How ubiquitinated proteins are recognized by proteasomes is not fully understood. Ubiquitin receptor protein has an ubiquitin-like domain and one or more ubiquitin-binding domains at the N-terminal. Ubiquitin-like domains can be recognized by 19S regulatory particles, while ubiquitin-binding domains can bind ubiquitin by forming triple helix bundles. These receptor proteins may be able to bind to ubiquitinated proteins and carry them to proteasome, but the specificity and regulatory mechanism of this binding are still unclear. Recently, however, some researchers found that regulating the subunit Rpn 13 on particles can play the role of ubiquitin receptor.
Ubiquitin protein itself consists of 76 residues, so it is named "Ubiquitin" because it exists widely in organisms: it has a highly conserved sequence and exists in all known eukaryotes. Genes encoding ubiquitin in eukaryotes are repeatedly arranged in series, which may be because a lot of transcription is needed to produce enough ubiquitin for cells. It has been suggested that Ubiquitin is the slowest protein ever discovered. The ubiquitous signal path. Where "Ub" stands for ubiquitin.
Ubiquitin protein (hereinafter referred to as substrate protein) is recognized by 19S regulatory particles, which is an ATP-dependent binding process. Then, the substrate protein must enter the internal pores of the 20S core particles in order to contact with the hydrolytic active sites located therein. Because the pore channels of 20S particles are relatively narrow and both ends are controlled by the N-terminal of α-cyclic subunit, the substrate protein must be at least partially unfolded before entering the core particles. The process of transferring unfolded protein to core particles is called "translocation", which must occur after depolymerization. However, the mechanism of ubiquitination and unfolding of substrate protein is still unknown. In the whole degradation process, which step is the rate-limiting step depends on the type of substrate protein; For some protein, the unfolding process is a speed-limiting step, while for other protein, it may be a speed-limiting factor. As for which substrate proteins must be unfolded before translocation, it is still inconclusive, but the tertiary structure of solids and some special non-local interactions, such as disulfide bonds, can inhibit degradation.
The "gate" formed by α subunit can prevent peptides with a length of more than four residues from entering the 20S particle. Before the recognition step, the bound ATP molecule is hydrolyzed before the shift occurs, but whether the energy generated by hydrolysis is used for protein unfolding or "opening the door" is still controversial. 26S proteasome can still degrade unfolded protein, but can't degrade folded protein when ATP analogues can't be hydrolyzed (that is, the energy generated by hydrolysis can't be obtained). This result shows that the energy generated by ATP hydrolysis is at least partially used for protein unfolding. When 19S hat is in ATP binding state, the unfolded substrate protein can be transferred through the open "gate" through facilitated diffusion.
The mechanism of globulin unfolding is basically similar, but it also depends on the amino acid sequence of protein to some extent. The researchers found that containing longer glycine or alanine sequences can inhibit unfolding, thus reducing the degradation efficiency of proteasome; The result is a mixture containing partially unfolded protein, which may be caused by the disconnection between ATP hydrolysis and unfolding steps. Some protein in nature also have this glycine-alanine repeat sequence, such as fibroin); Silk; It is worth mentioning that the expression products of specific human herpesvirus genes also contain such sequences, which can prevent the antigen from being presented to the main histocompatibility complex by inhibiting the role of proteasome, thus contributing to virus reproduction.
The cross section of 20S core particle shows the position of active site. Among them, the α subunit is represented by green balls, the protein skeleton of the β subunit is shown as a band, and different polypeptide chains are represented by different colors. Pink balls indicate the position of threonine residues at the active site of each subunit. The light blue chemical structure is bortezomib, an inhibitor that binds to the active site. Protein was degraded by β subunit in 20S core particles, and its mechanism was thought to be threonine-dependent nucleophilic attack. This mechanism may require the combined water molecules to participate in the deprotonation of hydroxyl groups on active threonine. Degradation occurs in the pore channel between the two β rings of the core particles, and generally does not produce partial degradation products, but completely degrades the substrate protein into peptide segments with a certain length; The length of peptide is usually 7-9 residues, but it can vary from 4-25 residues according to different organisms and substrate proteins. At present, the mechanism of determining the length of peptide fragments in decomposition products is not fully understood. Although the three β subunits with catalytic activity have the same degradation mechanism, they have slightly different specificity for substrates, namely chymotrypsin, tryptase and peptidyl-glutamyl hydrolysis. This difference in substrate specificity comes from the interaction between local residues near the active site and the substrate. Each β subunit with catalytic activity also contains conserved lysine necessary for degradation.
Although proteasome usually produces very short degradation fragments, in some cases, these degradation products are functional molecules with biological activity. Specific transcription factors, including a component of mammalian NF-κB complex, exist as inactive precursor molecules after synthesis, and are converted into active molecules after ubiquitination and protease degradation. This degradation requires proteasome to cut the middle part of protein, instead of cutting from one end of protein as usual. It has been suggested that the middle part to be cut is a long ring, which is located on the surface of protein, so that it can be used as the substrate of proteasome to enter its internal pores, while the other parts of protein are still outside the pores and will not be degraded. A similar phenomenon was also found in yeast protein. This selective degradation is called "regulated ubiquitin/proteasome-dependent processing". Although most proteasome substrates must be ubiquitinated before degradation, there are still some exceptions, especially proteasomes involved in post-translational processing of proteins. A major example is that proteasome activates NF-κB by cleaving p 105 protein into p50 protein. Some protein, which is considered unstable due to the existence of unstructured protein, can also be degraded by ubiquitin-independent pathway. Ornithine decarboxylase is the most famous proteasome substrate in ubiquitin-independent pathway. Although p53 protein can also be degraded by ubiquitin-dependent pathway, the ubiquitin-independent degradation mechanism of key cell cycle regulators such as p53 protein has been reported. In addition, under some cell stress conditions, protein with abnormal structure, misfolding or over-oxidation will also enter the ubiquitin-independent and 19S particle-independent degradation pathways.