Endoplasminen retikulum: laskostavien proteiinien toiminnasta

Endoplasmic Reticulum

In Cell Biology (Third Edition), 2017

Protein Folding and Oligomerization in the Endoplasmic Reticulum

Once nascent polypeptides translocate across the ER bilayer through the Sec61 translocon, they emerge into the ER lumen and interact with a wealth of proteins.

https://www.jbc.org/content/276/14/F1.medium.gif 

https://www.genecards.org/cgi-bin/carddisp.pl?gene=SEC61A1&keywords=Sec61

 Those proteins remove the signal sequence, add oligosaccharides, and direct folding by catalyzing disulfide bond formation and oligomerization. 

One such factor, the Hsp70 chaperone BiP, binds unfolded polypeptides by interacting with hydrophobic regions that are normally sequestered in the protein interior (see Fig. 12.12). These interactions prevent newly synthesized proteins from aggregating and promote their folding. Cycles of BiP binding also bias the movement of the polypeptide into the ER lumen but not back out. 

Another enzyme called oligosaccharyl transferase adds core sugars to the growing chain when an asparagine in an appropriate sequence context is detected.

 A fourth enzyme, protein disulfide isomerase (PDI), catalyzes disulfide exchange between sulfhydryl groups on cysteines allowing the formation of disulfide (S-S) bonds. The oxidizing equivalents to form disulfide bonds flow from flavin adenine dinucleotide (FAD) through two pairs of cysteines of an ER membrane protein that oxidizes a pair of cysteines in the active site of PDI. PDI then mediates correct formation of disulfide bonds by forming and breaking mixed disulfides with polypeptide substrates until the correct disulfides are formed.

Retention of these folding factors in the ER depends on the sequence lysine–aspartic acid–glutamic acid–leucine (KDEL) at the C-termini of these enzymes. If this sequence is deleted, the mutated protein is transported to the Golgi apparatus and secreted from the cell. Remarkably, addition of KDEL to a normally secreted protein results in its accumulation in the ER.

Folding and assembly factors interact with proteins throughout their lifetimes in the ER. The following sections describe the machinery that controls protein folding and assembly in the ER, mechanisms for sensing correctly folded or misfolded proteins, and pathways for disposing of misfolded proteins that accumulate in the ER.

ERAD koneisto on monen viruksen kaappaama, myös SARS2 CoV omaa tekijöitä, jotka tekevät siihen interaktiota

•  https://www.researchgate.net/profile/Timothy_Bergmann/publication/309439006/figure/fig3/AS:716405699784706@1547815837565/Folding-and-quality-control-of-glycosylated-proteins-within-the-ER-N-glycans-are.ppm

 

• ERAD koneistosta  artikkeleita.
•  ERAD  tarkoittaa endoplasmiseen retikulumiin(ER)  assosioitunutta hajoitusjärjestelmää, joka on solutalouden kannalta eräänlainen tarkka hyödynnyslaitos  tekovikamaterialle, jota ei onnistuta korjaamaan. 
• Virukset ja toksiinit kaappaavat tätä  järjestelmää.

https://www.sciencedirect.com/science/article/pii/S1097276515004499

ER-associated degradation (ERAD) is a protein clearance mechanism by which misfolded, misassembled, or metabolically regulated proteins are specifically dislocated from the ER into the cytosol and degraded by the ubiquitin proteasome system. 

 ERAD very likely evolved to maintain proteostasis and sterol homeostasis in the ER. 

However, the ironic truth is that membrane-penetrating transportation and protein degradation machineries in ERAD are preferably hijacked by exogenous pathogens such as viruses and toxins for their invasion and evasion from immunological surveillance. In this Review, we provide an overview of our current understanding of the pathogenic hijacking of the host cell ERAD, in which pathogens exploit the complex ERAD machinery in a variety of manners for their own use, suggesting flexibility and plasticity of the molecular machinery of ERAD.

• ERAD koneisto  toimii endoplasmisen retikulumin (ER)    syntetisoimien  proteiinin laadunkontrollijärjestelmänä (QC)  (Quality Control ) 
• Tämä ERQC käsittää  useita vaiheita ja lukuisia  komponentteja.

ERAD functions in ER protein quality control (ERQC) and sterol regulation (Ruggiano et al., 2014). Over the past two decades, it has been elucidated that ERQC-related ERAD is a multi-step and branched process that has numerous components (Araki and Nagata, 2011, Christianson and Ye, 2014).

 ERQC kolme akselia.

Three Axes of ERQC

I akseli  on proteiinien laskostuminen.  Useimmat  Endoplasmisessa retikulumissa (ER)  syntetisoituneista proteiineista  ovat modifioitu kovalenttisesti  molekyylien nsisäisten ja välisten disulfidisiltojen (-S-S-)   ja oligosakkaridiketjujen avulla  (OS) samanaikaisesti kun peptidiä syntetuisoidaan ja laskostetaan. Näitten modifikaatioitten avulla proteiinit saavat  sellaista kestokykyä, jolla  ne pysyvät elossa myös solun ulkopuolisessa miljöössä.  Tähän modifikaatioon kytkeytyneeseein proteiinien laskostumiseen osallistuu  kolme molekulaarista kaitsijaproteiiniryhmää
(Chaperoneproteiini = kaitsijaproteiini).
I chaperone-proteiiniryhmä  on immunoglobuliinin raskaan ketjun sisältävä BiP, joka kuuluu  HSP70-proteiiniperheeseen ja se edistää proteiinin laskostumista sisäisellä ATP.ta hydroplysoivalla aktiivisuudella.
II chaperoneproteiiniryhmä  ovat ER oxidoreduktaasit, kuten
PDI, proteiinidisulfidi-isomeraasi;
ERO1, ER oxidoreductiini-1.
ja ERp57, joka oksidoi cysteiinitähteitä , siis muodostaa niissä  disulfidisiltoja (-S-S-).
III chaperoniproteiiniryhmä ovat kalnexiini (CNX)  ja kalretikuliini(CRT). Ne ovat lektiinien kaltaisia kaitsijoita ja ne tunnistavat  oligosakkaridiketjuja vastasyntetisoituneissa  glykoproteiineissa ja edistävät proteiinin laskostumista.
Eräät näistä mainituista tekijöistä osallistuvat myös ERAD.iin ja moni patogeeni hyödyntää niitä. ( Esim SARSc  tekee interaktion ER- kalvoon integroituneeseen   kalnexiiniin , mutta  välttää interaktiota luminaaliseen  kalretikuliiniin, jolloin se häiritsee  CRT/CNX toimintasykliä). 
https://kyushu-u.pure.elsevier.com/en/publications/monitoring-of-s-protein-maturation-in-the-endoplasmic-reticulum-b

