myeloma(MM=  through inhibition of NF Kappa B pathway and proteasome activity. Gatt ME, et al. Br J Haematol, 2013 Jul. PMID 23647456, Free PMC Article
     Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis.
    Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.

TRIM13 (RFP2)  geenin pieni  deleetio B-CLL leukemiassa

  • RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia. van Everdink WJ, et al. Cancer Genet Cytogenet, 2003 Oct 1. PMID 14499696 Occurrence of 13q14 deletions between D13S273 and D13S25 in B-cell chronic lymphocytic leukemia (B-CLL) suggests that the region contains a tumor suppressor gene. We constructed a PAC/cosmid contig largely corresponding to a 380-kb 13q14 YAC insert that we found deleted in a high proportion of B-CLL patients. We found seven genes by exon trapping, cDNA screening and analysis/cDNA extension of known expressed sequence tags. One appeared to originate from another region of 13q. Recent publications have focused on two of the genes that most likely do not have a tumor suppressor role. This study evaluates the remaining four genes in the region by mutation scanning and theoretical analysis of putative encoded products. No mutations suggestive of a pathogenic effect were found. The 13q14 deletions may be a consequence of an inherent instability of the region, an idea supported by our finding of a considerable proportion of AluY repeats. Deletion of putative enhancer sequences and/or genes in the region may result in an inactivation of tumor suppression by a haploinsufficiency mechanism. We conclude that RFP2, c13ORF1, and a chromosome 13-specific ST13-like gene, FAM10A4, are the most likely candidates for such a type of B-CLL TSG.

TRIM3, LEU5, Tarkempi tutkimus instabiilista geenikohdasta

  • A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5. Kapanadze B, et al. FEBS Lett, 1998 Apr 17. PMID 9599022 B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

TRIM13 herkistää solua stressissä; K63polyubikitinaatio kaspaasi 8:aan. Fagolysosomitie.

  • TRIM13 regulates caspase-8 ubiquitination, translocation to autophagosomes and activation during ER stress induced cell death. Tomar D, et al. Biochim Biophys Acta, 2013 Dec. PMID 24021263 The emerging evidences suggest that endoplasmic (ER) stress is involved in onset of many pathological conditions like cancer and neurodegeneration. The persistent ER stress results in misfolded protein aggregates, which are degraded through the process of autophagy or lead to cell death through activation of caspases. The regulation of crosstalk of autophagy and cell death during ER stress is emerging. Ubiquitination plays regulatory role in crosstalk of autophagy and cell death. In the current study, we describe the role of TRIM13, RING E3 ubiquitin ligase, in regulation of ER stress induced cell death. The expression of TRIM13 sensitizes cells to ER stress induced death. TRIM13 induced autophagy is essential for ER stress induced caspase activation and cell death. TRIM13 induces K63 linked poly-ubiquitination of caspase-8, which results in its stabilization and activation during ER stress. TRIM13 regulates translocation of caspase-8 to autophagosome and its fusion with lysosome during ER stress. This study first time demonstrated the role of TRIM13 as novel regulator of caspase-8 activation and cell death during ER stress.

TRIM, CAR, Leu5, minimaalisen deleetioalueen (MDR) tarkempaa selvitystä

  • Nucleotide sequence, transcription map, and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia. Migliazza A, et al. Blood, 2001 Apr 1. PMID 11264177  Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses.
    The MDR contains 2 pseudogenes and 3 transcribed genes:
     CAR, encoding a putative RING-finger containing protein;
     1B4/Leu2, generating noncoding transcripts;
     and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons).
    These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion.
     The nondeleted allele of the CAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status.
    These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL.
    However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis. (Blood. 2001;97:2098-2104).

TRIM13 on endoplasmiseen retikulumiin(ER) ankkuroitunut ubikitiiniligaasi , joka säätelee solujen klonogeenista aktiivisuutta.  NEMO-ubikitinaatio.

  • TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation.TRIM13 mediated NF-kappaB repression is essential for negative regulation of clonogenic ability of the cells. The NF-κB family of transcription factors is activated in response to various intracellular or extracellular stimuli and its dysregulation leads to pathological conditions like infection, cancer, neurodegenerative disorders. 
  • The post-translational modification by ubiquitination regulates various steps of NF-κB pathway. In the current study, we have described the role of TRIM13, an endoplasmic reticulum (ER) membrane anchored E3 ligase in regulation of NF-κB. The expression of TRIM13 represses TNF induced NF-κB while the knockdown has the opposite effect. The E3 ligase activity and ER localization is essential for NF-κB suppression whereas TRIM13 regulated autophagy is not essential. TRIM13 interacts with NEMO and modulates its ubiquitination and turnover, hence may regulate IKK complex activity. TRIM13 mediated NF-κB repression is essential for negative regulation of clonogenic ability of the cells. This study for the first time demonstrated the role of TRIM13, ER resident RING E3 ligase as a novel regulator of NEMO ubiquitination, negative regulator of NF-κB signaling and its role as a tumor suppressor.

Endoplasminen reticulum (ER) on laadunkontrollipaikka proteiineille

Monet proteiinit, jotka eivät läpäise tätä endoplamisen verkoston kontrollia, siirretään syrjään sytoplasman puolelle ja siellä ne käsittelee monipuolinen proteosomi-silppurointijärjestelmä ja niiden peruselementit hyödynnetään esim johonkin synteesiin. ( Endoplasmiseen retikulumiin assosioitunut degradaatio: ERAD-L, ERAD-M).
 Kuitenkin tämä laadunkontrolli joissain tapauksissa säästää puuteellisenkin proteiinin funktiota. Turha proteiinien poisto edustaa patogeneettisiä piirteitä erilaisissa geneettisissä taudeissa, näistä on tunnetuin esimerkki CFTCR cystisen fibroosin transmembraanisen johtuvuuden säätelijän DF508 mutaatio. (https://www.ncbi.nlm.nih.gov/gene/1080 Kloridikanava)
Juovikkaan lihaksen sarkoglygaanin ( alfa, beta, gamma) epäasianmukainen proteosomaalinen hajoittaminen missense- mutaatiossa on arveltu LGMD-lihasdystrofia- taudin taustaksi (Siitä tunnetaan tyypit 2D, 2E, 2C, 2F). Sargoglykaanimutantin hajoittamisesta vastaava proteiinintarkkailutie ei ole vielä selvitetty. Tässä artikkelissa esitetään V247M- alfa-sarkoglykaanimutantin hajoamisreitti – tämä on toiseksi yleisimmin raportoitu val247met- alfa-sarkoglykaanimutaatio LGMF 2D- lihasdystrofia alatyypissä.
E3 ubikitiiniligaasit HRD1 (kr.11q13.1 synovioliini1) ja TRIM13 (RFP2, Kr. 13q14) avustavat tämän tien paljastamisessa ja ehkä voidaan löytää terapeuttisia kohteita tässä taudissa, johon ei nykyään vielä ole mitään parantavaa terapiaa. Mainittava seikka on, että E3 ubikitiiniligaasi HRD1 :n inhibitiolla säästyy V247 alfa-sarkoglykaanilaatua heterologisissa solumalleissa ja LMGD- D2- potilaiden myotuubeissa, kun kyse on  geenimutaatiosta L31P/V247M . Tämä on ensimmäinen osoitus siitä, että E3-ligaasit, jotka ovat mutatoituneen proteiinin hajoittamisesta vastuussa, voivat olla sarkoglykanopatiassa lääkeinterventiolla tavoitettavia kohteita.
  • Unveiling the degradative route of the V247M α-sarcoglycan mutant responsible for LGMD-2D. Results show that HRD1 and RFP2 contributes are required for the disposal of V247M alpha-sarcoglycan mutant. Many membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated into the cytosol and degraded by the proteasome. In applying rigid rules, however, quality control sometimes discharges proteins that, even though defective, retain their function. The unnecessary removal of such proteins represents the pathogenetic hallmark of diverse genetic diseases, in the case of ΔF508 mutant of cystic fibrosis transmembrane conductance regulator being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle sarcoglycans (α, β, γ and δ) with missense mutation has been proposed to be at the bases of mild-to-severe forms of limb girdle muscular dystrophy (LGMD) known as type 2D, 2E, 2C and 2F, respectively. The quality control pathway responsible for sarcoglycan mutant disposal, however, is so far unexplored. Here we reveal key components of the degradative route of V247M α-sarcoglycan mutant, the second most frequently reported mutation in LGMD-2D. The disclosure of the pathway, which is led by the E3 ligases HRD1 and RFP2, permits to identify new potential druggable targets of a disease for which no effective therapy is at present available. Notably, we show that the pharmacological inhibition of HRD1 activity rescues the expression of V247-α-sarcoglycan both in a heterologous cell model and in myotubes derived from a LGMD-2D patient carrying the L31P/V247M mutations. This represents the first evidence that the activity of E3 ligases, the enzymes in charge of mutant fate, can be eligible for drug interventions to treat sarcoglycanopathy.

