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37-491: Linear ubiquitin chain assembly complex (LUBAC) is a multi-protein complex and the only known E3 ubiquitin ligase able to conjugate ubiquitin in a head-to-tail manner to generate linear (M1-linked) polyubiquitin chains. The complex is currently known to be composed of three proteins: heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1), HOIL-1-interacting protein (HOIP), and Shank-associated RH domain-interacting protein (SHARPIN) . HOIL-1 and HOIP are both E3 ubiquitin ligases, however,

74-491: A post-translational modification such as phosphorylation of a tyrosine , serine or threonine residue. In this case, the ubiquitin ligase exclusively recognizes the phosphorylated version of the substrate due to stabilization within the binding site . For example, FBW7 , the F-box substrate recognition unit of an SCF ubiquitin ligase, stabilizes a phosphorylated substrate by hydrogen binding its arginine residues to

111-412: A "raft" form, researchers suggest that caveolae formation also follows this mechanism since caveolae are also enriched in raft constituents. When caveolin proteins bind to the inner leaflet via cholesterol , the membrane starts to bend, leading to spontaneous curvature. This effect is due to the force distribution generated when the caveolin oligomer binds to the membrane. The force distribution then alters

148-424: A different extent by their appropriate ubiquitin ligase (N-recognin), influencing the half-life of the protein. For instance, positively charged ( Arg , Lys , His ) and bulky hydrophobic amino acids ( Phe , Trp , Tyr , Leu , Ile ) are recognized preferentially and thus considered destabilizing degrons since they allow faster degradation of their proteins. A degron can be converted into its active form by

185-738: A peptide bond with ubiquitin. Humans have an estimated 500-1000 E3 ligases, which impart substrate specificity onto the E1 and E2. The E3 ligases are classified into four families: HECT, RING-finger, U-box, and PHD-finger. The RING-finger E3 ligases are the largest family and contain ligases such as the anaphase-promoting complex (APC) and the SCF complex ( Skp1 - Cullin -F-box protein complex). SCF complexes consist of four proteins: Rbx1, Cul1, Skp1, which are invariant among SCF complexes, and an F-box protein, which varies. Around 70 human F-box proteins have been identified. F-box proteins contain an F-box, which binds

222-781: A result, a number of these proteins are involved in a variety of cancers, including famously MDM2, BRCA1 , and Von Hippel-Lindau tumor suppressor . For example, a mutation of MDM2 has been found in stomach cancer , renal cell carcinoma , and liver cancer (amongst others) to deregulate MDM2 concentrations by increasing its promoter’s affinity for the Sp1 transcription factor , causing increased transcription of MDM2 mRNA. Several proteomics-based experimental techniques are available for identifying E3 ubiquitin ligase-substrate pairs, such as proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, and tandem ubiquitin-binding entities (TUBEs). Endocytosis Endocytosis

259-454: A role in the assembly of caveolae. Besides caveolae assembly, researchers have also discovered that CAV1 proteins can also influence other endocytic pathways. When CAV1 binds to Cdc42 , CAV1 inactivates it and regulates Cdc42 activity during membrane trafficking events. The process of cell uptake depends on the tilt and chirality of constituent molecules to induce membrane budding. Since such chiral and tilted lipid molecules are likely to be in

296-454: A specific E3 partner and transfers the ubiquitin to the target protein . The E3, which may be a multi-protein complex , is, in general, responsible for targeting ubiquitination to specific substrate proteins. The ubiquitylation reaction proceeds in three or four steps depending on the mechanism of action of the E3 ubiquitin ligase. In the conserved first step, an E1 cysteine residue attacks

333-414: Is hydroxylated . Under hypoxia , on the other hand, HIF-a is not hydroxylated, evades ubiquitination and thus operates in the cell at higher concentrations which can initiate transcriptional response to hypoxia. Another example of small molecule control of protein degradation is phytohormone auxin in plants. Auxin binds to TIR1 (the substrate recognition domain of SCF ubiquitin ligase) increasing

