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The discovery of the Ubiquitin-proteasome system (UPS), a process critical to many biological mechanisms including antigen processing, immune response and DNA repair1, has specifically revolutionised the concept of intracellular protein degradation. The UPS is the primary non-lysosomal method responsible for the turnover of regulatory proteins, the change in levels of which regulate specific cellular processes. 1,2. The regulatory proteins may comprise transcription factors, enzymes and signal molecules which in turn control cell proliferation and signalling via influencing cell cycle progression, signal transduction, protein transport plus degradation and apoptosis. Most of this post-translational modification through ubiquitin addition is necessary for cell viability. In this highly conserved mechanism ubiquitin (UB), a small 8.5KDa and 76 amino acids long protein, dictates the fate of the protein via its post-translational addition to the protein. Due to the presence of 7Lys residues in UB, it can both monoubiquitinate or polyubiquitinate, hence directing the different fates of proteins and illustrating the different roles of the Ub.
Studies with EGFRs(Epidermal Growth Factor Receptor) and the chimera protein**shows monoubiquitination to serve as both an internalization and a sorting signal3,6.
(REFs::**Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.)â€¦ALSO Urbeâ€¦.Also Acconia..
In effect, the UPS maintains cell function via damaged or misfolded protein trafficking and destruction through a two-stage mechanism. Initially, target proteins conjugate with a conserved ubiquitin protein monomer (ubiquitination) initiating endocytic trafficking. Following this, the protein-ubiquitin complex (ubiquitinated protein) is recognised and degraded by the 26S proteasome, a 2.5 MD proteolytic macromolecular complex, localised to the cytoplasm and nucleus of eukaryotic cells1,2..
Ubiquitination is also used as an initiation signal for a range of proteins with diverse functionality, mostly cellular in nature. Specifically membrane proteins employ a pathway not mainly leading to proteasomal degradation6. This is through the use of both mono and poly-ubiquitination, which act as tags directing internalisation and endocytic sorting of membrane protein following which they are either recycled or degraded. Use of monoubiquitination tags also regulates processes such as sorting of newly synthesised proteins to the trans-Golgi network, as well as histone modification and viral budding.6
The sequential activities of the ESCRT complexes are required for the recognition and sorting ofÂ ubiquitin-modified cargo proteins into the internal vesicles ofÂ multivesicular bodiesÂ (MVBs). Sorting of membrane cargo proteins into the internal vesicles of a MVB requires one or more ubiquitin tags, which are added to theÂ cytosolicÂ domains of membrane proteins. These ubiquitin tags are recognized by the ESCRT complexes, which bind sequentially and work in concert to pass along the cargo proteins from one complex to the next, sorting the ubiquitylated cargo proteins into subregions of theÂ endosomalÂ membrane for inclusion in an intralumenal MVB vesicle.
InvaginationÂ of the membrane into an internal vesicle also depends on PI(3)P, resulting from theÂ phosphorylationÂ ofÂ phosphatidylinositolÂ by a lipidÂ kinase, and functioning as another docking site for the ESCRT complexes. The complexes require PI(3)P and ubiquitylated cargo proteins to attach to the endosomal membrane. When anotherÂ phosphateÂ group is added to PI(3)P, producing PI(3,5)P2, the ESCRT-III complex is able to form a large multimeric aggregation on the membrane, enabling the invagination and pinching-off processes required to form the internal vesicles.
The ubiquitination pathway comprises of a multi-step sequential and ATP-dependent transfer of ubiquitin amongst three sets of enzymes; a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme and a Ubiquitin-protein ligase (E3) (Figure 1).
Figure 1 The Ubiquitination pathway representing the multi-step action of enzymes E1,E2 and E3 for protein degradation.3
The initial Ub-activating step is ATP-dependent, forming a thiolester bond between a sulphydryl group in the E1 enzyme and the glycine residue (G76) in the UB molecule. Following this, the activated Ubiquitin is transferred to the UB-conjugating enzymes (E2), subsequent to which the activated Ub is conjugated to the target protein with the help of Ubiqutin-ligases (E3).
