Proteasomes are giant tubular protein complexes that break peptide bonds to degrade unneeded or damaged proteins in cells. Enzymes that help such reactions are called proteases.
The proteasome is the main mechanism that cells use to regulate the concentration of specific proteins and to remove misfolded proteins. Proteins that need to be degraded are first tagged (i.e. linked) by a small protein called ubiquitin. This labeling reaction is catalyzed by ubiquitin ligase. Once a protein is tagged with a ubiquitin molecule, it triggers other ligases to add more ubiquitin molecules; This creates a "polyubiquitin chain" that can bind to the proteasome, thus bringing the proteasome to the labeled protein and begin its degradation process. After being degraded by the proteasome, the protein is cleaved into peptides about 7-8 amino acids long. These peptides can be further degraded into individual amino acid molecules, which are then used to synthesize new proteins.
Three-dimensional view of the proteasome
Proteasomes are present in all known eukaryotes and archaea, and also in some prokaryotes. In eukaryotes, it is located in the nucleus and cytoplasm.
In terms of protein structure, the proteasome is a barrel complex, consisting of a "core" of four stacked rings, which is hollow and forms a cavity. Each ring consists of seven protein molecules. The two middle loops each consist of seven beta subunits and contain six active sites for proteases. These sites are on the inner surface of the ring, so the protein must enter the "cavity" of the proteasome in order to be degraded. The two outer rings each contain seven alpha subunits, which act as "gates" and are essential for proteins to enter the cavity. These alpha subunits, or "gates", are controlled by "cap" -like structures, that bind to them; Regulatory particles recognize polyubiquitin chain tags attached to proteins and initiate the degradation process. The whole system involving ubiquitination and proteasome degradation is called the "ubiquitin-proteasome system".
The proteasome degradation pathway is essential for many cellular processes, including the cell cycle, regulation of gene expression, and response to oxidative stress. The importance of intracellular proteasome degradation and the role of ubiquitin proteolytic pathways was recognized by the 2004 Nobel Prize in Chemistry, which was shared by Aaron Cihanover, Avram Hershko, and Irving Ross.
Structural diagram of the proteasome
Structure and Composition
The components of the proteasome are usually named according to their Svedberg sedimentation coefficient, which is denoted by "S". The most common form of proteasome is the 26S proteasome, which has a molecular weight of about 2000kDa and contains a 20S core particle and two 19S regulatory particles. The core particle is a hollow structure, which encloses the active site of the shear protein in the "hole". By leaving the ends of the core particle open, the target protein can enter the "hole". Each end of the core particle is connected to a 19S regulatory particle, and each regulatory particle contains multiple ATPase active sites and ubiquitin-binding sites. Regulatory particles recognize polyubiquitin proteins and deliver them to core particles. In addition to 19S regulation particle, there is another regulation particle,, namely 11S particles; 11S regulatory particles can bind to the core particles in a similar way as 19S particles. 11S particles may play a role in degrading exogenous peptides, such as those produced after viral infection. In addition, PA200 (Blm10 in yeast) protein can also act alone as an activating protein to regulate 20S particle opening.
The Assembly Mechanism
The assembly of the proteasome is a complex process because all the numerous subunits must be correctly combined to form an active core particle complex. When the β subunit is synthesized, it has a "propeptide" at the N-terminal. During the assembly of 20S particles, the "propeptide" acts through post-translational modifications to expose the active site. The whole assembly process is complex, but also very orderly. First, the alpha subunit is assembled into a seven-membered ring to provide a template for the corresponding pre-β ring, and then the pre-β ring assembly is completed, such that a seven-subunit pre-β ring and a seven-subunit α-ring form half a core particle. The mechanism of α-ring assembly has not yet been determined. The two β-loops between the two half-core particles then bind and trigger the threonine-dependent autodegradation of the "propeptide", thereby exposing the active site, which assembles into an active 20S core particle. The interaction between β-loops is mediated by salt bridges and hydrophobic interactions between conserved α-helix residues. By mutating these conserved residues, proteasome assembly can be destroyed, thus confirming the importance of these residues for assembly on the other hand.