• Monitoring of s protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus  Masaya Fukushi, Yoshiyuki Yoshinaka et al    Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. . These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.

The first axis of ERQC is protein folding (Araki and Nagata, 2011). Most newly synthesized proteins in the ER are covalently modified with intra- and/or inter-molecular disulfide bonds and oligosaccharide chains in parallel with their synthesis and folding. These modifications reinforce the structure of these proteins such that they can survive in the extracellular environment. Three groups of molecular chaperones and folding enzymes majorly contribute to modification-coupled protein folding. The first group includes classic molecular chaperones, such as immunoglobulin heavy chain-binding protein (BiP), which belongs to the heat shock protein 70 family and promotes protein folding with an intrinsic ATP-hydrolyzing activity (Otero et al., 2010). The second group consists of ER oxidoreductases, such as protein disulfide isomerase (PDI), ER oxidoreductin 1 (ERO1), and ERp57, which oxidize substrate cysteine residues (synonymous with the formation of a disulfide bond) and isomerize improperly oriented disulfide bonds in parallel with de novo protein synthesis and folding (Ellgaard and Ruddock, 2005). The third group consists of lectin-like chaperones such as calnexin and calreticulin, which recognize oligosaccharide chains attached to newly synthesized glycoproteins and promote protein folding (Helenius and Aebi, 2004). 

Some of these factors also participate in ERAD and are occasionally utilized by pathogens, as discussed later.

II akseli on vaste proteiinin laskostumattomuuteen. (UPR,  Unfolded protein response). 

Jos jostain syystä proteiinit eivät laskostu  tai laskostuvat väärin ja jos  tällaista materiaalia kertyy endoplasmiseen verkostoon  ylen määrin, ylittyy   kapasiteetti  saada  tätä materiaalia  pois endoplasmisesta   verkostosta.
 Näitä proteiineja, joiden  tehtäviin kuluu tunnistaa  laskostumattomien proteiinien   ylikuormitusta,   ovat  PERK, ATF6 ja IRE1, ja ne pystyvät  kukin  tiettyä  tietään  aloittamaan alavirran tapahtumia kuten translaation vaimentamista ja  laskostavien entsyymien ERAD- tekijöiden   ylössäätämistä jotta ER  ylikuormitus tila korjautuisi ja ER  proteostaasi palautuisi. ESIM: https://www.frontiersin.org/articles/10.3389/fendo.2018.00210/full
https://www.frontiersin.org/files/Articles/364990/fendo-09-00210-HTML/image_m/fendo-09-00210-g001.jpg 

The second axis of ERQC is the unfolded protein response (UPR). When the level of unfolded or misfolded proteins accumulated in the ER exceeds the folding and clearance capacity of the ER for any reason, particular ER membrane proteins such as PERK, ATF6, and IRE1 sense the overloaded state and initiate downstream events, including translation attenuation and upregulation of folding enzymes and ERAD factors, which comprehensively reduce the protein burden in the ER and restore ER proteostasis (see previous review articles, e.g., Walter and Ron, 2011). The UPR also involves an apoptotic pathway, which is activated when the cell fails to resolve the stress conditions (Walter and Ron, 2011).

III akseli on varsinainen ERAD. 

Finally, the third axis of ERQC is ERAD, which is described in the next section.

(Tähän laitan  ERAD  kohtaan kuuluvia SARS interaktioproteiineja sitä mukaa kuin löydän. Selenos eli VIMP on yksi näitä.

https://www.researchgate.net/publication/307442236/figure/fig1/AS:403388127825920@1473186632494/ER-associated-degradation-ERAD-pathway-ERAD-is-an-ER-quality-control-pathway-that.png

SELENOS (15q26.2 Aliases for SELENOS Gene, VIMP

Selenoprotein S ^{2 3 4 5}
VCP-Interacting Membrane Protein ^{2 3 4} Valosin-Containing Protein-Interacting Membrane Protein ^{2 3} VCP Interacting Membrane Selenoprotein ^{2 3}
VIMP ^{3 4}, AD-015 ³, ADO15 ³, SBBI8 ³ SEPS1 ³, Tanis ³ , SelS ⁴, SELS ^{3 4}

 https://api.intechopen.com/media/chapter/65506/media/F2.png
 

This gene  SELENOS encodes a transmembrane protein that is localized in the endoplasmic reticulum (ER). It is involved in  ERAD, the degradation process of misfolded proteins in the ER, and may also have a role in inflammation control. This protein is a selenoprotein, containing the rare amino acid selenocysteine (Sec). Sec is encoded by the UGA codon, which normally signals translation termination. The 3' UTRs of selenoprotein mRNAs contain a conserved stem-loop structure, designated the Sec insertion sequence (SECIS) element, that is necessary for the recognition of UGA as a Sec codon, rather than as a stop signal. Two additional phylogenetically conserved stem-loop structures (Stem-loop 1 and Stem-loop 2) in the 3' UTR of this mRNA have been shown to function as modulators of Sec insertion. An alternatively spliced transcript variant, lacking the SECIS element and encoding a non-Sec containing shorter isoform, has been described for this gene (PMID:23614019). [provided by RefSeq, Jul 2017]

FYVE- Znf proteiineja on SARS CoV. interaktioproteiinien joukossa.

