Introduction

Ubiquitination is the biochemical process in which proteins are marked by ubiquitin, a 76 amino acid protein. It occurs intracellularly in eukaryotes and regulates a wide variety of biological processes. The ubiquitination of a protein most commonly results in the degradation of the protein via the ubiquitin-proteasome pathway but can also serve to alter protein-protein interaction. Ubiquitination is an extremely complex, temporally controlled, and highly regulated process that can play major roles in various pathways during cell life and death as well as in health and disease. This review will explore the fundamentals, function, pathophysiology, and clinical significance of ubiquitination.[1]

Fundamentals

Ubiquitination is a form of post-translational modification in which the ubiquitin-protein is attached to a substrate protein. It is a three-step process involving three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The result of this cascade of reactions is the linkage of one molecule of ubiquitin to a protein, known as mono-ubiquitination. Additional molecules can be attached to any of the seven lysine residues or the N-terminus of the ubiquitin molecule to form ubiquitin chains, resulting in polyubiquitination. Polyubiquitination subsequently leads to the initiation of proteolysis of the substrate by serving as the recognition signal for the 26S proteasome.[2][3]

Issues of Concern

The ubiquitin-proteasome pathway is a well-known pathway taught in many college biology and medical school classes. Its significance is its regulation of protein degradation in eukaryotes. Recent studies of ubiquitination have also hinted at its participation in various other cellular processes, including protein-protein regulation and DNA repair. Such functions of ubiquitination were unknown before due to the restrictions of the methods used to study ubiquitination. Scientists have tried approaching this by preventing ubiquitination of a protein involved in a certain pathway to determine the role of ubiquitination in other cellular processes. However, multiple attempts at doing so have failed due to:

• Difficulties in preventing ubiquitination of a protein due to the presence of multiple lysine residues and the inability to predict which residue will be ubiquitinated
• Existence of multiple ubiquitin E3 ligases that ubiquitinate a protein, which attempts to inhibit E3 ligases at preventing ubiquitination a challenge
• Inhibiting an E3 ligase may prevent the ubiquitination of other proteins that are not of point of interest, resulting in a change in phenotype.

Thus, specificity and isolation of a particular protein of interest remain the major issues in the study of ubiquitination. It is difficult to study the effects of ubiquitination in DNA repair when alterations of ubiquitination may lead to changes in other pathways.[3][4]

Cellular

Ubiquitination is a tightly regulated, highly specific, ATP-dependent biological process carried out by a complex cascade of chemical reactions. The ubiquitin pathway is involved in regulating many basic cellular processes, including cell division and differentiation, response to environmental stress, cell differentiation, immune response, DNA repair, and apoptosis. Ubiquitination is an essential player in maintaining the balance of proteins in eukaryotic organisms, serving to rapidly remove misfolded proteins.[5][6]

Molecular

Ubiquitin is a regulatory protein found in nearly all eukaryotic organisms. It is highly conserved and virtually identical across all forms of life, whether it be human, yeast, or plant. Ubiquitin consists of 76 amino acids with a molecular weight of 8.5 kDa. The main feature of ubiquitin is its seven Lys residues and N-terminus, all of which are potential sites for ubiquitination. Lys48-linked chains (also referred to as K48-polyubiquitin chains) are the most common, with their primary role being the targeter of proteins for degradation.[1][3][7]

Function

Ubiquitination plays a crucial role in everyday cellular functions. This pathway targets proteins to the proteasome, which degrades and recycles the substrates. As noted previously, it has a wide range of functions that include cell signaling, apoptosis, protein processing, immune response, and DNA repair.

The ubiquitination pathway is key in many cellular signaling pathways. Polyubiquitin chains regulate the signal activation of IkB-alpha in the inflammatory signaling pathway. Activation of this pathway via phosphorylation of IkB-alpha leads to subsequent ubiquitination and degradation. Degradation of IkB-alpha results in the release of the transcription factor NFkB to the nucleus, which causes an inflammatory response. 

Protein processing requires ubiquitination. Polyubiquitination is also recognizable as processing signals rather than degradation signals. Studies have shown ubiquitination to possibly play a role in the recruitment of BRCA1 to damaged sites. BRCA1 is crucial for cell survival because of its role in the repair of double-strand DNA break repairs. Mutation of the BRCA1 tumor suppressor gene is involved in the pathogenesis of breast and ovarian cancers. 

Ubiquitination plays an important role in regulating apoptosis by regulating the levels of pro- and anti-apoptotic proteins. The anti-apoptotic protein, Bcl-2, can be polyubiquitinated and subsequently degraded through the ubiquitin-proteasome pathway. Degradation of the Bcl-2 will result in the progression of apoptosis. The degradation of pro-apoptotic proteins, such as Bax and Bak, will prevent apoptosis. 