Little is known about the assembly and maturation of 19S-regulated particles. The current view is that the 19S regulatory particle is assembled from two distinct parts, the ATPaATPase-containing part and the ubiquitin recognition cap part. Of these, six ATPase in the basal part can be bound together in pairs by the interaction of coiled-coil helices. This sequence of assembly of the 19 subunits in the regulatory particle is most likely a regulatory mechanism to prevent exposure of the active site before assembly is complete.
Protein Degradation Process
Step 1: Ubiquitination and targeting
Proteins that need to be degraded by the proteasome are first labeled with ubiquitin, a lysine on the protein that forms a covalent link to ubiquitin. This process is a three-enzyme cascade, which requires the occurrence of a series of reactions catalyzed by three enzymes, and the whole process is called the ubiquitination signaling pathway. In the first reaction, ubiquitin activase, also known as E1, hydrolyzes ATP and adenylates a ubiquitin molecule. Next, ubiquitin is transferred to the cysteine residue of E1's active center, which is accompanied by adenylation of the second ubiquitin molecule. The adenylated ubiquitin molecule is then transferred to the cysteine residue of a second enzyme, ubiquitin cross-linking enzyme (E2). Finally, a member of the highly conserved ubiquitin ligase (E3) family (which varies according to the substrate protein) recognizes specific target proteins that need to be ubiquitinated and catalyzes the transfer of ubiquitin molecules from E2 to target proteins. The target protein must be labeled with at least four ubiquitin monomer molecules (in the form of polyubiquitin chains) before it can be recognized by the proteasome. Thus, it is E3 that makes this system substrate specific. The amount of E1, E2, and E3 proteins depends on the organism and cell type, and the presence of a large number of different E3 proteins in the human body suggests that the ubiquitin-proteasome system can act on a large number of target proteins.
Cartoon model of ubiquitin
How polyubiquitin proteins are recognized by the proteasome is not fully understood. The N terminus of the ubiquitin receptor protein has a ubiquitin-like domain and one or more ubiquitin-binding domains. The ubiquitin domain can be recognized by the 19S regulatory particle, and the ubiquitin-binding domain can bind ubiquitin by forming a triple helix bundle. These receptor proteins may be able to bind polyubiquitinated proteins and carry them to the proteasome, but the specificity and regulatory mechanisms of this binding are unknown. Recently, however, researchers have found that Rpn13, a subunit of regulatory particles, can function as a ubiquitin receptor.
The ubiquitin protein itself is made up of 76 residues and is named "ubiquitin" because it is ubiquitous in living organisms: it has a highly conserved sequence and is present in all known eukaryotic organisms. The genes encoding ubiquitin in eukaryotes are arranged in tandem repeats, probably because of the need for extensive transcription to produce enough ubiquitin for cells. It has been proposed that ubiquitin is the slowest evolving protein yet discovered.
Step 2: Unfolding and translocation
Ubiquitinated proteins (hereafter referred to as substrate proteins) are recognized by 19S regulatory particles in an ATP-dependent binding process. The substrate protein must then enter the inner pore of the 20S core particle in order to make contact with the hydrolytic active site located therein. Because the 20S particle has a relatively narrow pore and is switched at both ends by the N-terminus of the α-loop subunit, the substrate protein must be at least partially unfolded before entering the core particle. The process of delivering the folded protein into the core particle is called translocation, and the translocation must occur after deubiquitination. However, the mechanism of substrate protein deubiquitination and unfolding is still unknown. In the whole degradation reaction, which step is the rate-limiting step depends on the type of substrate protein; For some proteins, the unfolding process is a rate-limiting step, while for others, it may be de-ubiquitination to a rate-limiting factor. It has not been determined which substrate proteins must be unfolded before translocation, but the strong tertiary structure and some special nonlocal interactions, such as disulfide bonds, inhibit degradation.
Ubiquitination signaling pathway. Ub stands for ubiquitin.