ZFYVE sinkkisormiproteiinit, joihin SARS2 CoV tekee interaktion: FYCO1 ja PLEKFH2. Mahdollisesti niiden kautta laajempiinkin komplekseihin.

SARS CoV nsp13 helikaasi  tekee interaktion FYCO1 proteiiniin (ZFYVE7), (3p21.31). 

FYCO1. https://www.genecards.org/cgi-bin/carddisp.pl?gene=FYCO1&keywords=FYCO1

• This gene encodes a protein that contains a RUN domain, FYVE-type zinc finger domain and Golgi dynamics (GOLD) domain. The encoded protein plays a role in microtubule plus end-directed transport of autophagic vesicles through interactions with the small GTPase Rab7, phosphatidylinositol-3-phosphate (PI3P) and the autophagosome marker LC3. Mutations in this gene are a cause of autosomal recessive congenital cataract-2 (CATC2). [provided by RefSeq, Dec 2011]
  FYCO1 (FYVE And Coiled-Coil Domain Autophagy Adaptor 1) is a Protein Coding gene. Diseases associated with FYCO1 include Cataract 18 and Cataract 44. Gene Ontology (GO) annotations related to this gene include nucleotide binding. An important paralog of this gene is RUFY4.
  FYVE domain found in FYVE and coiled-coil domain-containing protein 1 (FYCO1) and similar proteins
  FYCO1, also termed zinc finger FYVE domain-containing protein 7, is a phosphatidylinositol 3-phosphate (PtdIns3P or PI3P)-binding protein that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport. It acts as an effector of GTP-bound Rab7, a GTPase that recruits FYCO1 to autophagosomes and has been implicated in autophagosome-lysosomal fusion. FYCO1 also interacts with two microtubule motor proteins, kinesin (KIF) 5B and KIF23, and thus functions as a platform for assembly of vesicle fusion and trafficking factors. FYCO1 contains an N-terminal alpha-helical RUN domain followed by a long central coiled-coil region, a FYVE domain and a GOLD (Golgi dynamics) domain in C-terminus.putative Zn binding site [ion binding site], 8 residue positions
  Conserved feature residue pattern:C C C C C C C C

 https://www.researchgate.net/figure/Graphical-representation-of-diverse-domain-fold-and-metal-coordination-within-multiple-ZF_fig4_324280754

SARS COV  ORF7 tekee interaktion PLEKHF2 proteiiniin (ZFYVE18)(8q22.1) 

 

PLEKHF2 (Pleckstrin Homology And FYVE Domain Containing 2) is a Protein Coding gene. An important paralog of this gene is PLEKHF1.May play a role in early endosome fusion upstream of RAB5, hence regulating receptor trafficking and fluid-phase transport. Enhances cellular sensitivity to TNF-induced apoptosis (PubMed:18288467).PKHF2_HUMAN,Q9H8W4  Size:249 amino acidsMolecular mass: 27798 Da    (Quaternary structure: May interact with EEA1 (ZFYVE2, 2q22) https://www.genecards.org/cgi-bin/carddisp.pl?gene=EEA1&keywords=EEA1  Among its related pathways are Innate Immune System and Phagosome.  Quaternary structure: Homodimer. Binds STX6. Binds RAB5A, RAB5B, RAB5C and RAB22A that have been activated by GTP-binding. Interacts with RAB31. Interacts with ERBB2. Interacts with SAMD9 AND SAMD9L (PubMed:24029230). May interact with PLEKHF2.. Miscellaneous Antibodies against EEA1 are found in sera from patients with subacute cutaneous lupus erythematosus and other autoimmune diseases.

FYVE domeenin merkityksestä: 

FYVE domains bind Phosphatidylinositol 3-phosphate (PI3P) , in a way dependent on its metal ion coordination and basic amino acids. 

The FYVE domain inserts into cell membranes in a pH-dependent manner. 

The FYVE domain has been connected to vacuolar protein sorting and endosome function.

https://www.ncbi.nlm.nih.gov/pubmed/25985087

 (Suom.) FYVEsinkkisormidomeeni sinkkiproteiinissa linkitsee erityisesti mRNA-kuljetuksen endosomaaliseen liikennöintijärjestelmään. Lähetti RNA:lla on lähiyhteys kalvokuljetukseen. Hyvänä esimerkkinä on mRNA:n ja endosomiin assosioituneiden ribosomien mikrotubuluksista riippuva kuljetus. Tämä koordinoitunut prosessi on ratkaiseva korrektissa septiinifilamentaatiossa ja polarisoituneiden solujen tehokkaassa kasvussa, esim sienirihmastossa. Vaikka tiedetäänkin yksityiskohtia avainasemassa olevista RNA:ta sitovista proteiineista ja molekulaarisista moottoripoteiineista, on kuitenkin epäselvä, miten mRNA on liittynyt kalvoihin kuljetuksen aikana. Tutkijat ovat tunnistaneet uuden tekijän, jossa on FYVE sinkkisormidomeeni, jolla tapahtuu interaktio endosomilipideihin sekä uuden PAM2-kaltaisen domeenin, jota tarvitaan avain asemassa olevaan RNA.ta sitovan proteiiniN MLLE- domeenin kanssa. Johdonmukaisesti FYVE-domeeni proteenin menetys johtaa mRNA:- , ribosomi- ja septiinikuljetuksessa spesifisiin puutteisiin, vaikka endosomein ja niiden liikkeen yleisfunktiot eivät vaikutu. Tämä on ensimmäinen havaittu endosomaalinen komponentti, joka on mRNP-kuljetukselle spesifinen , mistä selviää, että on olemassa eräs uusi mekanismi, jolla mRNA liittyy endosomeihin.