Membrane trafficking also requires mono-ubiquitination to recruit proteins for vesicular trafficking and endo or exocytosis. In the cell, ubiquitination acts as a signal for endocytosis and transporting of these vesicles to lysosomes. Disruptions in this pathway have implications in many oncogenic formations. Ubiquitination for membrane trafficking has also shown associations with various viruses such as Ebola and HIV that make their way to the cell surface after replicating within the cell.[8][9][10][11]

Mechanism

Ubiquitination ultimately breaks down into three essential steps that are catalyzed by the enzymes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The three steps are:

1. Activation: Ubiquitin activating enzyme E1 uses an ATP dependent process to establish a thioester bond between the C-terminal carboxyl group of ubiquitin and the cysteine group of the ubiquitin-activating enzyme.
2. Conjugation: The E2 ubiquitin-conjugating enzyme then binds to both the activated ubiquitin and E1 enzyme complex. E2 catalyzes the transfer of ubiquitin from the E1 site to the active site on E2 by way of a transesterification reaction.
3. Ligation: In the final step of the ubiquitination pathway, E3 ubiquitination ligase creates an isopeptide bond with the lysine of the target protein and the C-terminal glycine of ubiquitin. 

The result is the formation of a ubiquitin-substrate complex.[1]

Testing

Identifying the E3 ligase substrate is critical to understand its implications in malignancies and human diseases. Deregulation of E3-substrate interactions are often indicators of many of these pathologies. To identify the substrates of the E3 Ligase, a system called the Global Protein Stability (GPS) profiling was created. The GPS system made use of reporter proteins fused with hundreds of potential substrates independently. By inhibiting the ligase activity (causing ubiquitination not to occur), increased reporter activity shows that the identified substrates are accumulating. These results demonstrate the potential of these technologies as basic platforms for the global discovery of E3-substrate regulatory networks.[12]

Pathophysiology

Ubiquitination occurs throughout eukaryotic cell signaling and has been implicated in many malignancies through the gain of function and loss of function mutations. Loss of function mutation on the tumor suppressor gene can lead to inhibition or activation of ubiquitination. The gain of function mutations has mainly been implicated with increased activation of ubiquitination. 

In von-Hippel Lindau (VHL) disease, the loss of function mutation in the VHL tumor suppressor results in hemangioblastoma formation in multiple organs, renal cell carcinoma, and pheochromocytoma. The VHL tumor suppressor gene codes for the VHL protein. The VHL protein is a type of E3 ubiquitin ligase that catalyzes the ubiquitination of hypoxia-inducible transcription factor-alpha (HIF1-alpha). HIF-alpha regulates erythropoietin (EPO) production and vascular endothelial growth factor (VEGF). Under normal physiologic conditions, HIF1-alpha remains hydroxylated. In the hydroxylated form, it can be recognized and degraded by the VHL protein. This results in the prevention of EPO and VEGF induction under normoxic conditions. In VHL disease, the gene mutation results in the VHL protein’s inability to bind HIF-alpha, leading to uncontrolled growth.

Another way ubiquitination has been implicated in malignancy is by the way that uncontrolled proliferation has been able to evade the ubiquitin-proteasome protein degradation pathway. The ubiquitin-proteasome system plays a vital role in colorectal cells in regulating the APC (adenomatous polyposis coli)/beta-catenin signaling pathway, which regulates the growth of colorectal epithelial cells. Mutations in APC result in the failure in the degradation of beta-catenin, which results in inhibited cell proliferation.

Ubiquitination also correlates with several genetic disorders. Angelman syndrome is a rare genetic disorder affecting the nervous system that results from a mutation in UBE3A, which codes for an E3 ubiquitin ligase. Previous genetic studies have proposed that the UBE3A encoded E3 ubiquitin ligase is important for normal human cognitive function. 3-M syndrome is a disorder characterized by intrauterine growth retardation that results from mutations in CUL7, which is important in the assembly of an E3 ubiquitin ligase complex and promotion of ubiquitination. Disruption of this pathway plays a role in the pathogenesis of 3-M syndrome.[13][14][15][16]

Clinical Significance

As discussed in previous sections, the ubiquitin-proteasome pathway plays an important role in maintaining cell homeostasis. Changes in this process can lead to the formation of tumors and neurodegenerative disorders. Thus, pharmacological treatments targeted at the ubiquitin-proteasome pathway provide potential opportunities to treat tumors and neurodegenerative disorders. For example, the ubiquitin ligase MDM2 plays an essential role in regulating P53 stability, and research is focusing on developing an inhibitor that disrupts this interaction. However, creating therapeutic targets against a specific ubiquitination enzyme is challenging because multiple ubiquitin E3 ligases ubiquitinate one protein, and targeting one enzyme may not be adequate. Proteasome inhibition is the only validated therapeutic target of the ubiquitin system. Bortezomib, a proteasome inhibitor, was approved in 2003 by the FDA to treat relapsing and refractory multiple myeloma.

Ubiquitin immunohistochemistry has played a crucial role in understanding the pathophysiology of neurodegenerative diseases and aiding in diagnosing these disorders. Inclusions (protein aggregates) containing ubiquitinated proteins are present in Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease.[4][17][18][19][20]