As much as the proteasome is complex, it does not work alone. There is an orchestra of proteins involved in the regulation of the proteasome activity. An increasing number of PIPs have been reported, which indicates that the 20S proteasome CP is a dynamic structure that interacts with specific proteins for specific functions. Therefore, any interference with the 20S proteasome and with its interacting proteins would affect the proteasome specific functions.
It has been a decade since Verma et al[23
] have reported and analyzed PIPs. These authors used a one-step affinity method to purify intact 26S proteasome and its interacting proteins from budding yeast cells, and reported the existence of PIPs. These newly discovered PIPs were classified in four groups that include proteasome subunits, chaperone, transcription, and ribosomal proteins. New methods to purify the proteasome were then developed. Scanlon et al[24
] have used the GST UBL (ubiquitin-like domain as an affinity chromatography matrix), and purified the proteasome from human cell lines. These authors have classified the 26S PIPs into four groups of proteins that include de-ubiquitinases, ubiquitin ligases, ubiquitin domain-containing proteins and conjugating/ubiquitin, and proteins involved in DNA repair. Using the GST UBL matrix to purify the PIPs has helped find proteins dominantly related to the proteasome and ubiquitination systems; probably because of the affinity of UBL for the ubiquitin-proteasome pathway.
Among identified PIPs are a number of abundant cellular proteins, such as heat shock proteins (Hsps), elongation factors, and ribosomal proteins. The Hsps interaction with the proteasome is highly specific because they are induced to compensate for the proteasome failure, when the proteasome activity is decreased. In higher eukaryotes, it has been shown that Hsc70/Hsp70 members facilitate the delivery of aggregation-prone substrates for degradation by interacting with the proteasome through an adaptor protein[25,26
]. Along with Hsp70, Hsp27 assists in the unfolding of the proteins designated for degradation by the proteasome[27
]. In addition, Hsp90 family members have been suggested to play a role in the proteasome structural integrity and assembly through their interactions with the 26S proteasome[28
Other proteins work upstream of the ubiquitin system to recognize, unfold, shuttle, dock, and deubiquitinate the protein substrate designated for degradation. The chaperone system Bip/PDI is associated with endoplasmic reticulum (ER)-associated degradation[29
]. Other proteins that contain a ubiquitin-associated domain (UBA), such as Rad23 and p62, are involved in carrying and docking the protein substrates at the proteasome[30
]. These interacting proteasome partners differ in their biological roles, and are chosen to interact with the proteasome according to their specific cellular function. Therefore, different chaperone members may play distinct roles in modulating protein degradation by the proteasome.
A great number of yet-to-be-identified proteins can mediate ubiquitin recognition at the proteasome. UBL-UBA domain-containing proteins associate with substrates designated for degradation, as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. The best known UBL-UBA proteins of PIPs are Cdc48/p97/valosin-containing protein (VCP), which present misfolded ER-proteins to the proteasome. P62, also called sequestosome 1, is also involved in presenting ubiquitinated proteins to the proteasome (Figure ).
Proteasome interacting proteins (PIPs) involved in unfolding and docking of protein substrates designated for degradation by the proteasome. VCP: Valosin-containing protein.
