Proteasome plasticity

The 26S proteasome is responsible for regulated proteolysis of most intracellular proteins yet the focus of intense regulatory action itself. Proteasome abundance is responsive to cell needs or stress conditions, and dynamically localized to concentrations of substrates. Proteasomes are continually assembled and disassembled, and their subunits subject to a variety of posttranslational modifications. Furthermore, as robust and multi-tasking as this complex is, it does not function alone. A spattering of closely associating proteins enhances complex stability, fine-tunes activity, assists in substrate-binding, recycling of ubiquitin, and more. HEAT repeat caps activate proteasomes, yet share remarkable features with nuclear importins. Fascinating cross talk even occurs with ribosomes through common maturation factors. The dynamics of proteasome configurations and how they relate to diverse activities is the topic of this review.     

Proteasome dynamics

Proteasomes are highly conserved multisubunit protease complexes and occur in the cyto- and nucleoplasm of eukaryotic cells. In dividing cells proteasomes exist as holoenzymes and primarily localize in the nucleus. During quiescence they dissociate into proteolytic core and regulatory complexes and are sequestered into motile cytosolic clusters. Proteasome clusters rapidly clear upon the exit from quiescence, where proteasome core and regulatory complexes reassemble and localize to the nucleus again. The mechanisms underlying proteasome transport and assembly are not yet understood. Here, I summarize our present knowledge about nuclear transport and assembly of proteasomes in yeast and project our studies in this eukaryotic model organism to the mammalian cell system. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Proteasome activators

Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential for many aspects of cellular function. Because the proteolytic sites are sequestered in a closed barrel-shaped structure, activators are required to facilitate substrate access. Structural and biochemical studies of two activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although the biological functions of these factors remain obscure. Recent advances have improved our understanding of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degradation. Here we present a structural perspective on how proteasomes are activated and how substrates are delivered to the proteolytic sites.     

Proteasome and myogenesis

In this report, we examine the involvement of the ubiquitin-proteasome pathway during fusion and differentiation of myoblast primary cell cultures. Up-regulation of proteasome was observed at the maximum fusion rate and was preceded by an increase of unidentified ubiquitin-conjugates. Cell permeable proteasome inhibitors prevent fusion as do antisense oligodesoxyribonucleotides targetted to three proteasome subunits. Identical results were obtained using E3 ubiquitin-ligases dipeptide inhibitor. Involvement of the ubiquitin-proteasome pathway in the regulation of myogenic factors was hypothetized.     

Proteasome and proteolysis

The maintenance of cellular homeostasis and the ability of cells to respond to their environment depend on the degradation of bulk proteins and orderly degradation of key regulatory proteins and their inhibitors. The 26S proteasome plays an essential role in these degradations. It is involved in the activation and inactivation of many cellular processes such as cell cycle progression, apoptosis and regulation of gene expression. It presents unique structural and functional properties. It degrades proteins by an unusual mechanism. Several series of proteasome inhibitors have been developed, useful to elucidate the biological roles of this multicatalytic enzyme. Velcade (bortezomid) was the first proteasome inhibitor to undergo, in may 2003, clinical trials in cancer patients.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

蛋白酶体与神经原纤维缠结

蛋白酶体在真核生物体内选择性识别、清除错误折叠和异常聚积的蛋白质。神经原纤维缠结是阿尔茨海默病患典型的病理特征之一,主要由异常磷酸化的微管相关蛋白tau聚积而成,但其形成机制尚未阐明。越来越多的研究表明蛋白酶体功能异常和神经原纤维缠结的形成密切相关。     

Proteasome inhibition by peptide-semicarbazones

Peptide-semicarbazones derived from Z-Trp-Trp-Phe-aldehyde inhibit the chymotryptic activity of the human proteasome at nanomolar concentrations, but are less active in a NFkappaB reporter gene assay. Cyclic semicarbazones, in contrast, combine a strong inhibitory effect on the enzyme with an inhibition of NFkappaB signaling in the nanomolar range. In addition, a practical synthesis for scale-up of such compounds was developed.     

Caspase-3 Cleaves Specific 19 S Proteasome Subunits in Skeletal Muscle Stimulating Proteasome Activity

With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically to increase the degradation of muscle proteins. Whether proteasome activity is also elevated in response to catabolic conditions is unknown. We find that caspase-3 increases proteasome activity in myotubes but not in myoblasts. This difference is related to the cleavage of specific 19 S proteasome subunits. In mouse muscle or myotubes, caspase-3 cleaves Rpt2 and Rpt6 increasing proteasome activity. In myoblasts, caspase-3 cleaves Rpt5 to decrease proteasome activity. To confirm the caspase-3 dependence, caspase-3 cleavage sites in Rpt2, Rpt6, or Rpt5 were mutated. This prevented the cleavage of these subunits by caspase-3 as well as the changes in proteasome activity. During differentiation of myoblasts to myotubes, there is an obligatory, transient increase in caspase-3 activity, accompanied by a corresponding increase in proteasome activity and cleavage of Rpt2 and Rpt6. Therefore, differentiation changes the proteasome type from sensitivity of Rpt5 to caspase-3 in myoblasts to sensitivity of Rpt2 and Rpt6 in myotubes. This novel mechanism identifies a feed-forward amplification that augments muscle proteolysis in catabolic conditions. Indeed, we found that in mice with a muscle wasting condition, chronic kidney disease, there was cleavage of subunits Rpt2 and Rpt6 and stimulation of proteasome activity.     

