ERAD tiestä asiaa -Siihen kuuluvia TM-RNF proteiineja ja muuta proteiinikoneistoa

Moni transmembraaninen  RNF- molekyyli toimii ERAD- tiessä, joten  otan tähän mainintaa ERADista tänään:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021871/

3.1. Protein Degradation at the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is an interconnected network of membranous tubes, vesicles and sacs, which carries out the synthesis, assembly and processing of secreted proteins and integral membrane proteins. Approximately 30% of the genes in eukaryotes encode secreted and transmembrane proteins [43,44,45].

 Nascent polypeptide chains are co-translationally translocated across the ER membrane through the Sec61 translocon into the lumen and, in the case of transmembrane proteins, inserted into the ER membrane [46].

 Subsequent folding and assembly are assisted by molecular chaperones and folding enzymes. Nevertheless, protein folding is an intricate and error-prone process that often results in misfolding. Impaired protein folding is also caused by genetic mutations as well as cellular stresses, such as heat shock and oxidative stress. Improperly folded proteins are retained in the ER and their accumulation induces stress in the ER. Increased ER stress induces cellular dysfunction and cell death. 

Cells are equipped with an evolutionarily conserved protective mechanism that reduces further accumulation of misfolded proteins, which is referred to as the unfolded protein response (UPR). The UPR includes (1) attenuation of protein translation, (2) degradation of ER-associated mRNA, (3) transcriptional induction of chaperones to increase the capacity of protein folding and (4) degradation of misfolded proteins by ER-associated degradation (ERAD) [47,48].

ERAD serves to eliminate misfolded proteins by targeting them for proteasomal degradation [49]. 

ERAD substrates also include native ER proteins, such as hydroxymethylglutaryl-CoA reductase (HMGR) and inositolo 1,4,5-triphosphate (IP₃) receptors (IP₃R), indicating that ERAD participates in both metabolism and intracellular signaling [50,51]. 

 Proteins undergoing ERAD are transported back through the Sec61-based protein channel into the cytosol, where they are subjected to ubiquitination [52,53,54,55,56]. 

 Poly-Ub tags on ERAD substrates are recognized by a cytosolic multimeric complex composed of the AAA-ATPase valosin-containing protein (VCP)/p97, Ufd1 and Npl4 (Cdc48p, Ufd1p and Npl4p in yeast). The VCP complex drives extraction of ERAD substrates from the ER and escorts them to the proteasome [57,58,59,60,61,62]. In yeast, at least three ERAD pathways (ERAD-C, ERAD-M and ERAD-L) have been proposed. ERAD-L degrades luminal proteins, and ERAD-C and ERAD-M recognize cytoplasmic and membrane domains, respectively [63,64,65,66]. 

Yeast contains two ER-resident E3 enzymes involved in three ERAD pathways: Hrd1p for ERAD-L and -M and Doa10p for ERAD-C [67,68,69,70]. In mammals, the ERAD system is conserved but more complicated, as reflected by the increased number of ER-localized E3 enzymes, which has been extensively reviewed elsewhere [71,72,73].

HRD1/Synoviolin, a mammalian ortholog of Hrd1p, is an ER-resident E3 enzyme with multiple transmembrane segments [74,75]. HRD1 forms a complex with the adaptor protein SEL1L, which recruits the ERAD machinery, including the E2 enzyme Ubc7, HERP, OS9, XTP3 and the Derlin proteins [75,76,77,78,79,80,81,82,83]. OS9 and XTP3 are ER luminal proteins containing mannose 6-phosphate receptor (MPR) homology domains, and they are required for, at least, the recognition of misfolded luminal glycoproteins [76,77,83]. The Derlins facilitate retrotranslocation of ERAD substrates [78,79,84]. 

Gp78/AMFR/RNF45 has a moderate level of sequence homology in the transmembrane domains with HRD1. Gp78 associates with the E2 enzyme Ubc7 through the C-terminal CUE domain [85,86]. RMA1/RNF5 is a C-terminally anchored ER protein that associates with the E2 enzyme Ubc6 and Derlin-1 [87,88]. 

 Numerous studies have demonstrated that HRD1, gp78 and RMA1 play an integral role in the ERAD pathway by mediating the ubiquitination of misfolded and native ER proteins, including cystic fibrosis transmembrane conductance receptor (CFTR), HMGR and CD3δ [74,75,85,86,87,88,89,90,91]. 

RMA1 cooperates with gp78 to target mutant CFTR for ERAD [92]

 MARCH6/TEB4/RNF176, a mammalian ortholog of Doa10p, is a 14-transmembrane ER protein that catalyzes Lys48-linked ubiquitination with Ubc7 [70,93,94], but its precise biological function is less clear. The possible involvement of MARCH6 in the ERAD pathway in liver disease and metabolism has been suggested by recent studies demonstrating that MARCH6 ubiquitinates type 2 iodothyronine deiodinase (DIO2) and the mutant bile salt pump (Bsep)/ABCB11 associated with familial intraheoatic cholestasis type I for proteasomal degradation [95,96].

Kf-1/RNF103 was identified as a gene highly expressed in the cerebral cortex of Alzheimer disease patients [97]. Kf-1 has E3 activity and interacts with Derlin-1 and VCP [98]. Kf-1 mRNA expression is predominantly detected in the hippocampus and cerebellum, and is elevated in the frontal cortex of rats after antidepressant treatment [97,99,100]. Kf-1 knockout mice exhibit increased anxiety behavior [101]. Kf-1 may be involved in the regulation of neuronal activity and homeostasis in the central nervous system.

RFP2 is associated with VCP and Derlin-1, and regulates the turnover of CD3δ and the L-type calcium channel by mediating their ubiquitination [102,103].