The E1 is at the summit of the ubiquitination cascade, activating the UB via adenylation of the C-terminal glycine carboxyl group of UB. Hence the activated UB is able to react ,forming a thiol-ester link between the glycine residue and the cysteine in the E1active site. This high energy link formation is very energy demanding and depends on the dephosphorylation of ATP into ADP + PPi. Following the formation of the high energy intermediate, the bound E1-activated UB complex, interacts with the E2 facilitating activated UB transfer. 1
Unlike E1s a variety of E2 enzymes are present, in all of which a highly conserved core-catalytic domain, UBC domain, is integrated. It is this domain that defines the function of the E2 in the cascade. The E2s continue the ubiquitination cascade via the trans-thiolation process, where the activated-UB molecule is transferred from the E1 active site to a cysteine residue in the active site of the E2. The trans-thiolation process being ATP-independent, transfers to one or many E2s. Ultimately, the UB is transferred either directly, by the E2 to the lysine Îµ-amino group3 of the target protein substrate, or via the E3 mediators. The specificity of this amplification cascade is dictated principally by the E3s, as one E1 interacts with several E2, which consecutively interacts with numerous E3s. Therefore it is the foremost factor in substrate protein recognition, multi-ubiquitin chain topology and consecutively the fate of the protein. The E3s complete the ubiqutination process, via supporting the transfer of UB to the substrate protein.
The numerous E3 enzymes can be categorised into two distinct groups, based on their binding features: the HECT and the RING groups.
The RING (Really Interesting New Gene) family of UB-Ligases, are the largest of the two and function as scaffolds for direct transfer of the activated-UB to the E3-bound targets.3 The thiol-ester E2-UB complex and the E3-substrate moiety are brought together in close proximity by the E3 enzyme. They exist as single chains or as a part of multimeric complexes with a key feature, the RING finger domain. This domain is distinguished as a crossbrace motif consisting of eight highly conserved cysteine or histidine residues binding two zinc ions.3,4
The RING domain illustrates the cross-brace motif. Created via the distinct sequence "CX2CX9-39CX1-3HX2-3(C/H)
X2CX4-48CX2C", it is stabilised through the binding of 2 Zinc2+ ions amongst the histidine and cysteine residues. Image A adapted from the
Image B crystal structure of RING finger domain of c-CBL, an E3 that binds to the E2 ubc7. created using Uniprot.REFERENCE
The HECT (homology to E6-AP carboxy terminus) family of E3s however, contain a highly conserved cysteine residue, present in the HECT E2-binding domainat the C-terminus (C-lobe). An evolutionary conservation in E3s, of the HECT domain across species,from yeast to humans, and various N-terminal motifs across most family members can be seen. The catalytic active site of this domain containing the cys residue interacts with the cognate E2 enzymes forming a thiol-ester link with the C-terminus of the complexed UB. However, the N-terminal (N-lobe) of the protein intercedes protein or phospholipid interactions and binds the E2 enzyme. Hence an E3-Ub-E2 thio-ester complex is formed prior to catalysing substrate ubiquitination.
The HECT family can further be divided into three types derived from the N-terminal design: Nedd4, the HERC and other HECTs. The Nedd 4 family distinctly contains a C2 domain6 that interact with molecules such as phospholipids, reinforcing adapter-mediated recruitment to the specific membranes, henceresponsible in intracellular targeting and protein sorting, to bodies such as MVBs (multivesicular bodies) and endosomes.14
2-4 WW domains, accountable for protein-protein interaction via two conserved Trp residues, and a HECT domain can also be observed. Although an unidentified function the HERC subtype illustrates the presence of the HECT-domain alongside many regulating chromosomal condensation (RCC1) domains. They localise to the golgi and endosomes, whilst also interacting with the ARF, Rab, UBL( ubiquitin like proteins) proteins and clathrin. Hence, it is presumed to partake in membrane trafficking, and regulate vesicular transport 3,4
In recent years a complex known as ESCRT (endosomal sorting complex required for transport) has been discovered. It has been found to be involved in facilitating trafficking of ubiquitylated proteins from endosomes to lysosomes through the formation of intra lumenal vesicles within endosomal vesicles (10). There are 4 ESCRT complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III respectively. Each of these are recruited to endosomes via interactions with the cell membrane, ubiquitin, the coat protein clathrin and between themselves (10).
