Proteasome Totally Explained

Everything about Proteasome totally explained

Proteasomes are large protein complexes inside all eukaryotes and archaea, as well as in some bacteria. In eukaryotes, they're located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases. Proteasomes are part of a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into amino acids and used in synthesizing new proteins. Proteins are tagged for degradation by a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that's bound by the proteasome, allowing it to degrade the tagged protein.

Discovery

Before the discovery of the ubiquitin proteasome system, protein degradation in cells was thought to rely mainly on lysosomes, membrane-bound organelles with acidic and protease-filled interiors that can degrade and then recycle exogenous proteins and aged or damaged organelles. Later work on modification of histones led to the identification of an unexpected covalent modification of the histone protein by a branched bond between a histone lysine residue and the C-terminal glycine residue of the protein ubiquitin, which had no known function. It was then discovered that a previously-identified protein associated with proteolytic degradation, known as ATP-dependent proteolysis factor 1 (APF-1), was the same protein as ubiquitin.
Much of the early work leading up to the discovery of the ubiquitin proteasome system occurred in the late 1970s and early 1980s at the Technion in the laboratory of Avram Hershko, where Aaron Ciechanover worked as a graduate student. Hershko's year-long sabbatical in the laboratory of Irwin Rose at the Fox Chase Cancer Center provided key conceptual insights, though Rose later downplayed his role in the discovery. The three shared the 2004 Nobel Prize in Chemistry for their work in discovering this system. the first structure of the proteasome core particle wasn't solved by X-ray crystallography until 1994. As of 2006, no structure has been solved of the core particle in complex with the most common form of regulatory cap.

Structure and organization

The proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient (denoted S). The most common form of the proteasome is known as the 26S proteasome, which is about 2000 kilodaltons (kDa) in molecular mass and contains one 20S core particle structure and two 19S regulatory caps. The core is hollow and provides an enclosed cavity in which proteins are degraded; openings at the two ends of the core allow the target protein to enter. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites; it's this structure that recognizes polyubiquitinated proteins and transfers them to the catalytic core. An alternative form of regulatory subunit called the 11S particle can associate with the core in essentially the same manner as the 19S particle; the 11S may play a role in degradation of foreign peptides such as those produced after infection by a virus.

20S core particle

The number and diversity of subunits contained in the 20S core particle depends on the organism; the number of distinct and specialized subunits is larger in multicellular than unicellular organisms and larger in eukaryotes than in prokaryotes. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits; α subunits are structural in nature, whereas β subunits are predominantly catalytic. The outer two rings in the stack consist of seven α subunits each, which serve as docking domains for the regulatory particles and form an exterior gate blocking unregulated access to the interior cavity. The inner two rings each consist of seven β subunits and contain the protease active sites that perform the proteolysis reactions. The size of the proteasome is relatively conserved and is about 150 angstroms (Å) by 115 Å. The interior chamber is at most 53 Å wide, though the entrance can be as narrow as 13 Å, suggesting that substrate proteins must be at least partially unfolded to enter.
In archaea such as Thermoplasma acidophilum, all the α and all the β subunits are identical, while eukaryotic proteasomes such as those in yeast contain seven distinct types of each subunit. In mammals, the β1, β2, and β5 subunits are catalytic; although they share a common mechanism, they've three distinct substrate specificities considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH). Alternative β forms denoted β1i, β2i, and β5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory signals such as cytokines, in particular, interferon gamma. The proteasome assembled with these alternative subunits is known as the immunoproteasome, whose substrate specificity is altered relative to the normal proteasome. ATP hydrolysis is required for the assembled complex to degrade a folded and ubiquitinated protein, although it isn't yet clear whether that energy is used mainly for substrate unfolding, opening of the core channel, or some combination of processes.