TRIM13  on osoitettu kaspaasi8:n aktivaation säätelijäksi (solustressissä)

TRIM13 osuus multippelissa myeloomassa selvitetty

Multippelissa myeloomassa luuytimen ( B-soluista erikoistuva) plasmasolu on alkanut pahanlaatuisesti kasvaa. Kromosomideleetio on usein taustalla. On ajateltu jopa,  että TRIM13 olisi tuumorisupressiivinen geeni (TSG) (?); kromosomideleetiossa sen määrä on matala. TRIM13:n  alassäätö kuitenkin vähensi tuumorin solusyklin etenemistä ja  tuumorisolun kasvua.NF-kB tie inaktivoitui,  samoin 20S proteosomin aktiivisuus aleni. Tämän työn  tieto  ei tue TRIM13:n osuutta tuumorisuppressorina, vaan TRIM13  näytti  edistävän  sensijaan  myeloomasolun elossapysymistä ja on täten uusi terapeuttisen intervention kohde.
  1. Shows important roles of TRIM13 in MM tumour survival and proliferation in multiple myeloma. Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.

TRIM13 autofagian aloittajana

(Suomennosta) Autofagia on solun adaptiivisia prosesseja ja antaa suojaa monia patologisia tiloja kohtaan kuten infektioita, syöpää, neurodegeneraatiota ja ikääntymistä kohtaan. Ubikitinaatio hajoittaa viallisia organelleja joko proteosomisilppurissa tai autofagialla. TRIM13 on endoplasmiseen retikulumiin (ER) ankkuroitunut E3-ligaasi ja indusoi autofagiaa ja osallistuu ER- stressiin. Ektooppinen TRIM13 indusoi autofagiaa tutkituissa soluissa. TRIM13 :n coiled-coil domeeni(C-C) vaaditaan tähän autofagiafunktioon. ER-stressi stabiloi TRIM13- proteiinin ja se  tekee vuorovaikutuksen  p62/SQSTM1:n kanssa ja ko-lokalisoituu DFCP1:n kanssa.

  • Autophagy is one of the cellular adaptive processes that provide protection against many pathological conditions like infection, cancer, neurodegeneration, and aging.
    Recent evidences suggest that ubiquitination plays an important role in degradation of proteins or defective organelle either through proteasome or autophagy. In this study, we describe the role of TRIM13, ER resident ubiquitin E3 ligase in induction of autophagy and its role during ER stress. The ectopic expression of TRIM13 in HEK-293 cells induces autophagy. Domain mapping showed that coiled-coil (CC) domain is required for induction of autophagy. TRIM13 is stabilized during ER stress, interacts with p62/SQSTM1 and co-localizes with DFCP1. TRIM13 regulates initiation of autophagy during ER stress and decreases the clonogenic ability of the cells. This study for the first time demonstrates the role of TRIM13 in induction of autophagy which may play an important role in regulation of ER stress and may act as tumor suppressor.

TRIM13 “reseptoriregulaattorina” täsmäautofagiassa (Precision autophagy )(Kuva selvityksenä)

TRIM13 kuuluu myös RET- finger proteiineihin, mistä nimi RFP2. Aktivoituu säteilystä, stabiloi p53:n. Omaa erikoisen  4G Quadruplex-toistorakenteen.

