Comparison of human COP9 signalosome and 26S proteasome ‘lid’

The human core COP9 signalosome consists of eight subunits which have been identified, cloned and sequenced. The components of COP9 signalosome possess homologies with eight non-ATPase regulatory subunits of the 26S proteasome. These polypeptides of the 19S regulator form a reversibly binding subcomplex called the lid. We isolated the lid from human red blood cells and compared it with the COP9 signalosome complex. In addition to the non-ATPase regulatory polypeptides, we found a high molecular mass ATPase copurifying with the human lid. The COP9 signalosome-associated kinase activity is either not at all or only weakly affected by common kinase inhibitors such as 1-(5-Isoquinolinesulfonyl)-2-methyl-piperazine (H7), 5,6-dichloro-1--D-ribofuranosyl-benzimidazole (DRB) or Wortmannin. Curcumin, a tumor suppressor and effector of AP-1 activation, is a potent inhibitor of the COP9 signalosome kinase activity with a Ki of about 10 M. Since curcumin is known as an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway acting upstream of the MAP kinase kinase kinase level, one site of action of the COP9 signalosome might be proximal to regulators on that level.     

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.     

The CBM signalosome: Potential therapeutic target for aggressive lymphoma?

The CBM signalosome plays a pivotal role in mediating antigen-receptor induced NF-κB signaling to regulate lymphocyte functions. The CBM complex forms filamentous structure and recruits downstream signaling components to activate NF-κB. MALT1, the protease component in the CBM complex, cleaves key proteins in the feedback loop of the NF-κB signaling pathway and enhances NF-κB activation. The aberrant activity of the CBM complex has been linked to aggressive lymphoma. Recent years have witnessed dramatic progresses in understanding the assembly mechanism of the CBM complex, and advances in the development of targeted therapy for aggressive lymphoma. Here, we will highlight these progresses and give an outlook on the potential translation of this knowledge from bench to bedside for aggressive lymphoma patients.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.     

What curves alpha-solenoids? Evidence for an alpha-helical toroid structure of Rpn1 and Rpn2 proteins of the 26 S proteasome

The alpha-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved alpha-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an alpha-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an "antechamber" of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.     

Trails: an alternative to the field demonstration?

For many students, the traditional field demonstration has several disadvantages. The trail is suggested as an alternative technique, which may be appropriate for students who are not biology specialists, and for senior pupils. The advantages of this method include simpler presentation of information and questions, freedom for the tutor to assist weaker or more questioning students, flexibility in the timing of the exercise, and the need for careful preparation. The use of trails in assessment is suggested. Examples from trails used with Landscape Architecture students are given.     

Unemployment rates: an alternative to the Jarman index?

OBJECTIVE--To compare the Jarman index with alternative deprivation measures with regard to its usefulness to district health authorities as an indicator of need at small area level. DESIGN--The Jarman index (UPA (8)), Townsend''s index of material deprivation, the Scottish Development Department''s index, the Department of the Environment''s basic index, and unemployment rates were compared in respect of their correlation with measures of morbidity by electoral ward in a typical English district health authority. Measures of morbidity comprised standardised mortality ratios, admission rates (standardised and non-standardised), and permanent sickness rates. Spearman rank correlation coefficients were calculated for each combination of measures and were then ranked for each of the deprivation indices. SETTING--The 59 electoral wards of the Central Nottinghamshire Health Authority. RESULTS--The Jarman index consistently ranked lower in respect of its correlation with measures of morbidity than did the other deprivation measures. Current unemployment rates correlated well with morbidity measures, in particular with hospital admission rates, with correlations ranging from 0.669 to 0.830 for average and standardised all age admission rates. CONCLUSIONS--The Jarman index seems to be the least appropriate of these indices for health authority use. Unemployment rate merits further consideration as a simple, up to date marker for deprivation and consequent need for health service provision.     

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.     

Lepidopteran cells,an alternative for the production of recombinant antibodies?