Sinkkisormidomeeni voidaan  kaavamaisesti   ilmaista : CCCCCCCC

tai selkeämmin C-x-C-x-C-x-C-xxx-C-x-C-x-C-x-C, mistä näkee että  ensimmäiset  neljä cysteiiniä ovat  ryhmittyneet  sinkin ympärille  aika lähekkäin, sitten on enemmn aminohappoväliä ja toinen ryväs neljä cysteiiniä (ja niihin koordinoitunut Zn). Maininta FYVE,ilmaisee  mikä  sinkkikooridaatiomalli on kyseessä  ( tässä cross-brace topologia)

Elife. 2015 May 18;4. doi: 10.7554/eLife.06041.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

Pohlmann T¹, Baumann S¹, Haag C¹, Albrecht M², Feldbrügge M¹.Abstract

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

KEYWORDS: FYVE; PAM2; RRM; Ustilago maydis; cell biology; endosome; infectious disease; mRNA transport; microbiology

SARS2 CoV tekee ERAD evaasion S-proteiinin, ORF8, ORF9 ja nsp15 avulla.

 SARS2 tekee  ERAD- evaasion.

ERAD järjestelmässä  on chaperone molekyylejä kalretikuliini (CRT) , joka on luminaalinen,  ja kalnexiini (CNX)  sijaitsee integraalisesti  ER kalvossa.
https://www.springer.com/gp/book/9783662062050

Myös SARS2 virus  kohdistaa epätasapainottavan  vaikutuksensa  tähän  CRT/CNX sykliin, joka näiden  kaitsijaproteiinien kesken vallitsee ja vaikuttaa että  virusproteiinit eivät joudu hajoitustiehen ERADissa. 

Niiden vastuulla on muodostuvien glykoproteiinien  korrekti laskostuminen. Ne ovat kvaliteetin kontrollitekijöitä uusille syntetisoiduille glykoproteiineille. Niiden vaikutusalue ulottuu sekä endoplasmisen verkoston sisään että ulkopuolelle. Kalretikuliinista sanotaan että se on multiprosessimolekyyli.  Se säätelee  kalsiumjonin homeostaasia ja puskurointia endoplasmisessa verkostossa. Nämä ovat hydrofobisia lektiinejä ja  laskostusta  auttavat  proteiinit BIP ja PDI   tunnistavat  niitä.

KDEL on motiivi, joka estää proteiineja  sekretoitumistiestä .
KDEL-reseptorien kanssa ligandi-interaktiot on havaittu ER luminaalisella  puolella  proteiinit kuljetetaan takaisin ER.än päin COP I rakkuloiden muodostuksella.  Näiden rakkuloiden silmukointi ja kuljetus on riippuva  GTPaasi ADP- ribosylaatiofaktorista 1, ARF1.

BIP molekyylissä on kauttaaltaan  runsaasti K, D, E L  aminohappoja ja samoin C-terminaali  on KDEL. 
https://www.ncbi.nlm.nih.gov/protein/NP_005338.1
 P4HB eli PDI omaa myös KDEL päädyn ja runsaasti K, D, E ja L aminohappoja.

SARS2 CoV nsp15  tekee interaktion toisen ARF - ryhmän entsyymin kanssa: ARF6.
 ARF6 taas  on ADP-ribosylaatio faktori 6, joka lokalisoituu  er plasmamembraaniin ja säätelee  vesikulaarista liikennöintiä,  membraanilipidien  muokkausta  ja signalointiteitä, jotka  johtavat  aktiinin uudelleen muokkaukseen.

ORF9 rakenteessa on  myös runsaasti  K, E, D ja L aminohappoja, mutta pääty ei ole tuo KDEL.
Se voi toimia jonkinlaisena hämäysmolekyylinä,oy  chaperonen kaltaisena joutumatta kuitenkaan  KDEL-tielle.

Mutta  Sars S  sitoutuu kalnexiiniin ja laskostuu hyvin.

 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486308/

Monitoring of S Protein Maturation in the Endoplasmic Reticulum by Calnexin Is Important for the Infectivity of Severe Acute Respiratory Syndrome Coronavirus

Masaya Fukushi,^a,b,c,d Yoshiyuki Yoshinaka 

We have shown here that the binding of SARS-CoV S protein to the cellular molecular chaperone calnexin plays a critical role in SARS-CoV infection. Calnexin strictly managed the folding of glycosylated S protein in viral production, with the result that daughter viruses acquired the infectious ability.

The interaction between S protein and calnexin was analyzed here, because calnexin provided the strongest signal in our pulldown assays using S2-N. These pulldown assays, however, showed that S2-N also bound to other cellular proteins containing actin. Recently, palmitoylated calnexin, which forms a supercomplex with the ribosome-translocon complex, was reported to interact with actin (25), suggesting that S protein may indirectly interact with actin through calnexin. Calnexin bound to all truncated S proteins except for S2-C, consistent with a previous report that 23 putative N-linked glycosylation sites are scattered throughout S protein (34, 40, 48). Calnexin associates with monoglycosylated N-glycan chains on polypeptides/proteins. The presence of many glycosylation sites in S protein is also consistent with our Western blot results with cell lysates, showing that S protein is present as two major bands with smearing. This result is consistent with a previous report using a full glycan analysis (39).

ORF8  tekee interaktion EDEM3 kanssa. https://www.ncbi.nlm.nih.gov/gene/80267

Official Symbol EDEM3

Official Full Name ER degradation enhancing alpha-mannosidase like protein 3 

Also known asC1orf22

SummaryQuality control in the endoplasmic reticulum (ER) ensures that only properly folded proteins are retained in the cell through recognition and degradation of misfolded or unassembled proteins. EDEM3 belongs to a group of proteins that accelerate degradation of misfolded glycoproteins in the ER (Hirao et al., 2006 [PubMed 16431915]).[supplied by OMIM, Mar 2008]

Expression Ubiquitous expression in stomach (RPKM 15.9), colon (RPKM 13.5) and 25 other tissues See more

SARS2 CoV ORF10 interaktiossa CLR2 välitteiseen HIF1alfa turnover säätelyjärjestelmään

Mitäkomponentteja  kuuluu CLR2 kompleksiin ja millä alueella se toimii ?
SARS2 CoV pitää interaktioproteiineina  CUL2, ELOB2  ja RBX1, jotka ovat CLR2 komponentteja.