ESCRT-0 is involved initially in recognition of the ubiquitylated cargo present at the endosomal membrane. This is achieved through binding phosphatidylinositol 3 phosphate, (PI(3)P) (11) and ubiquitin interaction motifs present in the Hrs (Hepatocyte growth factor receptor tyrosine kinase substrate). These interactions link the ubiquitylated receptors to clathrin lattices (12) and STAM (signal transducing adaptor molecule) components (7) (10). Having recognised the ubiquitylated cargo ESCRT-0 interacts with ESCRT-I in order to recruit it to the membrane, via a Pro-(Ser/Thr)-X-Pro motif and a domain known as UEV (ubiquitin E2 variant) that is present in the Vps23 component of ESCRT-I. This also interacts with the ubiquitylated cargo by the same UEV domain (10) (13) (1). ESCRT-I subsequently recruits ESCRT-II and ESCRT-II recruits ESCRT-III (11). ESCRT-III coordinates the final stages of ILV (intra-lumenal vesicle) formation (11). It consists of 6 200-250 residue proteins in yeast which increases to 11 proteins in humans which are either equivalents to those present in yeast or members of a group of proteins known as CHMPS or chromatin modifying proteins (10) (13) (11). It has been found that proteins in the ESCRT-III complex are able to form large polymers (14) (15) (referenced in (11)) (13). These polymers are then able to incarcerate the ubiquitylated cargo and remain associated with ESCRT-I/II and allow DUBs to remove ubiquitin from the targeted cargo while preventing their escape (11). The formation of these polymers also stabilises or creates negative membrane curvature required for ESCRT mediated events prior to ILV budding from the membrane (13). Dissociation of the ESCRT complexes is thought to be mediated by the ATPase known as Vps4 and upon completion of this process ILVs are able to form (10) (13) (11).
The remnant members of the HECT family enclose proteins consisting of a variety of domains, such as a ubiquitin-associated domain (UBA), and also ones containing ankyrin repeats.3,4 Hence, the structure of the E3 portrays the function encompassed, which they fulfil either alone or alongside accessory proteins.
The variety of HECT-Ligases with the varied domains and motifs present on the N-terminal, propagate several protein-protein or protein-lipid interactions. Processes that modulate HECT-E3 function or aid substrate binding are mediated by a number of proteins that interact with the ligases. Others such as intramolecular interactions inhibit the autoubiquitination of the E3s, hence illustrating the need for interaction with accessory proteins which when activated initiate phosphorylation. In the case of the ligase E6AP, adaptor proteins like Smud interact and support its function, regulation of p53.3
In this study we characterise a novel interacting protein, lifeguard (LFG), of the HECT family of E3 ligases. The protein being evolutionarily conserved, is expressed as different homologues in several species. Initially the rat homologue of LFG, Neural Membrane Protein 35(NMP35), a 35Kda protein was discovered. This was later characterised in humans as the lifeguard.
The LFG, 316 amino acids in length, is encoded by the LFG gene which has been mapped to chromosome 12, sublocation q13.5 Studies have shown LFG to contain seven trans-membrane domains and predicted localisation to the endoplasmic reticulum and the plasma membrane7, 810.
It is known to function as an inhibitor of the Fas-mediated apoptosis pathway due to its resemblance in structure with the anti-apoptotic protein Bax Inhibitor-1 and as it contains the BAX inhibitor motif.7,810 Hence it has also been given the designation of FAIM 2 (Fas apoptotic inhibitory molecule 2). The Fas ligand, a type 2 transmembrane protein of the tumour necrosis family, plays a vital role in the immune system via the initiation of apoptosis upon receptor binding. The binding of a complementary FAS-ligand to the receptor activates caspase 8 which leads to a cascade of caspase (cysteine-dependent aspartate-directed proteases) activation which in turn direct apoptosis as well as necrosis and inflammation5.
LFG is known to exhibit anti-apoptotic quality by decreasing or stopping caspase activation and via interactions with FAS in the lipid-raft microdomains, hence preventing the initiation of the cascade in turn preventing FasL activated apoptosis. 5,9
Similar to the rat homologue neural membrane protein 35, lifeguard is expressed most highly in the neural tissues. Exclusively in the case of LFG, most of the localisation is seen in the hippocampus and the cerebellum9 of the human brain. Therefore, LFG is presumed to play a role in pathologies concerned with the cell death principle via FAS ligand mediation. Lifeguard is connected to cancers5,7, 9, 10 such as breast cancer, leukaemia, lipomas as well as neurodegenerative disorders like Alzheimer's disease and Parkinsons.9 Therefore characterisation of this novel interacting protein of the HECT family of E3s would benefit both research and medicine, by entailing use of the lifeguard protein in practical therapeutic treatmentsDrug production to protect from FAS and keep viability as well as save biological activity of other death receptors.LOOK UP there is an article about this
Lots still to do! You have said nothing about the role of UB in trafficking, endocytosis and MVB formation at a molecular level. What about ESCRT complexes, receptor ubiquitylation etc etc?? You could write plenty about this. Given what you have said about the role of LFG, you certainly need to describe the FAS signalling pathway at a molecular level and should be aware of the overlap and similarities with, say, canonical (i.e. TNF-ï¡ dependent) NFï«B signalling.
CHECK AND DELETE any repetitions
LYS63 and 48 RUBICON