VCP and P62 have been reported to be involved in cytokeratin 8/cytokeratin 18 aggregate sequestration and Mallory-Denk body (MDB) formation in alcoholic liver disease[31
] (Figure ). Both proteins are significantly upregulated when the proteasome activity is inhibited using PS-341, thus indicating that these proteins play a crucial role in proteasome activity[32
The 26S stabilizing protein, Ecm29, has been found in the fraction of the purified proteasome, and is well established as a PIP. Ecm29 tethers the proteasome CP to the 19S regulatory particle, and confers stability of 19S-20S binding in yeast[33
]. It may play a crucial role in the 26S proteasome dysfunction in alcoholic liver disease[34
More recently, Kautto et al[35
] have developed a rapid method of 26S proteasome isolation using chromatography, and have identified over 100 proteins in the proteasome purified fraction, including the 26S proteasome and 32 proteasome subunits. 14-3-3-like proteins have been identified to interact with the proteasome, and also have been classified as PIPs. In our laboratory, when chromatography and high salt concentration purification was used, only a few proteins were identified by mass spectrometry, which indicates that the salt disrupted the proteasome interactions with its associating proteins. The identified proteins included the 14-3-3 proteins, the kinases protein kinase A (PKA) and transglutaminase, and the phosphatases PP2A and PP1[36
The role and function of the 14-3-3 proteins, in the regulation of proteasome function, remain to be elucidated. This protein has been identified by mass spectrometry in the 20S fraction purified chromatographically with a high salt gradient, which reflects the strength of 14-3-3 interaction with the 20S proteasome. 14-3-3, which is a major scaffolding protein and a phosphobinding protein in the cell, plays a major role in cellular mechanisms, such as signal transduction and regulation of transcription factors[37-39
]. A pilot study has shown that chronic ethanol feeding increases the interaction of 14-3-3 with the 20S proteasome, probably to regulate the 20S proteasome ratio of phosphorylation/dephosphorylation to modulate the proteasome activity changes due to ethanol feeding[36
The proteins kinases associated with the 20S proteasome, such as casein kinase II[40
], transglutaminase (TG2), and PKA also co-isolated with the 20S proteasome through multiple chromatographic steps and high salt concentration, as well as the phosphatases PP2A and PP1[41,42
]. These proteins regulate the 20S proteasome activity via
phosphorylation/dephosphorylation and are believed to regulate also the 20S proteasome binding to its regulatory complexes. TG2 has been found in the fraction of highly purified 20S proteasome[36
], and is known to be responsible for stabilizing macromolecular assemblies[43,44
]. It is possible that TG2 is involved in the stabilization of the proteasome macromolecules via
its kinase activity. These kinases and phosphatases are crucial for the function of the different types of proteasomes because they regulate the phosphorylation of the α type subunit of the proteasome, thus determining the binding of the 20S proteasome to its regulatory complexes (Figure ).
Figure 4 Illustration of hypothetical chronic ethanol feeding effects in 26S dysfunction. As a result of phosphorylation deregulation, 20S and 19S binding is altered, which causes 26S dysfunction. Total dephosphorylation of proteasome subunits by phosphatases (more ...)
Similarly to PKA, TG2 and PP2A, the enzyme δ-aminolevulinate dehydratase (ALAD) has been identified by mass spectrometry in the highly purified 20S proteasome fraction[45
]. ALAD, also called porphobilinogen synthase, is a cytosolic sulfhydryl-containing enzyme that catalyzes the condensation of two molecules of aminolevulinic acid (ALA). It has been reported that blood ALAD activity is significantly decreased when rats are chronically fed ethanol, which indicates that ethanol feeding causes an alteration in blood[46
] as well as liver ALAD activity[47
When this enzyme is inhibited, the ALA accumulates, which may impair heme biosynthesis and cause porphyria in the liver and pro-oxidant activity in the brain[48,49
]. ALAD was among the first PIPs to be identified and its role in the proteasome pathway still needs to be clarified[50
The recent and most productive method to investigate the PIPs is the QTAX-based tag-team technique, as used by Guerrero et al[51,52
]. These authors have identified at least 471 proteins in the network of the proteasome from yeast. The first group of proteins that have been characterized with high affinity was the ubiquitin receptor proteins. The rest of the identified proteins were grouped in the 35 distinct gene ontology protein complexes that are involved in various biological processes, such as chromatin remodeling, metabolism, translation, DNA replication, endocytosis, and protein folding.
Another method to purify the proteasome is to use multiple centrifugation with ATP and a final glycerol gradient zonal centrifugation. This procedure separates the proteasomes and preserves its binding to its regulatory complexes and interacting proteins[34
]. Then, mass spectrometry analysis is used to identify the PIPs, as well as to quantify their levels, and thus their interaction with the proteasome. The PIPs identified with the other above-mentioned method have also been characterized by our approach, and in addition, the effects of chronic ethanol feeding have been analyzed[34
Proteasome activity is regulated at multiple levels. Chronic ethanol feeding, which causes dysfunction of the proteasome pathway, may also occur at the level of PIPs, which could lead to the failure of the proteasome pathway in the liver of alcoholic patients.