Esimerkki löytyy  ihan Google haun avulla.
 CRL2^VHL ligase complex,
E3 ubikitiiniligaasi MUL1, joka  tunnistaa mitokondriaalisen stressin.
 https://www.genecards.org/cgi-bin/carddisp.pl?gene=MUL1&keywords=MUL1
Adaptori UBXN7
Järjestelmä, jota CLR2 säätelee tässä: HIF-1alfa pitoisuus normoxian ja hypoxian vallitessa
PHD2 on happisensori entsyymi, joka normoksian vallitessa  hydrolysoi  HIF.1alfa:n  ja tämä joutuu koneistoon, joka johtaa  proteosomisilppuriin. https://www.nature.com/articles/s41598-020-58484-8#Fig7

UBXN7 toimii adaptorina.  UBXN7 (UBX Domain Protein 7) is a Protein Coding gene.

Ubiquitin-binding adapter that links a subset of NEDD8-associated cullin ring ligases (CRLs) to the segregase VCP/p97, to regulate turnover of their ubiquitination substrates.UBXN7_HUMAN,O94888

https://www.nature.com/articles/s41598-020-58484-8
Article Open Access  Published: 31 January 2020

Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein

Lucia Cilenti, Jacopo Di Gregorio,  Camilla T. Ambivero, Thomas Andl, Ronglih Liao & Antonis S. Zervos  Scientific Reports volume 10, Article number: 1609 (2020) Cite this article

 Abstract

MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with its RING finger domain facing the cytoplasm. MUL1 participates in various biological pathways involved in apoptosis, mitochondrial dynamics, and innate immune response. The unique topology of MUL1 enables it to “sense” mitochondrial stress in the intermembrane mitochondrial space and convey these signals through the ubiquitination of specific cytoplasmic substrates.

 We have identified UBXN7, the cofactor protein of the CRL2^VHL ligase complex, as a specific substrate of MUL1 ligase.

CRL2^VHL ligase complex regulates HIF-1α protein levels under aerobic (normoxia) or anaerobic (hypoxia) conditions.

 Inactivation of MUL1 ligase leads to accumulation of UBXN7, with concomitant increase in HIF-1α protein levels, reduction in oxidative phosphorylation, and increased glycolysis.

We describe a novel pathway that originates in the mitochondria and operates upstream of the CRL2^VHL ligase complex.

 Furthermore, we delineate the mechanism by which the mitochondria, through MUL1 ligase, can inhibit the CRL2^VHL complex leading to high HIF-1α protein levels and a metabolic shift to glycolysis under normoxic conditions.

Introduction

Eukaryotic cells are dependent on oxygen levels as well as the presence of functional mitochondria in order to efficiently generate ATP through oxidative phosphorylation (OXPHOS). Cells respond quickly to changes in oxygen levels by activating several signaling pathways that provide metabolic and adaptive mechanisms to the new environment. Hypoxia-inducible factor 1α (HIF-1α) is the primary transcriptional regulator of the cell response to low oxygen levels (hypoxia)^1,2,3,4. Accumulation of HIF-1α protein and its translocation to the cell nucleus leads to the transcriptional activation of several hundred genes that carry a hypoxia response element (HRE) in their promoters^5,6. This leads to HIF-1α-dependent reprogramming of cellular metabolism that shifts the ATP production from oxidative phosphorylation, that is limited under low oxygen levels, to glycolysis^7,8. There is an important phenomenon associated with most cancer cells where glycolysis is predominantly used as the main source of ATP production, even under normal levels of oxygen (normoxia). This is known as the Warburg effect, originally described in 1923 and has since been extensively studied^9,10. Accumulating evidence indicates that induced aerobic glycolysis is not only the hallmark of cancer, but it is also important in many cellular processes including embryogenesis, innate and adaptive immunity, type 2 diabetes, starvation, as well as cardiomyopathy^{11,12,13,14,15,16,17}.
The mechanism that potentially “bypasses” the tight regulation of cellular metabolism by the HIF-1α transcription factor is unknown.
 Under normoxia, HIF-1α is continuously synthesized and degraded in the cytosol through a highly regulated process. The oxygen sensor propyl hydroxylase 2 (PHD2) hydroxylates HIF-1α which then binds the von Hippel-Lindau (VHL) tumor suppressor protein and gets ubiquitinated by the CRL2^VHL ligase complex¹⁸.
We have uncovered a new pathway that regulates HIF-1α protein levels and involves the mitochondrial MUL1 E3 ubiquitin ligase. MUL1 (also known as Mulan, MAPL, GIDE, and HADES) is one of three mitochondrial E3 ubiquitin ligases, the other two being MARCH5 and RNF185^{19,20,21,22,23,24,25,26,27,28}. Previous studies have shown MUL1 to be a major player in a number of pathways involved in apoptosis, mitophagy, and innate immune response^{21,25,26,29,30,31,32,33}. MUL1 is able to modify specific substrates through SUMOylation, as well as K63- or K48-ubiquitination^20,21,34,35. Our data show that MUL1, through K48-ubiquitination, directly regulates the level of UBXN7 protein, also known as UBX domain protein 7 (UBXD7)³⁶. UBXN7 serves as a substrate binding adaptor and interacts with several proteins including HIF-1α, CUL2, as well as AAA + ATPase p97, also known as VCP (Valosin-containing protein)^36,37. We identified lysine 14 (K14) and lysine 412 (K412) on the UBXN7 protein as the two major K48-ubiquitination sites for MUL1 ligase. Ubiquitination of UBXN7 targets the protein for proteasome degradation and inactivation of MUL1 leads to high levels of UBXN7 with concomitant increase in HIF-1α protein. The accumulation of HIF-1α is functional and is accompanied by activation of GLUT1, a known target of HIF-1α³⁸. This deregulation of HIF-1α affects the metabolic state of cells with glycolysis becoming the predominant energy production pathway even during aerobic conditions. In summary, we describe a new mitochondrial-initiated pathway that interferes with the process of HIF-1α regulation and reprograms cellular metabolism to induce aerobic glycolysis

