Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δrpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.     

26S蛋白酶体的结构生物学研究进展

26S蛋白酶体是真核细胞内负责蛋白质降解的主要分子机器,通过特异性降解目的蛋白质,几乎参与了生物体的绝大多数生命活动.26S蛋白酶体在结构上可分为19S调节颗粒和20S核心颗粒两部分.19S调节颗粒负责识别带有泛素链标记的蛋白质底物及对其进行去折叠,并最终将去折叠的蛋白质底物传送至20S核心颗粒中进行降解.由于26S蛋白酶体的结构组成复杂,分子量十分巨大,现有的X-ray技术和NMR技术对其完整结构的解析都无能为力,仅能解析出部分单个蛋白成员或分子量较低的亚复合物晶体结构.而冷冻电镜技术在相当一段时间内处于发展的初级阶段,导致其三维结构的研究进展曾经十分缓慢,严重阻碍了人们对其结构和功能的了解.近年来,随着在X-ray技术领域对大分子复合物结构解析的经验积累和冷冻电镜技术领域的技术革命,完整的26S蛋白酶体三维结构解析取得了飞速的发展.本文回顾了近几年在26S蛋白酶体结构生物学领域的重要进展,并展望了该领域未来的发展及面临的挑战.     

26S蛋白酶体组装的分子机制研究

26S蛋白酶体广泛分布于真核细胞中的胞质和胞核,主要是由20S核心复合物(coreparticle,CP)和19S调节复合物(regulatory particle,RP)组成,它负责细胞大多数蛋白质的降解,在几乎所有生命活动中具有关键的调控作用。26S蛋白酶体的组装是一个非常复杂且高度条理的过程,不同的分子伴侣,如PAC1-4、Ump1、p27、p28和s5b等,参与其中发挥识别及调节作用,以确保高效准确地完成蛋白酶体的组装。本文系统总结分析了20S核心复合物和19S调节复合物的组装过程及调控机制的最近研究进展。     

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.     

Structural organization of the 19S proteasome lid: insights from MS of intact complexes

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex.     

1H, 13C and 15N resonance assignments of Rpn9, a regulatory subunit of 26S proteasome from Saccharomyces cerevisiae

The 26S proteasome is an essential molecular machine for specific protein degradation in eukaryotic cells. The 26S proteasome is formed by a central 20S core particle capped by two 19S regulatory particle (RP) at both ends. The Rpn9 protein is a non-ATPase subunit located in the lid complex of the 19S RP, and is identified to be essential for efficient assembly of yeast 26S proteasome. Bioinformatics analysis of Saccharomyces cerevisiae Rpn9 suggested it contains a PCI domain at the C-terminal region. However, high-resolution structures of either the PCI domain or the full-length Rpn9 still remain elusive. Herein, we report the chemical shift assignments of ¹H, ¹³C and ¹⁵N atoms of the individual N- and C-domains, as well as full-length S. cerevisiae Rpn9, which provide the basis for further structural and functional studies of Rpn9 using solution NMR technique.     

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.     

20S proteasome prevents aggregation of heat-denatured proteins without PA700 regulatory subcomplex like a molecular chaperone

The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation.