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lördag 30 maj 2020

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.
 CRL2VHL 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:


Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein
Lucia Cilenti, Jacopo Di GregorioCamilla T. Ambivero, Thomas Andl, Ronglih Liao & Antonis S. Zervos  Scientific Reports volume 10, Article number: 1609 (2020)

 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 CRL2VHL ligase complex, as a specific substrate of MUL1 ligase.

CRL2VHL 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 CRL2VHL ligase complex.

 Furthermore, we delineate the mechanism by which the mitochondria, through MUL1 ligase, can inhibit the CRL2VHL 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 promoters5,6. 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 glycolysis7,8. 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 studied9,10. 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 cardiomyopathy11,12,13,14,15,16,17.
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 CRL2VHL 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 RNF18519,20,21,22,23,24,25,26,27,28. Previous studies have shown MUL1 to be a major player in a number of pathways involved in apoptosis, mitophagy, and innate immune response21,25,26,29,30,31,32,33. MUL1 is able to modify specific substrates through SUMOylation, as well as K63- or K48-ubiquitination20,21,34,35. 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 CRL2VHL 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 CRL2VHL 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 CRL2VHL complex. Without an active CRL2VHL 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.
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 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.

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