Monoclonal antibodies are used with great success in many different therapeutic domains. In order to satisfy the growing demand and to lower the production cost of these molecules, many alternative systems have been explored. Among them, the baculovirus/insect cells system is a good candidate. This system is very safe, given that the baculoviruses have a highly restricted host range and they are not pathogenic to vertebrates or plants. But the major asset is the speed with which it is possible to obtain very stable recombinant viruses capable of producing fully active proteins whose glycosylation pattern can be modulated to make it similar to the human one. These features could ultimately make the difference by enabling the production of antibodies with very low costs. However, efforts are still needed, in particular to increase production rates and thus make this system commercially viable for the production of these therapeutic agents.     

The pipe and the pinwheel: Is pressure an effective stimulus for the 9 + 0 primary cilium?

Almost universally, the effective stimulus for mammalian 9+0 primary cilia has been taken to be bending. In this article I point out that in several physiological contexts there is great advantage in detecting pressure differences across the cell wall, i.e. axially directed forces pushing fluid to and fro through the hollow cilium and its basal body beneath. The form of the cilium--a fluid-filled pipe that connects to an intricate pinwheel-shaped basal body--is well configured for detecting fluid flow. Pressure-detection calls for compressible elements within the cell, but it effectively matches form and function in a range of cases. The "pipe and pinwheel" scheme suggests that the bulbous swellings commonly found near the tip of some primary cilia are compliant, pressure-sensitive elements which act like the bulb of an eye-dropper. In looking exclusively at the bending of cilia, we might be missing aspects of a dual-stimulus system.     

Lepidopteran cells,an alternative for the production of recombinant antibodies?

Monoclonal antibodies are used with great success in many different therapeutic domains. In order to satisfy the growing demand and to lower the production cost of these molecules, many alternative systems have been explored. Among them, the baculovirus/insect cells system is a good candidate. This system is very safe, given that the baculoviruses have a highly restricted host range and they are not pathogenic to vertebrates or plants. But the major asset is the speed with which it is possible to obtain very stable recombinant viruses capable of producing fully active proteins whose glycosylation pattern can be modulated to make it similar to the human one. These features could ultimately make the difference by enabling the production of antibodies with very low costs. However, efforts are still needed, in particular to increase production rates and thus make this system commercially viable for the production of these therapeutic agents.     

The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes

Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28αβ regulator all exhibited substantially increased de novo synthesis during adaptation over 24?h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28αβ-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28αβ regulator blocked 50-100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.     

Probiotics and pharmabiotics: alternative medicine or an evidence-based alternative?

That commensal bacteria play an important role in human health is beyond doubt, and it is now widely accepted that humans function as super organisms, whose collective metabolic potential exceeds the sum of our individual eukaryotic and prokaryotic components. However, while it is has been established that the prokaryotic component of the human superorganism is amenable to manipulation by chemotherapeutic, dietary or microbial interventions, the significance of such alterations in terms of human health or well being is less well established. Prebiotics (non- digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system) and probiotics (live microorganisms that when administered in adequate amounts, confer a health benefit on the host) are often bracketed among 'alternative' approaches to influencing human health, such as homeopathy, naturopathy, acupuncture and hypnotherapy. Others believe that prebiotics and probiotics have proven their effectiveness in properly conducted, clinically controlled human trials and therefore can be considered as evidence-based alternatives or adjuncts to conventional medicines. My journey from a position of total skepticism to 'reluctant convert' is the basis of this article, which should not be considered in any sense as a review of the literature but simply a personal account of this transition. While I am not bent on converting other doubters, I will recount some of the thought processes and evidence that has helped to form my current opinion.     

The Arabidopsis clock: time for an about-face?

Three recent studies have altered thinking on the role of TIMING OF CAB EXPRESSION 1 (TOC1), a core element in the plant circadian oscillator.     

The COP9 complex: a link between photomorphogenesis and general developmental regulation?

Light signals perceived by photoreceptors are transduced into the nucleus to regulate gene expression and development. The COP9 protein complex localized in the nucleus has a central role in modulating light-regulated development, and may have a role in regulating development in general in all organisms. This review considers recent advances in the study of the COP9 complex and its sub-units in plant and animal systems, and discusses the possible functional implications.