Research Area

Proteasome


Introduction of proteasome

Proteasome is a highly organized protease complex located in the cytosol as well as the eukaryotic cell nucleus comprising a catalytic 20S core particle (CP) and two 19S regulatory particles (RP), which together form the 26S structure. The 26S proteasome is responsible for the degradation of most ubiquitylated proteins through a multistep process involving recognition of the polyubiquitin chain, unfolding of the substrate, and translocation of the substrate into the active site in the cavity of the CP.

In eukaryotic cells, the ubiquitin–proteasome system (UPS) controls almost all basic cellular processes – such as progression through the cell cycle, signal transduction, cell death, immune responses, metabolism, protein quality control, and development – by degrading short-lived regulatory or structurally aberrant proteins. Proteins destined for degradation are modified by a small degradation label called ubiquitin (Ub) modified by a series of ubiquitination synergies. Repeated rounds of ubiquitin conjugation lead to the formation of a polyubiquitin chain on the target protein. The polyubiquitin chains with four or more ubiquitin are targeted by the 26S proteasome and the target protein is degraded into an oligopeptide. Concomitantly, the polyubiquitin chains are disassembled by deubiquitylating enzymes, which cleave the ubiquitin molecules from the strand to allow ubiquitin recirculation.

The proteasome has recently emerged as a promising drug target for cancer therapy. For example, Velcade (bortezomib), a proteasome inhibitor, was first approved for refractory multiple myeloma and was extended to mantle cell lymphoma. In addition, the next-generation proteasome inhibitors, such as carfizomib and salinosporamide A, have already been developed and are used in clinical trials. Thus, new and extensive research has focused on the proteasome in both basic and clinical fields.

Structure and function of the proteasomes

The 26S Proteasome

The 26S proteasome is composed of at least 33 different subunits and arranged into two subcomplexes: a proteolytic core particle (CP; also known as the 20S proteasome) and one or two terminal 19S regulatory particle(s) (RP; also known as PA700) (Fig. 1).

 

Figure 1. Structure and function of the 26S proteasome.

The 26S proteasome consists of the catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The CP is formed by four stacked rings: two outside α -rings and two inner β -rings. The 19S ATPase subunits also form a double-ring structure, named CC–OB ring and ATPase ring. The polyubiquitin chains with four or more ubiquitin proteins serve as a targeting signal for the 26S proteasome. The substrate protein is unfolded, translocated into the CP, and degraded into oligopeptides. Concomitantly, the polyubiquitin chains are disassembled by deubiquitylating enzyme subunits.

The CP is a barrel-shaped structure of ~730 kDa consisting of four heptameric rings, whereas the 19S RP is a ~930 kDa complex constituting 19 different subunits. The 19S RP binds to one or both ends of the latent CP to form an enzymatically active proteasome.

The 20S CP (alias 20S proteasome) is a well-organized protein complex with a sedimentation coefficient of 20S and a molecular mass of approximately 730 kDa (Fig.1).The CP processively degrades substrate proteins, generating oligopeptides ranging in length from 3 to 15 amino-acid residues. The resulting peptide products are subsequently hydrolyzed to amino acids by oligopeptidases and/or amino-carboxyl peptidases. In higher eukaryotes, the oligopeptides generated by the proteasome can be used by major histocompatibility complex (MHC) class I molecules for the display of intracellular/endogenous antigens to the immune system.

The RP regulates substrate degradation by binding of polyubiquitylated substrates, removing the polyubiquitin chains, unfolding substrate proteins, opening the gate of the CP, and transferring the unfolded substrates into the CP where the catalytic sites are located. The 19S RP comprises at least 19 different integral subunits with molecular masses ranging from 10 to 110 kDa that can be subclassified into two groups, regulatory particle of triple-ATPase subunits (Rpt1–6) and regulatory particle of non-ATPase subunits (Rpn1–15). Although two Rpn subunits, Rpn4 and Rpn14, were incorrectly considered to be  integral subunits, the two proteins were found to be proteasome transcription factors and assembly factors, respectively. The RP can be divided into two subcomplexes, the lid and the base; the base includes six different AAA+ (ATPases Associated with diverse cellular Activities) ATPase subunits (Rpt1–Rpt6) and three non-ATPase subunits (Rpn1, Rpn2, and Rpn13), while the lid comprises nine non-ATPase subunits (Rpn3, 5–9, 11, 12, and 15). The connection between the lid and the base is stabilized by the Rpn10 subunit.

Proteasome assembly

In recent years, several groups have focused on the mechanisms involved in the organization of the complex structures of the 26S proteasomes. To ensure quick and complete degradation of the substrate, the 26S proteasome itself should be formed correctly and rapidly into the sophisticated structure from more than 66 subunits. In this regard, the 26S proteasome has three rings, the CP α -ring, the CP β -ring, and the 19S ATPase ring, each of which are formed correctly from six or seven structurally related but distinct subunits. It is now known that a series of proteasome-dedicated chaperones are involved in the efficient and correct assembly of the CP and the 19S base, respectively. Interestingly, both assemblies are multi-step processes initiated by the formation of specific subassemblies as described below. Although the mechanisms underlying CP assembly are well established, how the RP assembly proceeds is somewhat controversial. Nevertheless, the study of the proteasome assembly is fundamentally important and could provide the foundation for the design and development of novel anticancer drugs that target proteasome biogenesis.

Reference:

1. Simon Thompson, Liam Loftus, Michelle Ashley et al. Ubiquitin-Proteasome System as a Modulator of Cell Fate. Curr Opin Pharmacol. 2008; 8(1): 90–95.

2. Van G. Wilson. The Role of Ubiquitin and Ubiquitin-Like Modification Systems in Papillomavirus Biology.Viruses 2014, 6, 3584-3611

3. Robert Layfield, R. John Mayer. The ubiquitin proteasome system in human disease. Biophysica Acta 1782 (2008) 681–682

4. John M. Walker. Ubiquitin family modifiers and the proteasome. Methods in Molecular Biology. 2012; 313-348

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