Results

Schematic diagram of the proposed new pathway that operates upstream of the CRL2^VHL complex and involves the UBXN7 cofactor protein and its regulation by mitochondrial MUL1 E3 ligase. MUL1 ligase, through constant K48-linked polyubiquitination, maintains a steady low level of UBXN7 protein that is able to act as cofactor in assembling the active CRL2^VHL complex necessary for the regulation of HIF-1α during normoxia. When MUL1 becomes inactive or its activity is compromised, it leads to high levels of UBXN7 protein that function as an inhibitor of the CRL2^VHL complex. Without an active CRL2^VHL complex HIF-1α protein accumulates and drives glycolysis under normoxia. MUL1 protein levels are regulated by K48-autoubiquitination as well as by the action of the mitochondrial Omi/HtrA2 protease.
 ---------
 This is the first report to show the existence of a new pathway where mitochondria, through MUL1, regulate HIF-1α protein levels. Our data clearly show this pathway is very important under normal conditions and could potentially be involved in the mitochondrial induction of aerobic glycolysis.

nsp 13 helicase interactio proteiiineja sentrioleissa. Esimerkkejä

 https://www.researchgate.net/figure/Prototypic-vertebrate-centrosome-Salient-architectural-features-of-the-post-mitotic_fig3_258956529

PCNT. Pericentrin
 (21q22.3),
https://www.genecards.org/cgi-bin/carddisp.pl?gene=PCNT&keywords=PCNT
 Pericentrin ^{2 3 4 5} , Kendrin ^{2 3 4} , Pericentrin-B ^{3 4} , PCNT2 ^{3 4}

Pericentrin 2 (Kendrin) ² , Seckel Syndrome 4 ² , Pericentrin-380 ³
Pericentrin-2 ³  KIAA0402 ^4,  MOPD2 ³ , PCNTB ³ , PCTN2 ³ , SCKL4 ³
KEN ³ , PCN ³    

The protein encoded by this gene binds to calmodulin and is expressed in the centrosome. It is an integral component of the pericentriolar material (PCM). The protein contains a series of coiled-coil domains and a highly conserved PCM targeting motif called the PACT domain near its C-terminus. The protein interacts with the microtubule nucleation component gamma-tubulin and is likely important to normal functioning of the centrosomes, cytoskeleton, and cell-cycle progression. Mutations in this gene cause Seckel syndrome-4 and microcephalic osteodysplastic primordial dwarfism type II. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2015] 

 https://www.nature.com/articles/nrg1557/figures/1

CENTRL, CP110, Cep110, Centrosoma Coiled-Coil protein kDa 110

 (9q33.2) Centriolin

https://www.genecards.org/cgi-bin/carddisp.pl?gene=CNTRL&keywords=CNTRL

Centriolin ^{2 3 4 5} ,Centrosomal Protein Of 110 KDa ^{3 4} ,Centrosomal Protein 110kDa ^{2 3} ,Centrosomal Protein 1 ^{2 4}  ,CEP110 ^{3 4} ,CEP1 ^{3 4} 

This gene encodes a centrosomal protein required for the centrosome to function as a microtubule organizing center. The gene product is also associated with centrosome maturation. One version of stem cell myeloproliferative disorder is the result of a reciprocal translocation between chromosomes 8 and 9, with the breakpoint associated with fibroblast growth factor receptor 1 and centrosomal protein 1. [provided by RefSeq, Jul 2008]

Among its related pathways are FGFR1 mutant receptor activation and Regulation of PLK1 Activity at G2/M Transition. Gene Ontology (GO) annotations related to this gene include nucleotide binding.

  CEP250,

(2q11.22)  Centrosome-Associated Protein CEP250-  , C-Nap1

 ^{https://www.genecards.org/cgi-bin/carddisp.pl?gene=CEP250&keywords=CEP250}

Centrosomal Protein 250 ^{2 3 5}

Centrosomal Nek2-Associated Protein 1 ^{3 4} , 
250 KDa Centrosomal Protein ^{3 4} , Centrosomal Protein 2 ^{2 4} , CNAP1 ^{3 4} , CEP2 ^{3 4}

 This gene encodes a core centrosomal protein required for centriole-centriole cohesion during interphase of the cell cycle. The encoded protein dissociates from the centrosomes when parental centrioles separate at the beginning of mitosis. The protein associates with and is phosphorylated by NIMA-related kinase 2, which is also associated with the centrosome. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Dec 2015).

 CENPF 

(1q41), Centromere protein F

https://www.genecards.org/Search/Keyword?queryString=CENPF

Centromere Protein F ^{2 3 4 5}

Mitosin ^{2 3 4}
Centromere Protein F, 350/400kDa ^{2 3}
Kinetochore Protein CENPF ^{3 4}
AH Antigen ^{3 4}
Centromere Protein F, 350/400kDa (Mitosin) ²
Cell-Cycle-Dependent 350K Nuclear Protein ³

CENP-F Kinetochore Protein ³ ,PRO1779 ³ ,CILD31 ³ ,STROMS ³ ,CENP-F ⁴ ,Hcp-1 ³ ,CENF ³

