Ubiquitination



Ubiquitin is a highly conserved small regulatory protein that is ubiquitous in eukaryotes. Ubiquitination (or Ubiquitylation) refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. The most prominent function of Ubiquitin is labeling proteins for proteasomal degradation (see: Proteasome). Besides this function, ubiquitination also controls the stability, function, and intracellular localization of a wide variety of proteins.

Identification
Ubiquitin (originally, Ubiquitous Immunopoietic Polypeptide) was first identified in 1975 as an 8.5 kDa protein of unknown function expressed universally in living cells. The basic functions of ubiquitin and the components of the ubiquitination pathway were elucidated in the early 1980s in groundbreaking work performed by Aaron Ciechanover, Avram Hershko and Irwin Rose for which the Nobel Prize in Chemistry was awarded in 2004.

The ubiquitylation system was initially characterised as an ATP-dependent proteolytic system present in cellular extracts. A heat-stable polypeptide present in these extracts, ATP-dependent proteolysis factor 1 (APF-1), was found to become covalently attached to the model protein substrate lysozyme in an ATP and Mg2+-dependent process. Multiple APF-1 molecules were linked to a single substrate molecule by an isopeptide linkage and conjugates were found to be rapidly degraded with the release of free APF-1. Soon after APF-1-protein conjugation was characterised, APF-1 was identified as ubiquitin. The carboxyl group of the C-terminal glycine residue of ubiquitin (Gly76) was identified as the moiety conjugated to substrate lysine residues.

The protein
Ubiquitin is a small protein that occurs in all eukaryotic cells. Its main known function is to mark other proteins for destruction, known as proteolysis. At least four ubiquitin molecules attach to a lysine residue on the condemned protein, in a process called polyubiquitination, and the protein then moves to a proteasome, a barrel-shaped structure where the proteolysis occurs. Apparently, at least four ubiquitins are required on a substrate protein in order for the proteasome to bind and therefore degrade the substrate, though there are examples of non-ubiquitinated proteins being targeted to the proteasome. Ubiquitin can also mark transmembrane proteins (for example, receptors) for removal from membranes and fulfill several signalling roles within the cell. Monoubiquitination has been associated with targeting of membrane proteins to the lysosome, for example.

Ubiquitin consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Key features include its C-terminal tail and the Lys residues. It is highly conserved among eukaryotic species: Human and yeast ubiquitin share 96 % sequence identity. The human ubiquitin sequence is: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG



Ubiquitination (Ubiquitylation)


The process of marking a protein with ubiquitin (ubiquitylation or ubiquitination) consists of a series of steps:
 * 1) Activation of ubiquitin - Ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme in a process requiring ATP as an energy source. The initial step involves production of a ubiquitin-adenylate intermediate. The second step transfers ubiquitin to the E1 active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group.
 * 2) Transfer of ubiquitin from E1 to the active site cysteine of a ubiquitin-conjugating enzyme E2 via a trans(thio)esterification reaction.  Mammalian genomes contain 20-30 UBCs.
 * 3) The final step of the ubiquitylation cascade generally requires the activity of one of the hundreds of E3 ubiquitin-protein ligases (often termed simply ubiquitin ligase). E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and substrate. E3 enzymes possess one of two domains:
 * 4) *The HECT (Homologous to the E6-AP Carboxyl Terminus) domain
 * 5) *The RING (Really Interesting New Gene) domain (or the closely related U-box domain)
 * Transfer can occur in two ways:
 * Directly from E2, catalysed by RING domain E3s.
 * Via an E3 enzyme, catalysed by HECT domain E3s. In this case, a covalent E3-ubiquitin intermediate is formed before transfer of ubiquitin to the substrate protein.

In many cases, ubiquitin molecules are further added on to previously-conjugated ubiquitin molecules to form a polyubiquitin chain. If the chain is longer than 3 ubiquitin molecules, the tagged protein is rapidly degraded by the 26S-proteasome into small peptides (usually 3-24 amino acid residues in length). Ubiquitin moieties are cleaved off the protein by deubiquitinating enzymes and are recycled for further use.

Cell-surface transmembrane molecules that are tagged with ubiquitin are often mono-ubiquitinated, and this modification alters the subcellular localization of the protein, often targeting the protein for destruction in lysosomes.

The Anaphase-promoting complex (APC) and the SCF complex (for Skp1-Cullin-F-box protein complex) are two examples of multi-subunit E3s involved in recognition and ubiquitination of specific target proteins for degradation by the proteasome.

Genetic disorders

 * The gene whose disruption causes Angelman syndrome, UBE3A, encodes a ubiquitin ligase (E3) enzyme termed E6-AP.
 * The gene disrupted in Von Hippel-Lindau syndrome encodes a ubiquitin E3 ligase termed the VHL tumor suppressor or VHL gene.
 * The gene disrupted in Liddle's Syndrome results in disregulation of an epithelial Na+ channel (ENaC) and causes hypertension.

Immunohistochemistry
Antibodies to ubiquitin are used in histology to identify abnormal accumulations of protein inside cells that are markers of disease. These accumulations are called inclusion bodies. Examples of such abnormal inclusions in cells are
 * Neurofibrillary tangles in Alzheimer's disease
 * Lewy body in Parkinson's disease
 * Pick bodies in Pick's disease
 * Inclusions in motor neuron disease
 * Mallory's Hyalin in alcoholic liver disease
 * Rosenthal fibres in astrocytes