Ubiquitylation



Ubiquitin is a highly-conserved regulatory protein that is ubiquitously expressed 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. 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. It is observed that 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 signaling 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, in general, 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 monoubiquitinated, 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.

Disease association
Protein modification by ubiquitin also has unconventional (non-degradative) functions such as the regulation of DNA repair and endocytosis. These non-traditional functions are dictated by the number of ubiquitin units attached to proteins (mono- versus poly-ubiquitination) and also by the type of ubiquitin chain linkage that is present. As such, the Ubiquitin Proteasome Pathway has been linked to diseases involving all types of cellular activity including:
 * Antigen processing
 * Apoptosis
 * Biogenesis of organelles
 * Cell cycle and division
 * DNA transcription and repair
 * Differentiation and development
 * Immune response and inflammation
 * Neural and muscular degeneration
 * Morphogenesis of neural networks
 * Modulation of cell surface receptors, ion channels and the secretory pathway
 * Response to stress and extracellular modulators
 * Ribosome biogenesis
 * Viral infection

Genetic disorders
Some genetic disorders associated with ubiquitin are:
 * 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.
 * Eight of the thirteen identified genes whose disruption causes Fanconi anemia encode proteins that form a large ubiquitin ligase (E3) complex.
 * mutations of the Cullin7 E3 ubiquitin ligase gene are linked to 3-M syndrome, an autosomal-recessive growth retardation disorder

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

Ubiquitin hydrolase
Human ubiquitin hydrolase has the most complicated knot structure yet discovered for a protein, with five knot crossings. It is speculated that a knot structure increases a protein's resistance to degradation in the proteasome.

Ubiquitin derivatives
Chemical modification of ubiquitin protein results in several useful derivatives. Modifying the C-terminal glycine carboxyl of ubiquitin to an aldehyde thus results in a highly potent inhibitor of de-ubiquitinating enzymes(also known as DUBs for "DeUBiquitinating enzymes"). Alternatively, a fluorogenic substrate for DUBs is generated by synthetically conjugating AMC to the C-terminus of ubiquitin. In addition, reductive methylation of the amine groups prevents the formation of poly-ubiquitin chains via lysine linkages. Ubiquitin can also be coupled to agarose via its primary amines, leaving the C-terminus free and available to purify ubiquitin binding proteins.


 * Ubiquitin Aldehyde - A potent and specific inhibitor of all ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs) and deubiquinating enzymes (DUBs). This protein blocks the hydrolysis of poly-ubiquitin chains on substrate proteins in vitro and thus enhances poly-ubiquitin chain accumulation.
 * Methylated Ubiquitin - Reductive methylation of lysine residues renders ubiquitin unable to form poly-ubiquitin chains via lysine linkages with other Ub molecules. Methylated Ub can form an E1-catalyzed active thioester at the C-terminus allowing the molecule to be transferred to the lysines of substrate proteins (which can be mono-ubiquitinated). Ideal for the reduction in poly-ubiquitin chain length and rates of ubiquitin conjugation.
 * Ubiquitin AMC - Fluorogenic substrate for ubiquitin hydrolases based on the C-terminus derivatization of ubiquitin with 7-amido-4-methylcoumarin (AMC). Ubiquitin-AMC is an exquisitely sensitive substrate for UCH-L3 (Km = 0.039 μM) and for the Isopeptidase-T (Km = 0.17 - 1.4 μM). Ub-AMC is useful for studying ubiquitin hydrolases when detection sensitivity or continuous monitoring of activity is essential.
 * Ubiquitin Vinyl Sulfone - A potent, irreversible and specific inhibitor of all ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs) and deubiquitinating enzymes (DUBs). Useful for inhibiting the hydrolysis of poly-ubiquitin chains on substrate proteins in vitro and thus enhances polyubiquitin chain accumulation.

Ubiquitin-like Modifiers
Although ubiquitin is the most well understood post-translation modifier, there is a growing family of ubiquitin-like proteins (UBLs) that modify cellular targets in a pathway that is parallel to but distinct from that of ubiquitin. These alternative modifiers include: SUMO (Sentrin, Smt3 in yeast),  NEDD8 (Rub1 in yeast),  ISG15 (UCRP), APG8, APG12, FAT10, Ufm1 URM1 & Hub1.

These related molecules have novel functions and influence diverse biological processes. There is also cross-regulation between the various conjugation pathways since some proteins can become modified by more than one UBL, and sometimes even at the same lysine residue. For instance, SUMO modification often acts antagonistically to that of ubiquitination and serves to stabilize protein substrates. Proteins conjugated to UBLs are typically not targeted for degradation by the proteasome, but rather function in diverse regulatory activities. Attachment of UBLs might alter substrate conformation, affect the affinity for ligands or other interacting molecules, alter substrate localization and influence protein stability.

UBLs are structurally similar to ubiquitin and are processed, activated, conjugated and released from conjugates by enzymatic steps that are similar to the corresponding mechanisms for ubiquitin. UBLs are also translated with C-terminal extensions that are processed to expose the invariant C-terminal LRGG. These modifiers have their own specific E1 (activating), E2 (conjugating) and E3 (ligating) enzymes that conjugate the UBLs to intracellular targets. These conjugates can be reversed by UBL-specific isopeptidases that have similar mechanisms to that of the deubiquitinating enzymes.