Säteilytys aiheuttaa RFP2 (TRIM13):n yli-ilmenemää, mikä lisää jonisoivan säteilyn indusoimaa apoptoosia kohottamalla p53:n stabiiliutta ja vähentämällä AKT-kinaasiaktiivisuutta ( hajoitamalla  MDM2:n ja AKT:n). Monissa tuumorityypeissä on tämä RFP2-geeni usein deletoituna – ja se on juuri TRIM13-proteiinin koodaaja. Tässä osoitetaan RFP2:n osuus trimien funktioissa ( joita ovat apoptoosi, proliferaatio, differentiaatio ja transkriptionaalinen säätely). Omalta osaltaan TRIM13 indusoi apoptoosin hajoittamalla proteosomisilppurissa MDM2- ja AKT- proteiinit. Radioaktiivisen säteilyn annoksesta ja altistusajasta riippuvalla tavalla lisääntyi RFP2:n expressio. RFP2:n yli-ilmenemä indusoi solukuoleman ja nosti apoptoottisten molekyylien ilmenemää (p53, p21 ja Bax).
 Jos RFP2:n RING-domeeni oli mutatoitunut, se ei kyennyt aiheuttamaan apoptoosia. Solutasossa apotoosin inhibition säätelijä on AKT.  MDM2 on p53-tuumorisuppressorin negatiivinen säätelijä. TRIM13 ( RFP2) teki näiden kanssa interaktionsa ( eli ubikitinoi ne ja johti ne silppuroitumaan proteosomissa)- sekä in vivo että in vitro ( elävässä kehossa ja koeputkessa).
Näistä tiedoista päätellen säteily aiheuttaa RFP2:n yliekspression, joka lisää jonisoivan säteilyn aiheuttamaa apoptoosia kohottamalla p53:n ( Guardian of Genome) ) stabiliteettia ja alentamalla AKT-kinaasin aktiiviteettia hajoittamalla MDM2:n ( Zn finger RANB 2- tyyppinen E3-ubikitiiniligaasin) ja AKT:n.
    These results depended on the E3 ubiquitin ligase activity of RFP2 because mutant RFP2, which contains a mutated RING domain, failed to drive apoptosis compared with wild-type RFP2. We observed that RFP2 formed a complex with MDM2, a negative regulator of the p53 tumor suppressor, and AKT, a regulator of apoptosis inhibition at the cellular level. Additionally, we found that the interaction of RFP2 with MDM2 and AKT resulted in ubiquitination and proteasmal degradation of MDM2 and AKT in vivo and in vitro. Thus, these data suggest that irradiation causes RFP2 overexpression, which enhances ionizing radiation-induced apoptosis by increasing p53 stability and decreasing AKT kinase activity through MDM2 and AKT degradation.

RFP2 rakenne on ainutlaatuinen: monia aktiiveja G4 guadruplex GGGGA -toistoja ja elektrostaattista kenttää - tätä jaksoa puuttuu monista tuumoreista

Human RFP2 gene promoter: unique structure and unusual strength. Skoblov M1, Shakhbazov K, Oshchepkov D, Ivanov D, Guskova A, Ivanov D, Rubtsov P, Prasolov V, Yankovsky N, Baranova A.Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells. 

TRIM13 on ERAD E3 ubikitiiniligaasi 

Endoplsmiseen retikulumiin assosioituvassa  degradaatiossa välttämätön proteiini 

(Suomennosta)RFP2 geeni puuttuu useissa pahanlaatuisissa taudeissa . Sen koodaama proteiini on RBCC/TRIM perhettä- Proteiinina Rfp2 on instabiili ja se pystyy autopolyubikitinoitumaan kehossa ja koeputkessa, mikä viittaa sen toimivan RING-tyyppisenä E3-ubikitiiniligaasina. Sen ubikitiiniligaasiaktiivisuudelle on intakti RING-domeeni välttämättömyys. RING-puutteiset mutantit eivät pysty tekemään polyubikitinaatiota koeputkessa ja kehossa ne ovat stabiileja. Endoplasmisesta verkostosta löydettiin useita proteiineja, jotka tekevät interaktion sen kanssa ( esim. valosiinia sisältävä proteiini VCP , joka on ER:iin assosioituvassa degradaatiossa (ERAD) välttämätön proteiini. Rfp2 säätelee ER:n proteolyyttistä substraattia CD3-delta. RFP2 sisältää C-terminaalissa TM-domaanin, joka on välttämätön sen ankkuroitumiselle endoplasmiseen retikulumiin - RFP2 kolokalisoituuu useihin ER:n proteiineihin. Tiedot viittaavat siihen, että TRIM13 on yksi ERAD E3 ubikitiiniligaaseista.
  1. RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
Muistiin 12.2. 2018. Tässä on  suomentamatonta  englantilaista tekstiä runsaaati muistissa, koska  TRIM13 rakenteen ja  siihen liittyvien tautien  assosioaatio on edelleen selvityksenalainen.

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