11S regulatory particle

20S proteasomes can also associate with a second type of regulatory particle, the 11S regulatory particle, a heptameric structure that doesn't contain any ATPases and can promote the degradation of short peptides, but not of complete proteins. It is presumed that this is because the complex can't unfold larger substrates. This structure is also known as PA28 or REG. The mechanisms by which it binds to the core particle through the C-terminal tails of its subunits and induces α-ring conformational changes to open the 20S gate suggest a similar mechanism for the 19S particle. The expression of the 11S particle is induced by interferon gamma and is responsible, in conjunction with the immunoproteasome β subunits, for the generation of peptides that bind to the major histocompatibility complex. The assembly of the half-proteasomes, in turn, is initiated by the assembly of the α subunits into their heptameric ring, forming a template for the association of the corresponding pro-β ring. The assembly of α subunits hasn't been characterized.
   In general, less is known about the assembly and maturation of the 19S regulatory particles. They are believed to assemble as two distinct subcomponents, the ATPase-containing base and the ubiquitin-recognizing lid. The six ATPases in the base may assemble in a pairwise manner mediated by coiled-coil interactions. The order in which the nineteen subunits of the regulatory particle are bound is a likely regulatory mechanism that prevents exposure of the active site before assembly is complete. This adenylated ubiquitin is then transferred to a cysteine of a second enzyme, ubiquitin-conjugating enzyme (E2). In the last step, a member of a highly diverse class of enzymes known as ubiquitin ligases (E3) recognizes the specific protein to be ubiquitinated and catalyzes the transfer of ubiquitin from E2 to this target protein. A target protein must be labeled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it's recognized by the proteasome lid. It is therefore the E3 that confers substrate specificity to this system. The number of E1, E2, and E3 proteins expressed depends on the organism and cell type, but there are many different E3 enzymes present in humans, indicating that there's a huge number of targets for the ubiquitin proteasome system.
   The mechanism by which a polyubiquitinated protein is targeted to the proteasome isn't fully understood. Ubiquitin-receptor proteins have an N-terminal ubiquitin-like (UBL) domain and one or more ubiquitin-associated (UBA) domains. The UBL domains are recognized by the 19S proteasome caps and the UBA domains bind ubiquitin via three-helix bundles. These receptor proteins may escort polyubiquitinated proteins to the proteasome, though the specifics of this interaction and its regulation are unclear.
   The ubiquitin protein itself is 76 amino acids long and was named due to its ubiquitous nature, as it has a highly conserved sequence and is found in all known eukaryotic organisms. The genes encoding ubiquitin in eukaryotes are arranged in tandem repeats, possibly due to the heavy transcription demands on these genes to produce enough ubiquitin for the cell. It has been proposed that ubiquitin is the slowest-evolving protein identified to date.

Unfolding and translocation

After a protein has been ubiquitinated, it's recognized by the 19S regulatory particle in an ATP-dependent binding step. Which of these processes is the rate-limiting step in the overall proteolysis reaction depends on the specific substrate; for some proteins, the unfolding process is rate-limiting, while deubiquitination is the slowest step for other proteins.
The gate formed by the α subunits prevents peptides longer than about four residues from entering the interior of the 20S particle. The ATP molecules bound before the initial recognition step are hydrolyzed before translocation, although there's disagreement whether the energy is needed for substrate unfolding Passage of the unfolded substrate through the opened gate occurs via facilitated diffusion if the 19S cap is in the ATP-bound state.
The mechanism for unfolding of globular proteins is necessarily general, but somewhat dependent on the amino acid sequence. Long sequences of alternating glycine and alanine have been shown to inhibit substrate unfolding decreasing the efficiency of proteasomal degradation; this results in the release of partially degraded byproducts, possibly due to the decoupling of the ATP hydrolysis and unfolding steps. Such glycine-alanine repeats are also found in nature, for example in silk fibroin; in particular, certain Epstein-Barr virus gene products bearing this sequence can stall the proteasome, helping the virus propagate by preventing antigen presentation on the major histocompatibility complex.

Proteolysis

The mechanism of proteolysis by the β subunits of the 20S core particle is through a threonine-dependent nucleophilic attack. This mechanism may depend on an associated water molecule for deprotonation of the reactive threonine hydroxyl. Degradation occurs within the central chamber formed by the association of the two β rings and normally doesn't release partially degraded products, instead reducing the substrate to short polypeptides typically 7–9 residues long, though they can range from 4 to 25 residues depending on the organism and substrate. The biochemical mechanism that determines product length isn't fully characterized. Although the three catalytic β subunits have a common mechanism, they've slightly different substrate specificities, which are considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH)-like. These variations in specificity are the result of interatomic contacts with local residues near the active sites of each subunit. Each catalytic β subunit also possesses a conserved lysine residue required for proteolysis. Similar effects have been observed in yeast proteins; this mechanism of selective degradation is known as regulated ubiquitin/proteasome dependent processing (RUP).