 ZNRF4 is also proposed to be an ER E3 enzyme that regulates the UPR and ERAD by controlling the stability of the ER chaperone calnexin [21].

Cellular cholesterol homeostasis is maintained through the actions of the sterol response element binding proteins (SREBPs), SREBP cleavage-activated protein (SCAP) and insulin-inducing gene (INSIG) [104]. SREBPs are synthesized as membrane proteins and bind to SCAP at the ER. Under low cholesterol conditions, SCAP escorts SREBPs to the Golgi where SREBPs are processed to release their N-terminal regions that act as transcription factors. Cleaved SREBPs enter the nucleus and transactivate genes related to choresterol and fatty acid metabolism. In contrast, high cholesterol conditions cause a conformational change in SCAP, which allows SREBP precursors to reside in the ER through their binding to the ER membrane protein INSIG [104]. 

Translocation in renal carcinoma, chromosome 8 gene (TRC8)/RNF139 was identified as a tumor suppressor gene product associated with renal carcinoma [105,106]. TRC8 is an ER protein with multiple transmembrane regions, including a sterol-sensing domain [106,107]. TRC8 is rapidly degraded by self-ubiquitination in the presence of sterols, while it becomes stable in the absence of sterols [107]. TRC8 interacts with SREBP-2 and SCAP. This interaction inhibits ER–Golgi transport and proteolytic processing of SREBP-2, thereby preventing SREBP-2 target gene expression [107]. TCR8 ubiquitinates INSIG-1, and probably SREBPs, thereby reducing their expression by proteasomal degradation [108]. Thus, TRC8 contributes to lipid metabolism by controlling the stability and trafficking of SREBPs.

RNF170 is implicated in the regulation of calcium signaling via the activity of IP₃R. RNF170 is stably associated with the ER lipid raft proteins, erlin1 and erlin2, that bind to IP₃R upon cell activation. Through this interaction, RNF170 recognizes and ubiquitinates activated IP₃R to target it for ERAD [109]. RNF170 also has pathogenic importance, since a mutation in the RNF170 gene is associated with autosomal dominant sensory ataxia [110]. Forced expression of zebrafish RNF170 with the mutation causes failure in embryonic development in zebrafish, suggesting that RNF170 is involved in neuronal transmission and differentiation [110].

The calcium sensing receptor (CaR) is a member of the G protein-coupled receptor family, and has an important role in calcium homeostasis through its effect on regulating parathyroid hormone secretion and renal calcium reabsorption [111]. The Dorfin E3 enzyme interacts with CaR through their C-termini, and this promotes ubiquitination of CaR for ERAD targeting [112]. The precursor forms of CaR, which reside in the ER, are more sensitive to Dorfin-mediated ubiquitination than mature CaR, suggesting that Dorfin controls the protein levels of active CaR during protein synthesis [112]. Overexpressed Dorfin is accumulated in an aggresome-like structure near the centrosome, which is characteristic of a variety of neurodegenerative diseases [113]. Dorfin is present in Lewy body-like inclusions in neurons from familial and sporadic amyotrophic lateral sclerosis (ALS) and Parkinson disease [113]. Dorfin ubiquitinates mutant Cu/Zn-superoxide dismutase-1 (SOD1) and accelerates its degradation, which in turn reduces protein aggregation and cell toxicity [113,114,115,116]. The biological features and association with neurodegenerative diseases suggest that Dorfin protects the nerve system by mediating protein quality control.

Bifunctional apoptosis regulator (BAR)/RNF47, a member of the B cell lymphoma (Bcl)-2 family, was originally identified as a regulator of apoptosis (see Section 3.3). BAR is an ER-resident E3 enzyme that interacts with Ubc7, and is itself an ERAD substrate that undergoes self-ubiquitination [117]. However, BAR is unlikely to be a central mediator of ERAD since it does not affect the turnover of typical ERAD substrates, including T cell receptor (TCR)α and CD3δ [117]. Rather, BAR contributes to the cellular adaptation to ER stress by regulating inositol-requiring protein-1 (IRP1) signaling, one of the UPR signaling pathways.

 IRP1 is an ER-anchored protein that mediates alternative splicing of the transcription factor XBP1 under ER stress conditions, which induces the transcriptional upregulation of chaperones and ERAD components [118,119,120]. Activated IRP1 interacts with the signaling adaptor tumor necrosis factor (TNF) receptor-associated factor (TRAF)2 and then activates downstream kinases such as the c-Jun N-terminal kinases (JNKs) [121]. BAR ubiquitinates Bax inhibitor-1 (BI-1), an ER-resident negative regulator of IRE1, targeting the protein for proteasomal degradation [117]. Depletion of BAR expression increases the BI-1 protein level, thereby suppressing IRE1 activity [117]. BAR thus controls directly cell viability by regulating UPR as well as apoptosis.

RNF122 is also very unstable due to its self-ubiquitination, but is stabilized by the interaction with calcium-modulating cyclophilin ligand (CAML), an ER transmembrane protein implicated in calcium signaling [122]. This interaction is likely to be mediated through the RNF domain [122], suggesting that CAML may interfere with RNF122 E3 activity. RNF122 may regulate calcium signaling at the ER.

Rines/RNF180 is a tail-anchored ER E3 enzyme highly expressed in the brain [123]. Through the basic coiled-coil domain, Rines interacts with the transcription factor Zic2, which is associated with holoprosencephaly, a congenital malformation of the forebrain. Overexpressed Rines promotes ubiquitination and proteasomal degradation of Zic2 in cultured cells. However, the Zic2 protein levels are not significantly altered in the brain of Rines knockout mice. The precise in vivo function of Rines is therefore still uncertain.