This gene encodes a protein that associates with the centromere-kinetochore complex. The protein is a component of the nuclear matrix during the G2 phase of interphase. In late G2 the protein associates with the kinetochore and maintains this association through early anaphase. It localizes to the spindle midzone and the intracellular bridge in late anaphase and telophase, respectively, and is thought to be subsequently degraded. The localization of this protein suggests that it may play a role in chromosome segregation during mitotis. It is thought to form either a homodimer or heterodimer. Autoantibodies against this protein have been found in patients with cancer or graft versus host disease. [provided by RefSeq, Jul 2008]

CEP68,
 (2p14),  Centrosomal Protein Of 68 KDa
https://www.genecards.org/cgi-bin/carddisp.pl?gene=CEP68&keywords=CEP68

Centrosomal Protein 68 ^{2 3 5} KIAA0582 ^{2 3}   ,
CEP68 (Centrosomal Protein 68) is a Protein Coding gene. Diseases associated with CEP68 include Retinitis Pigmentosa 28 and Leukodystrophy, Demyelinating, Adult-Onset, Autosomal Dominant. Gene Ontology (GO) annotations related to this gene include protein kinase binding and protein domain specific binding. An important paralog of this gene is AKAP6.

Involved in maintenance of centrosome cohesion, probably as part of a linker structure which prevents centrosome splitting (PubMed:18042621). Required for localization of CDK5RAP2 to the centrosome during interphase (PubMed:24554434, PubMed:25503564).CEP68_HUMAN,Q76N32

 Size: 757 amino acids Molecular mass: 81102 Da

Quaternary structure: Interacts with CNTLN; the interaction recruits CEP68 to the centrosome (PubMed:24554434). Interacts with the SCF(FBXW11) complex which contains SKP1, CUL1 and FBXW11; the interaction is probably mediated by FBXW11 and the complex also contains CDK5RAP2 and PCNT (PubMed:25503564). Also interacts with F-box protein BTRC (PubMed:25704143, PubMed:25503564). Interacts with serine/threonine-protein kinase PLK1; the interaction leads to phosphorylation of CEP68 and its subsequent degradation (PubMed:25503564). Interacts with NEK2; the interaction leads to phosphorylation of CEP68 (PubMed:24554434) 

NINL,

( 20p11.21), Ninein Like protein.

Ninein Like ^{2 3 5} ,Ninein-Like Protein ^{2 3 4} ,NLP ^{3 4} ,KIAA0980.
 https://www.genecards.org/cgi-bin/carddisp.pl?gene=NINL&keywords=NINLNINL (Ninein Like) is a Protein Coding gene. Diseases associated with NINL include Trichostrongylosis and Hodgkin's Lymphoma, Lymphocytic-Histiocytic Predominance. Among its related pathways are Regulation of PLK1 Activity at G2/M Transition and Cell Cycle, Mitotic. Gene Ontology (GO) annotations related to this gene include calcium ion binding. An important paralog of this gene is NIN.

Involved in the microtubule organization in interphase cells. Overexpression induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery; it also interferes with mitotic spindle assembly. May play a role in ovarian carcinogenesis. NINL_HUMAN,Q9Y2I6.  Size: 1382 amino acids.. Molecular mass: 156344 Da  . Quaternary structure: Interacts with gamma-tubulin and TUBGCP4. Interacts with anaphase promoting complex/cyclosome (APC/C). Interacts with CDC20 and FZR1. Isoform 2 interacts with LCA5 and USH2A. 

 NIN,(14q22.1) ,  Ninein. Glycogen Synthase Kinase 3 Beta-Interacting Protein
 https://www.genecards.org/cgi-bin/carddisp.pl?gene=NIN&keywords=NINNinein ^{2 3 4 5},
This gene encodes one of the proteins important for centrosomal function. This protein is important for positioning and anchoring the microtubules minus-ends in epithelial cells. Localization of this protein to the centrosome requires three leucine zippers in the central coiled-coil domain. Multiple alternatively spliced transcript variants that encode different isoforms have been reported. [provided by RefSeq, Jul 2008]
Names: Glycogen Synthase Kinase 3 Beta-Interacting Protein ^{3 4}Ninein (GSK3B Interacting Protein) ^{2 3} , HNinein ^{3 4}Ninein Centrosomal Protein ³ , GSK3B-Interacting Protein ⁴ KIAA1565 ⁴ ,SCKL7.
Centrosomal protein required in the positioning and anchorage of the microtubule minus-end in epithelial cells (PubMed:15190203, PubMed:23386061). May also act as a centrosome maturation factor (PubMed:11956314). May play a role in microtubule nucleation, by recruiting the gamma-tubulin ring complex to the centrosome (PubMed:15190203). Overexpression does not perturb nucleation or elongation of microtubules but suppresses release of microtubules (PubMed:15190203). Required for centriole organization and microtubule anchoring at the mother centriole (PubMed:23386061). NIN_HUMAN,Q8N4C6

 

 

CEP350,

 (1q25.2)  Centrosome-Associated Protein 350.

 

 https://www.genecards.org/cgi-bin/carddisp.pl?gene=CEP350&keywords=CEP350

The product of this gene is a large protein with a CAP-Gly domain typically found in cytoskeleton-associated proteins. The encoded protein primarily localizes to the centrosome, a non-membraneous organelle that functions as the major microtubule-organizing center in animal cells. The encoded protein directly interacts with another large centrosomal protein and is required to anchor microtubules at the centrosome. It is also implicated in the regulation of a class of nuclear hormone receptors in the nucleus. Several alternatively spliced transcript variants have been found, but their full-length nature has not been determined. [provided by RefSeq, Jul 2008] Names:  Centrosomal Protein 350 ^{2 3 5} ,Centrosome-Associated Protein 350 ^{3 4}  Centrosomal Protein 350kDa ^{2 3} ,CAP350 ^{3 4} Centrosome-Associated Protein Of 350 KDa ⁴ ,Centrosome Associated Protein 350 ² KIAA0480 ⁴ ,Cep350 ⁴ ,GM133 ³ . Size:3117 amino acids Molecular mass:350930 Da. Quaternary structure:   Part of a ternary complex that contains CEP350, FGFR1OP and MAPRE1. Interacts (via C-terminus) directly with FGFR1OP (via N-terminus) (PubMed:16314388, PubMed:28625565, PubMed:28428259). Interacts with NR1H3, PPARA, PPARD and PPARG (PubMed:15615782). Interacts directly with microtubules (PubMed:17878239). Interacts with the fusion protein FGFR1OP-FGFR1, and by doing so recruits and activates PI3K and PLC-gamma (PubMed:18412956). Interacts with CYLD (PubMed:25134987). Interacts with CFAP157 (By similarity). Interacts with CEP19 (via C-terminus) (PubMed:28659385). 