370-420: Is a cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane , which then buds off inside the cell to form a vesicle containing the ingested materials. Endocytosis includes pinocytosis (cell drinking) and phagocytosis (cell eating). It is a form of active transport. The term was proposed by De Duve in 1963. Phagocytosis

407-666: Is also able to catalyze formation of oxyester bonds between the C-terminus of ubiquitin and serine/threonine of substrate protein in TLR signaling. SHARPIN exhibits a significant sequence similarity to HOIL-1 and is important for LUBAC stability. Spontaneous point mutation in the Sharpin gene in mice leads to development of chronic proliferative dermatitis (cpdm). Both HOIL-1 and SHARPIN bind to HOIP through their ubiquitin-like (UBL) domain. LUBAC consisting of either HOIP-HOIL-1 or HOIP-SHARPIN

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444-478: Is also reversible through disassembly under certain conditions such as increased plasma membrane tension. These certain conditions then depend on the type of tissues that are expressing the caveolar function. For example, not all tissues that have caveolar proteins have a caveolar structure i.e. the blood-brain barrier . Though there are many morphological features conserved among caveolae, the functions of each CAV protein are diverse. One common feature among caveolins

481-494: Is functional in vitro , however the greatest activity of the complex has been observed in the presence of all three components. LUBAC modulates signaling complexes activating the canonical NF-kB pathway in response to various stimuli (e.g., TNF, IL-1, CD40L) by adding M1-linked polyubiquitin chains to signaling proteins. Additionally, LUBAC has been shown to interact with PKC and NLRP3/ASC inflammasome. Antagonistic to LUBAC are deubiquitinases such as OTULIN or CYLD, of which OTULIN

518-511: Is the 190-kD protein called clathrin heavy chain (CHC), which is associated with a 25- kD protein called clathrin light chain (CLC), forming three-legged trimers called triskelions. Vesicles selectively concentrate and exclude certain proteins during formation and are not representative of the membrane as a whole. AP2 adaptors are multisubunit complexes that perform this function at the plasma membrane. The best-understood receptors that are found concentrated in coated vesicles of mammalian cells are

555-596: Is the only deubiquitinase that removes M1-linked ubiquitin linkages exclusively. LUBAC components have been most widely studied in the context of TNF signaling. E3 Ubiquitin Ligase The ubiquitin ligase is referred to as an E3, and operates in conjunction with an E1 ubiquitin-activating enzyme and an E2 ubiquitin-conjugating enzyme . There is one major E1 enzyme, shared by all ubiquitin ligases, that uses ATP to activate ubiquitin for conjugation and transfers it to an E2 enzyme. The E2 enzyme interacts with

592-448: Is their hydrophobic stretches of potential hairpin structures that are made of α-helices . The insertion of these hairpin-like α-helices forms a caveolae coat which leads to membrane curvature. In addition to insertion, caveolins are also capable of oligomerization which further plays a role in membrane curvature. Recent studies have also discovered that polymerase I, transcript release factor, and serum deprivation protein response also play

629-455: The ERAD pathway, on the other hand, are recognized by Fbs1 and Fbs2, mammalian F-box proteins of E3 ligases SCF and SCF . These recognition domains have small hydrophobic pockets allowing them to bind high- mannose containing glycans . In addition to linear degrons , the E3 ligase can in some cases also recognize structural motifs on the substrate. In this case, the 3D motif can allow

666-534: The LDL receptor (which removes LDL from circulating blood), the transferrin receptor (which brings ferric ions bound by transferrin into the cell) and certain hormone receptors (such as that for EGF ). At any one moment, about 25% of the plasma membrane of a fibroblast is made up of coated pits. As a coated pit has a life of about a minute before it buds into the cell, a fibroblast takes up its surface by this route about once every 50 minutes. Coated vesicles formed from

