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]

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]

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 inhibition of 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]

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 the formation of hemangioblastomas 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 mutation of the gene results in the VHL protein’s inability to bind HIF-alpha, leading to uncontrolled growth.

Another way in which 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]

As discussed in previous sections, the ubiquitin-proteasome pathway plays an important role in maintaining the cell homeostasis, and 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 enzyme of ubiquitination 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 the diagnosis of 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]