CEP135 (4q12), 

 Centrosomal Protein Of 135 KDa,

https://www.genecards.org/cgi-bin/carddisp.pl?gene=CEP135&keywords=CEP135Centrosomal Protein 135 ^{2 3 5}  Centrosomal Protein 4 ^{2 3 4} ,KIAA0635 ^{2 3 4} ,Centrosomal Protein Of 135 KDa ^{3 4} Centrosomal Protein 135kDa ^{2 3} ,CEP4 ^{3 4} ,Centrosome Protein Cep135 ³  Cep135 ⁴ ,MCPH8 ³ Involved in early centriole assembly/duplication/biogenesis/formation/. Required for centriole elongation. Required for the recruitment of CEP295 to the proximal end of new-born centrioles at the centriolar microtubule wall during early S phase in a PLK4-dependent manner.
CP135_HUMAN,Q66GS9

Centrosomal protein involved in centriole biogenesis. Acts as a scaffolding protein during early centriole biogenesis. Required for the targeting of centriole satellite proteins to centrosomes such as of PCM1, SSX2IP and CEP290 and recruitment of WRAP73 to centrioles. Also required for centriole-centriole cohesion during interphase by acting as a platform protein for CEP250 at the centriole. Required for the recruitment of CEP295 to the proximal end of new-born centrioles at the centriolar microtubule wall during early S phase in a PLK4-dependent manner. Required for the recruitment of CEP295 to the proximal end of new-born centrioles at the centriolar microtubule wall during early S phase in a PLK4-dependent manner (PubMed:27185865) CP135_HUMAN,Q66GS9

 CEP112, (17q24.1)  Recommended name: Centrosomal protein of 112 kDa

https://www.genecards.org/cgi-bin/carddisp.pl?gene=CEP112&keywords=CEP112

Centrosomal Protein 112 ^{2 3 5}
Coiled-Coil Domain-Containing Protein 46 ^{3 4} , Centrosomal Protein Of 112 KDa ^{3 4} , Centrosomal Protein 112kDa ^{2 3} , CCDC46 ^{3 4} ,Coiled-Coil Domain Containing 46 ²  ,MACOCO
 This gene encodes a coiled-coil domain containing protein that belongs to the cell division control protein 42 effector protein family. In neurons, it localizes to the cytoplasm of dendrites and is also enriched in the nucleus where it interacts with the RNA polymerase III transcriptional repressor Maf1 to regulate gamma-aminobutyric acid A (GABA A) receptor surface expression. In addition, the protein has been identified as a component of the human centrosome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2014]

CEP43

Interaktioaffiniteettijärjestys:

PCNT,

CNTRL

CEP250

CENPF

CEP68

NINL

AKAP9 (7q26.2),  A-Kinase Anchoring Protein 9;   CG-Nap. Hyperion
https://www.genecards.org/cgi-bin/carddisp.pl?gene=AKAP9&keywords=AKAP9
A-Kinase Anchoring Protein 9 ^{2 3 5}
Centrosome- And Golgi-Localized Protein Kinase N-Associated Protein ^{2 3}
Centrosome- And Golgi-Localized PKN-Associated Protein ^{3 4}
Protein Phosphatase 1, Regulatory Subunit 45 ^{2 3} Protein Kinase A Anchoring Protein 9 ^{2 3}   Scaffolding protein that assembles several protein kinases and phosphatases on the centrosome and Golgi apparatus. Required to maintain the integrity of the Golgi apparatus (PubMed:10202149, PubMed:15047863). Required for microtubule nucleation at the cis-side of the Golgi apparatus (PubMed:15047863, PubMed:19242490). Required for association of the centrosomes with the poles of the bipolar mitotic spindle during metaphase (PubMed:25657325). In complex with PDE4DIP isoform 13/MMG8/SMYLE, recruits CAMSAP2 to the Golgi apparatus and tethers non-centrosomal minus-end microtubules to the Golgi, an important step for polarized cell movement (PubMed:27666745, PubMed:28814570). In complex with PDE4DIP isoform 13/MMG8/SMYLE, EB1/MAPRE1 and CDK5RAP2, contributes to microtubules nucleation and extension also from the centrosome to the cell periphery (PubMed:29162697).

• • AKAP9_HUMAN,Q99996
• [Isoform 4]: Associated with the N-methyl-D-aspartate receptor and is specifically found in the neuromuscular junction (NMJ) as well as in neuronal synapses, suggesting a role in the organization of postsynaptic specializations.

  • AKAP9_HUMAN,Q99996

GOLGB1

CDK5RAP2,  ( 9q33.2)  CEP215,  

gamma TuRC, centrosomin,  CDK5 regulatory subunit associated protein 2. https://www.genecards.org/cgi-bin/carddisp.pl?gene=CDK5RAP2&keywords=gamma-TuRC

PDE4DIP

GCC2

FYCO

GORASP1

CEP350

GOLGA2

JAKMIP1

PRKAR2B

CEP135

MIPOL1

CLIP4

TBK1

GRIPAP1

PRKAR2A

CEP112

NIN

ERC1

CIT

TBKBP1

HSBD1

(CEP43, FGFR  oncogene partner ?, ei löydy)

PRKACA

RDX

TLE1

TLE2

GOLGA3

USP13

C1orf50

HOOK1

TLE3

GCC1