Ubiquitin-independent degradation

Although most proteasomal substrates must be ubiquitinated before being degraded, there are some exceptions to this general rule, especially when the proteasome plays a normal role in the post-translational processing of the protein. The proteasomal activation of NF-κB by processing p105 into p50 via internal proteolysis is one major example. are degraded in a ubiquitin-independent manner. The most well-known example of a ubiquitin-independent proteasome substrate is the enzyme ornithine decarboxylase. Ubiquitin-independent mechanisms targeting key cell cycle regulators such as p53 have also been reported, although p53 is also subject to ubiquitin-dependent degradation. Finally, structurally abnormal, misfolded, or highly oxidized proteins are also subject to ubiquitin-independent and 19S-independent degradation under conditions of cellular stress.

Evolution

The 20S proteasome is both ubiquitous and essential in eukaryotes. Some prokaryotes, including many archaea and the bacterial order Actinomycetales also share homologs of the 20S proteasome, whereas most bacteria possess heat shock genes hslV and hslU, whose gene products are a multimeric protease arranged in a two-layered ring and an ATPase. The hslV protein has been hypothesized to resemble the likely ancestor of the 20S proteasome. In general, HslV isn't essential in bacteria, and not all bacteria possess it, whereas some protists possess both the 20S and the hslV systems. In vertebrate cells, "slippage" through the mitotic checkpoint leading to premature M phase exit can occur despite the delay of this exit by the spindle checkpoint.
Earlier cell cycle checkpoints such a post-restriction point check between G₁ phase and S phase similarly involve proteasomal degradation of cyclin A, whose ubiquitination is promoted by the anaphase promoting complex (APC), an E3 ubiquitin ligase. The APC and the Skp1/Cul1/F-box protein complex (SCF complex) are the two key regulators of cyclin degradation and checkpoint control; the SCF itself is regulated by the APC via ubiquitination of the adaptor protein, Skp2, which prevents SCF activity before the G1-S transition.

Regulation of plant growth

In plants, signaling by auxins, or phytohormones that order the direction and tropism of plant growth, induces the targeting of a class of transcription factor repressors known as Aux/IAA proteins for proteasomal degradation. These proteins are ubiquitinated by SCFTIR1, or SCF in complex with the auxin receptor TIR1. Degradation of Aux/IAA proteins derepresses transcription factors in the auxin-response factor (ARF) family and induces ARF-directed gene expression. The cellular consequences of ARF activation depend on the plant type and developmental stage, but are involved in directing growth in roots and leaf veins. The specific response to ARF derepression is thought to be mediated by specificity in the pairing of individual ARF and Aux/IAA proteins.

Apoptosis

Both internal and external signals can lead to the induction of apoptosis, or programmed cell death. The resulting deconstruction of cellular components is primarily carried out by specialized proteases known as caspases, but the proteasome also plays important and diverse roles in the apoptotic process. The involvement of the proteasome in this process is indicated by both the increase in protein ubiquitination, and of E1, E2, and E3 enzymes that's observed well in advance of apoptosis, During apoptosis, proteasomes localized to the nucleus have also been observed to translocate to outer membrane blebs characteristic of apoptosis.
Proteasome inhibition has different effects on apoptosis induction in different cell types. In general, the proteasome isn't required for apoptosis, although inhibiting it's pro-apoptotic in most cell types that have been studied. However, some cell lines — in particular, primary cultures of quiescent and differentiated cells such as thymocytes and neurons — are prevented from undergoing apoptosis on exposure to proteasome inhibitors. The mechanism for this effect isn't clear, but is hypothesized to be specific to cells in quiescent states, or to result from the differential activity of the pro-apoptotic kinase JNK. The ability of proteasome inhibitors to induce apoptosis in rapidly dividing cells has been exploited in several recently developed chemotherapy agents such as bortezomib and salinosporamide A.