Proteasome inhibitors in cancer therapy

The ubiquitin proteasome pathway plays a critical role in regulating many processes in the cell which are important for tumour cell growth and survival. Inhibition of proteasome function has emerged as a powerful strategy for anti-cancer therapy. Clinical validation of the proteasome as a therapeutic target was achieved with bortezomib and has prompted the development of a second generation of proteasome inhibitors with improved pharmacological properties. This review summarises the main mechanisms of action of proteasome inhibitors in cancer, the development of proteasome inhibitors as therapeutic agents and the properties and progress of next generation proteasome inhibitors in the clinic.     

Proteasome inhibitors for cancer therapy

Proteasome, a large multicatalytic proteinase complex that plays an important role in processing of proteins, has been shown to possess multiple catalytic activities. Among its various activities, the 'chymotrypsin-like' activity of proteasome has emerged as the focus of drug discovery efforts in cancer therapy. Herein we report chiral boronate derived novel, potent, selective and cell-permeable peptidomimetic inhibitors 6 and 7 that displayed activity against various rodent and human tumor cell lines (in vitro).     

Proteasome inhibitors against amelanotic melanoma

The incidence of malignant melanoma, the most aggressive skin cancer, is increasing constantly. Despite new targeted therapies, the prognosis for patients with metastatic disease remains poor. Thus, there is a need for new combinational treatments, and antineoplastic agents potentially valuable in this approach are inhibitors of the ubiquitin-proteasome system (UPS). In this work, we analyze the cytotoxicity mechanisms of proteasome inhibitors (MG-132, epoxomicin, and lactacystin) in a specific form of melanoma which does not synthesize melanin—the amelanotic melanoma (Ab cells). We found that the most cytotoxic of the compounds tested was epoxomicin. Caspase-9 activation as well as cytochrome C and AIF release from mitochondria indicated that exposure to epoxomicin induced the mitochondrial pathway of apoptosis. Epoxomicin treatment also resulted in accumulation of Bcl-2 family members—proapoptotic Noxa and antiapoptotic Mcl-1, which were postulated as the targets for bortezomib in melanoma. Inhibition of caspases by BAF revealed that cell death was partially caspase-independent. We observed no cell cycle arrest preceding the apoptosis of Ab cells, even though cdk inhibitors p21^Cip1/Waf1 and p27^Kip1 were up-regulated. The cell cycle was blocked only after inactivation of caspases by the pan-caspase inhibitor BAF. In summary, this is the first study exploring molecular mechanisms of cell death induced by epoxomicin in melanoma. We found that Ab cells died on the mitochondrial pathway of apoptosis and also partially by the caspase-independent way of death. Apoptosis induction was fast and efficient and was not preceded by cell cycle arrest.     

蛋白酶体相关翻译后修饰的研究进展

蛋白酶体是具有多种蛋白水解酶活性的蛋白质降解系统,由于细胞内许多关键信号调控分子都是蛋白酶体的降解底物,因此蛋白酶体在细胞周期调控、基因表达、炎症反应等各种关键的生物学活动中都发挥着极其重要的调节作用。蛋白酶体的降解活性也同时受多种机制的调控,其中的翻译后修饰是蛋白酶体降解途径中一个不可忽视的方面。着重阐述蛋白酶体自身及其底物的几种重要的翻译后修饰,探讨最新的进展及其生物学意义。     

Proteasome activity in experimental diabetes

Numerous studies have indicated that oxidative stress contributes to the development and progression of diabetes and other related complications. Since the ubiquitin-proteasome pathway is involved in degradation of oxidized proteins, it is to be expected that alterations in proteasome-dependent proteolysis accompany diabetes. This paper focuses on the role of the proteasome in alloxan-induced experimental diabetes. The changes in proteasomal activity and oxidative stress indices (protein oxidation and lipid peroxidation) were evaluated. The obtained results revealed increased protein oxidation and lipid peroxidation, as well as alterations in proteasomal activities in diabetic rats. Our data indicates a significant decrease in chymotryptic-like activity; increased tryptic-like activity; and unchanged post-glutamyl peptide hydrolytic-like activity. These findings suggest the presence of oxidative stress in diabetes that appears to result in changes to the ubiquitin-proteasome pathway.     

Proteasome degradation: enter the substrate

Cells depend upon the regulated destruction of their various proteins to maintain homeostasis and change their metabolic state. A key component of this process is the proteasome - a large multisubunit protease whose catalytic sites are sequestered within a central chamber. Entry of substrates into proteasomes is regulated by activators and is generally thought to proceed sequentially, starting from one end of the substrate polypeptide. This conventional view is expanded by a recent paper, which indicates that some unfolded substrates can open the entrance to the proteolytic chamber in the absence of an activator and can enter the proteasome in a hairpin conformation to allow limited proteolysis of internal segments.     

蛋白酶体的结构与功能

蛋白酶体又称26S蛋白酶体,由催化颗粒（CP）和调节颗粒（RP）构成。CP含α亚基和β亚基各7个,且各自成环,组成圆桶样结构。其中α环位于圆桶两端,β环位于中间。RP分为三倍体ATP酶亚基（Rpt)和非ATP酶亚基（Rpn)。基高级结构分为盖部和基底部。基底部位于CP的两端,由6个Rpt和Rpn组成。盖部由其他Rpn构成,位于基底部外侧。蛋白酶体具有蛋白水解酶活性,Rpt水解ATP供能。蛋白酶体水解作用需要泛蛋白参加,位于盖部的Rpn10是其受体。部分RP还具有与蛋白质水解无关的其他作用,如参与DNA修复工作等。蛋白酶体经不同的加工可以转变为免疫蛋白酶体,橄榄球蛋白酶体和杂化蛋白酶体,各有其重要的生理功能。     

Homopiperazine Derivatives as a Novel Class of Proteasome Inhibitors with a Unique Mode of Proteasome Binding

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.