703-547: The cell , and from other (ubiquitination-inactive) forms of the same protein. This can be achieved by different mechanisms, most of which involve recognition of degrons : specific short amino acid sequences or chemical motifs on the substrate. Proteolytic cleavage can lead to exposure of residues at the N-terminus of a protein. According to the N-end rule , different N-terminal amino acids (or N-degrons) are recognized to

740-495: The ATP-activated C-terminal glycine on ubiquitin, resulting in a thioester Ub-S-E1 complex. The energy from ATP and diphosphate hydrolysis drives the formation of this reactive thioester, and subsequent steps are thermoneutral. Next, a transthiolation reaction occurs, in which an E2 cysteine residue attacks and replaces the E1. HECT domain type E3 ligases will have one more transthiolation reaction to transfer

777-560: The CLIC/GEEC pathway (regulated by Graf1 ), as well as MEND and macropinocytosis . Clathrin-mediated endocytosis is the only pathway dependent on both clathrin and dynamin. The endocytic pathway of mammalian cells consists of distinct membrane compartments, which internalize molecules from the plasma membrane and recycle them back to the surface (as in early endosomes and recycling endosomes), or sort them to degradation (as in late endosomes and lysosomes). The principal components of

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814-580: The E3 ligase MDM2 ubiquitylates p53 either for degradation (K48 polyubiquitin chain), or for nuclear export (monoubiquitylation). These events occur in a concentration dependent fashion, suggesting that modulating E3 ligase concentration is a cellular regulatory strategy for controlling protein homeostasis and localization. Ubiquitin ligases are the final, and potentially the most important determinant of substrate specificity in ubiquitination of proteins . The ligases must simultaneously distinguish their protein substrate from thousands of other proteins in

851-745: The N-terminal methionine are used in chains in vivo. Monoubiquitination has been linked to membrane protein endocytosis pathways. For example, phosphorylation of the Tyrosine at position 1045 in the Epidermal Growth Factor Receptor (EGFR) can recruit the RING type E3 ligase c-Cbl, via an SH2 domain . C-Cbl monoubiquitylates EGFR, signaling for its internalization and trafficking to the lysosome. Monoubiquitination also can regulate cytosolic protein localization. For example,

888-523: The affinity of TIR1 for its substrates (transcriptional repressors : Aux/IAA), and promoting their degradation. In addition to recognizing amino acids, ubiquitin ligases can also detect unusual features on substrates that serve as signals for their destruction. For example, San1 ( Sir antagonist 1 ), a nuclear protein quality control in yeast , has a disordered substrate binding domain , which allows it to bind to hydrophobic domains of misfolded proteins . Misfolded or excess unassembled glycoproteins of

925-508: The caveolar formation process. More specifically, CAV1 and CAV2 are responsible for caveolae formation in non-muscle cells while CAV3 functions in muscle cells. The process starts with CAV1 being synthesized in the ER where it forms detergent-resistant oligomers . Then, these oligomers travel through the Golgi complex before arriving at the cell surface to aid in caveolar formation. Caveolae formation

962-528: The cytoplasm of the cell. In so doing, it brings into the cell not only a small area of the surface of the cell but also a small volume of fluid from outside the cell. Coats function to deform the donor membrane to produce a vesicle, and they also function in the selection of the vesicle cargo. Coat complexes that have been well characterized so far include coat protein-I (COP-I), COP-II, and clathrin. Clathrin coats are involved in two crucial transport steps: (i) receptor-mediated and fluid-phase endocytosis from

999-444: The electron microscope by Thomas F Roth and Keith R. Porter . The importance of them for the clearance of LDL from blood was discovered by Richard G. Anderson, Michael S. Brown and Joseph L. Goldstein in 1977. Coated vesicles were first purified by Barbara Pearse , who discovered the clathrin coat molecule in 1976. Caveolin proteins like caveolin-1 ( CAV1 ), caveolin-2 ( CAV2 ), and caveolin-3 ( CAV3 ), play significant roles in

1036-416: The endocytic pathway are: It was recently found that an eisosome serves as a portal of endocytosis in yeast. The major route for endocytosis in most cells, and the best-understood, is that mediated by the molecule clathrin . This large protein assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell. This pit then buds into the cell to form a coated vesicle in