The "gate" formed by the alpha subunit prevents peptides longer than four residues from entering the interior of the 20S particle. ATP molecules bound before the start of the recognition step are hydrolyzed before the shift occurs, and there is debate as to whether the energy produced by hydrolysis is used for protein unfolding or "gate" opening. The 26S proteasome can degrade the folded protein in the presence of an ATP analog that cannot be hydrolyzed (that is, the energy produced by hydrolysis is not available), but cannot degrade the folded protein. This result suggests that at least part of the energy generated by ATP hydrolysis is used for protein folding. When the 19S cap is in the ATP-bound state, the unfolded substrate protein can be transferred through the open "gate" by promoting diffusion.
The mechanism of globulin unfolding is similar, but also depends to some extent on the amino acid sequence of the protein. The researchers found that containing longer glycine or alanine sequences inhibited the unfolding, thus reducing the efficiency of proteasome degradation. The result is a mixture containing partially folded proteins, which may result from a disconnect between ATP hydrolysis and the unfolded steps. Such glycine-alanine repeats are also found in some proteins in nature, such as fibroin in silk; It is worth mentioning that the expression products of certain human herpesvirus genes also contain such sequences that inhibit proteasome action and prevent antigen presentation to the major histocompatibility complex, thus contributing to virus reproduction.
Step 3: Protein degradation
Protein degradation is carried out by the β subunit in the 20S core particle by a mechanism thought to be a threonine-dependent nucleophilic attack. This mechanism may require a bound water molecule to participate in the deprotonation of the hydroxyl group on the active threonine. The degradation occurs in the pores in the two β-rings in the middle of the core particles, and generally does not generate partial degradation products, but completely degrades the substrate proteins into peptides of a certain length. Peptides are typically 7-9 residues in length, but can range from 4-25 residues, depending on the organism and the substrate protein. The mechanisms that determine the length of the peptide in the decomposition products are not fully understood. Although the three catalytically active β subunits share a common degradation mechanism, their specificity for substrates is slightly different, namely chymotrypsin-like type, trypsin-like type, and peptide-glutamylpeptide-hydrolytic type. This difference in substrate specificity results from the different interactions between the local residues close to the active site and the substrate. Each catalytically active β subunit also contains a conserved lysine essential for degradation.
Although the proteasome usually generates very short degradation fragments, in some cases these degradation products are themselves biologically active functional molecules. Specific transcription factors, including a component of the mammalian NF-κB complex, are synthesized as inactive precursor molecules that are converted to active molecules after ubiquitination and protease degradation. This degradation requires the proteasome to cleave the middle of the protein, rather than the usual cleavage that starts at one end of the protein. It has been suggested that the middle portion that needs to be cleaved is a long loop (loop (Biochemistry)) that sits on the surface of the protein so that it can serve as a substrate for the proteasome to go inside its internal pores, while the rest of the protein remains outside the pores and is not degraded. A similar phenomenon has been found in yeast proteins; This selective degradation is known as regulated ubiquitin/proteasome-dependent processing.
Evolution of the Proteasome
20S proteasomes are ubiquitous and essential in eukaryotes. Some prokaryotes, including many archaea and actinomycetes in bacteria, also contain homologs of the 20S proteasome, the heat-shock genes hslV and hslU, which encode proteins that form bilayer circular polymerases and ATPase. Some researchers have suggested that the HslV protein probably resembles the ancestor of the 20S proteasome. In general, HslV proteins are not essential for bacteria and not all bacteria contain this protein, whereas protists contain both 20S proteasome and HslV protein systems.
Sequence analysis revealed that the catalytic beta subunit diverged earlier in evolution than the structural alpha subunit. In bacteria expressing 20S proteasomes, the β-subunit has a high degree of sequence similarity to the β-subunit of archaea and eukaryotes, while the α-subunit has a much lower degree of sequence similarity. The presence of 20S proteasomes in bacteria may be the result of horizontal gene transfer, while the differentiation of subunits in eukaryotes may be the result of multiple gene duplications.
HslV in Escherichia coli
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