Response to cellular stress

In response to cellular stresses - such as infection, heat shock, or oxidative damage - heat shock proteins that identify misfolded or unfolded proteins and target them for proteasomal degradation are expressed. Both Hsp27 and Hsp90—chaperone proteins have been implicated in increasing the activity of the ubiquitin-proteasome system, though they're not direct participants in the process. Hsp70, on the other hand, binds exposed hydrophobic patches on the surface of misfolded proteins and recruits E3 ubiquitin ligases such as CHIP to tag the proteins for proteasomal degradation. The CHIP protein (carboxyl terminus of Hsp70-interacting protein) is itself regulated via inhibition of interactions between the E3 enzyme CHIP and its E2 binding partner.
Similar mechanisms exist to promote the degradation of oxidatively damaged proteins via the proteasome system. In particular, proteasomes localized to the nucleus are regulated by PARP and actively degrade inappropriately oxidized histones. Oxidized proteins, which often form large amorphous aggregates in the cell, can be degraded directly by the 20S core particle without the 19S regulatory cap and don't require ATP hydrolysis or tagging with ubiquitin.
Impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases that share aggregation of misfolded proteins as a common feature, such as Parkinson's disease and Alzheimer's disease. In these diseases large insoluble aggregates of misfolded proteins can form and then result in neurotoxicity, through mechanisms that are not yet well understood. Decreased proteasome activity has been suggested as a cause of aggregation and Lewy body formation in Parkinson's. This hypothesis is supported by the observation that yeast models of Parkinson's are more susceptible to toxicity from α-synuclein, the major protein component of Lewy bodies, under conditions of low proteasome activity.

Role in the immune system

The proteasome plays a straightforward but critical role in the function of the adaptive immune system. Peptide antigens are displayed by the major histocompatibility complex class I (MHC) proteins on the surface of antigen-presenting cells. These peptides are products of proteasomal degradation of proteins originated by the invading pathogen. Although constitutively expressed proteasomes can participate in this process, a specialized complex composed of proteins whose expression is induced by interferon gamma produces peptides of the optimal size and composition for MHC binding. These proteins whose expression increases during the immune response include the 11S regulatory particle, whose main known biological role is regulating the production of MHC ligands, and specialized β subunits called β1i, β2i, and β5i with altered substrate specificity. The complex formed with the specialized β subunits is known as the immunoproteasome. This approach of selectively inducing apoptosis in tumor cells has proven effective in animal models and human trials. Bortezomib, a molecule developed by Millennium Pharmaceuticals and marketed as Velcade, is the first proteasome inhibitor to reach clinical use as a chemotherapy agent. Bortezomib is used in the treatment of multiple myeloma. Notably, multiple myeloma has been observed to result in increased proteasome levels in blood serum that decrease to normal levels in response to successful chemotherapy. Studies in animals have indicated that bortezomib may also have clinically significant effects in pancreatic cancer. Preclinical and early clinical studies have been started to examine bortezomib's effectiveness in treating other B-cell-related cancers, particularly some types of non-Hodgkin's lymphoma.
   The molecule ritonavir, marketed as Norvir, was developed as a protease inhibitor and used to target HIV infection. However, it has been shown to inhibit proteasomes as well as free proteases; to be specific, the chymotrypsin-like activity of the proteasome is inhibited by ritonavir, while the trypsin-like activity is somewhat enhanced. Studies in animal models suggest that ritonavir may have inhibitory effects on the growth of glioma cells.
   Proteasome inhibitors have also shown promise in treating autoimmune diseases in animal models. For example, studies in mice bearing human skin grafts found a reduction in the size of lesions from psoriasis after treatment with a proteasome inhibitor. Inhibitors also show positive effects in rodent models of asthma.
   Labeling and inhibition of the proteasome is also of interest in laboratory settings for both in vitro and in vivo study of proteasomal activity in cells. The most commonly used laboratory inhibitor is lactacystin, a natural product synthesized by Streptomyces bacteria.

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