1073-427: The phosphate, as shown in the figure to the right. In absence of the phosphate , residues of FBW7 repel the substrate. The presence of oxygen or other small molecules can influence degron recognition. The von Hippel-Lindau (VHL) protein (substrate recognition part of a specific E3 ligase), for instance, recognizes the hypoxia-inducible factor alpha (HIF-α) only under normal oxygen conditions, when its proline

1110-486: The plasma membrane have a diameter of about 100 nm and a lifetime measured in a few seconds. Once the coat has been shed, the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway. The actual budding-in process, whereby a pit is converted to a vesicle, is carried out by clathrin; Assisted by a set of cytoplasmic proteins, which includes dynamin and adaptors such as adaptin . Coated pits and vesicles were first seen in thin sections of tissue in

1147-468: The plasma membrane to early endosome and (ii) transport from the TGN to endosomes. In endocytosis, the clathrin coat is assembled on the cytoplasmic face of the plasma membrane, forming pits that invaginate to pinch off (scission) and become free CCVs. In cultured cells, the assembly of a CCV takes ~ 1min, and several hundred to a thousand or more can form every minute. The main scaffold component of clathrin coat

LUBAC - Misplaced Pages Continue

1184-476: The rest of the SCF complex, and a substrate binding domain, which gives the E3 its substrate specificity. Ubiquitin signaling relies on the diversity of ubiquitin tags for the specificity of its message. A protein can be tagged with a single ubiquitin molecule (monoubiquitylation), or variety of different chains of ubiquitin molecules (polyubiquitylation). E3 ubiquitin ligases catalyze polyubiquitination events much in

1221-470: The same way as the single ubiquitylation mechanism, using instead a lysine residue from a ubiquitin molecule currently attached to substrate protein to attack the C-terminus of a new ubiquitin molecule. For example, a common 4-ubiquitin tag, linked through the lysine at position 48 (K48) recruits the tagged protein to the proteasome, and subsequent degradation. However, all seven of the ubiquitin lysine residues (K6, K11, K27, K29, K33, K48, and K63), as well as

1258-503: The specific linear ubiquitin-ligating activity is enacted by HOIP. Mice deficient in HOIP are embryonically lethal. Two cases of mutated HOIP have been detected in humans. These patients presented with autoinflammation and immunodeficiency. HOIL-1 is required for LUBAC assembly and stability as demonstrated by embryonic lethality in HOIL-1 deficient mice. Recently, it has been noted, that HOIL-1

1295-513: The substrate to directly relate its biochemical function to ubiquitination . This relation can be demonstrated with TRF1 protein (regulator of human telomere length), which is recognized by its corresponding E3 ligase ( FBXO4 ) via an intermolecular beta sheet interaction. TRF1 cannot be ubiquinated while telomere bound, likely because the same TRF1 domain that binds to its E3 ligase also binds to telomeres. E3 ubiquitin ligases regulate homeostasis, cell cycle, and DNA repair pathways, and as

1332-450: The ubiquitin molecule onto the E3, whereas the much more common RING finger domain type ligases transfer ubiquitin directly from E2 to the substrate. The final step in the first ubiquitylation event is an attack from the target protein lysine amine group, which will remove the cysteine, and form a stable isopeptide bond. One notable exception to this is p21 protein, which appears to be ubiquitylated using its N-terminal amine, thus forming

1369-668: Was discovered by Élie Metchnikoff in 1882. Endocytosis pathways can be subdivided into four categories: namely, receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae , pinocytosis , and phagocytosis . More recent experiments have suggested that these morphological descriptions of endocytic events may be inadequate, and a more appropriate method of classification may be based upon whether particular pathways are dependent on clathrin and dynamin . Dynamin-dependent clathrin-independent pathways include FEME , UFE , ADBE , EGFR-NCE and IL2Rβ uptake. Dynamin-independent clathrin-independent pathways include

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