Alzheimer's disease meets the ubiquitin-proteasome system

Ubiquitin-positive deposits are histopathologically found in patients with Alzheimer's disease (AD). It is not understood why ubiquitin is accumulated in intra- and extra-cellular deposits or how it is involved in AD pathogenesis. Interestingly, recent evidence, including studies of E2-25K/Hip-2, has elucidated the molecular mechanism of the ubiquitin-proteasome system (UPS) malfunction in AD. The neurotoxicity and proteasome inhibition by Abeta, a main cause of AD pathogenesis, are mediated by increased E2-25K/Hip-2 in the brains of patients with AD. Furthermore, E2-25K/Hip-2 is required for the neurotoxicity that is mediated by a ubiquitin B mutant (UBB+1), which is a potent inhibitor of proteasomes that is found in patients with AD. Intensive research is required to identify the components of the UPS that are involved in AD pathogenesis.     

Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ub^ext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ub^ext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ub^ext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ub^ext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ub^ext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration.     

Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration

Local axon degeneration is a common pathological feature of many neurodegenerative diseases and peripheral neuropathies. While it is believed to operate with an apoptosis-independent molecular program, the underlying molecular mechanisms are largely unknown. In this study, we used the degeneration of transected axons, termed "Wallerian degeneration," as a model to examine the possible involvement of the ubiquitin proteasome system (UPS). Inhibiting UPS activity by both pharmacological and genetic means profoundly delays axon degeneration both in vitro and in vivo. In addition, we found that the fragmentation of microtubules is the earliest detectable change in axons undergoing Wallerian degeneration, which among other degenerative events, can be delayed by proteasome inhibitors. Interestingly, similar to transected axons, degeneration of axons from nerve growth factor (NGF)-deprived sympathetic neurons could also be suppressed by proteasome inhibitors. Our findings suggest a possibility that inhibiting UPS activity may serve to retard axon degeneration in pathological conditions.     

Functions of E3 Ubiquitin Ligase Hyd in Drosophila Tissues

Molecular Biology - The ubiquitin–proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle,...     

The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.     

The ubiquitin‐proteasome system in plant responses to environments

The ubiquitin‐proteasome system (UPS) is a rapid regulatory mechanism for selective protein degradation in plants and plays crucial roles in growth and development. There is increasing evidence that the UPS is also an integral part of plant adaptation to environmental stress, such as drought, salinity, cold, nutrient deprivation and pathogens. This review focuses on recent studies illustrating the important functions of the UPS components E2s, E3s and subunits of the proteasome and describes the regulation of proteasome activity during plant responses to environment stimuli. The future research hotspots and the potential for utilization of the UPS to improve plant tolerance to stress are discussed.     

Development of inhibitors in the ubiquitination cascade

The ubiquitin proteasome system (UPS) is essential in regulating myriad aspects of protein functions. It is therefore a fundamentally important regulatory mechanism that impacts most if not all aspects of cellular processes. Indeed, malfunction of UPS components is implicated in human diseases such as neurodegenerative and immunological disorders and many cancers. The success of proteasome inhibitors in cancer therapy suggests that modulating enzymes in the ubiquitination cascade would be clinically important for therapeutic benefits. In this review, we summarize advances in developing inhibitors of a variety of UPS components. In particular, we highlight recent work done on the protein engineering of ubiquitin as modulators of the UPS, a novel approach that may shed light on innovative drug discovery in the future.     

Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway

The ubiquitin proteasome system (UPS) comprises hundreds of different conjugation/deconjugation enzymes and multiple receptors that recognize ubiquitylated proteins. A formidable challenge to deciphering the biology of ubiquitin is to map the networks of substrates and ligands for components of the UPS. Several different receptors guide ubiquitylated substrates to the proteasome, and neither the basis for specificity nor the relative contribution of each pathway is known. To address how broad of a role the ubiquitin receptor Rpn10 (S5a) plays in turnover of proteasome substrates, we implemented a method to perform quantitative analysis of ubiquitin conjugates affinity-purified from experimentally perturbed and reference cultures of Saccharomyces cerevisiae that were differentially labeled with 14N and 15N isotopes. Shotgun mass spectrometry coupled with relative quantification using metabolic labeling and statistical analysis based on q values revealed ubiquitylated proteins that increased or decreased in level in response to a particular treatment. We first identified over 225 candidate UPS substrates that accumulated as ubiquitin conjugates upon proteasome inhibition. To determine which of these proteins were influenced by Rpn10, we evaluated the ubiquitin conjugate proteomes in cells lacking either the entire Rpn10 (rpn10delta) (or only its UIM (ubiquitin-interacting motif) polyubiquitin-binding domain (uimdelta)). Twenty-seven percent of the UPS substrates accumulated as ubiquitylated species in rpn10delta cells, whereas only one-fifth as many accumulated in uimdelta cells. These findings underscore a broad role for Rpn10 in turnover of ubiquitylated substrates but a relatively modest role for its ubiquitin-binding UIM domain. This approach illustrates the feasibility of systems-level quantitative analysis to map enzyme-substrate networks in the UPS.     

Illuminating the ubiquitin/proteasome system

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.     

The ubiquitin/26S proteasome system in plant–pathogen interactions: a never-ending hide-and-seek game

The ubiquitin/26S proteasome system (UPS) plays a central role in plant protein degradation. Over the past few years, the importance of this pathway in plant–pathogen interactions has been increasingly highlighted. UPS is involved in almost every step of the defence mechanisms in plants, regardless of the type of pathogen. In addition to its proteolytic activities, UPS, through its 20S RNase activity, may be part of a still unknown antiviral defence pathway. Strikingly, UPS is not only a weapon used by plants to defend themselves, but also a target for some pathogens that have evolved mechanisms to inhibit and/or use this system for their own purposes. This article attempts to summarize the current knowledge on UPS involvement in plant–microbe interactions, a complex scheme that illustrates the never-ending arms race between hosts and microbes.     

Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation

Substrate-specific protein degradation mediated by the ubiquitin proteasome system (UPS) is crucial for the proper function of the cell. Proteins are specifically recognized and ubiquitinated by the ubiquitin ligases (E3s) and are then degraded by the proteasome. BTB proteins act as the substrate recognition subunit that recruits their cognate substrates to the Cullin 3-based multisubunit E3s. Recently, it was reported that missense mutations in KLHL7, a BTB-Kelch protein, are related to autosomal dominant retinitis pigmentosa (adRP). However, the involvement of KLHL7 in the UPS and the outcome of the adRP causative mutations were unknown. In this study, we show that KLHL7 forms a dimer, assembles with Cul3 through its BTB and BACK domains, and exerts E3 activity. Lys-48-linked but not Lys-63-linked polyubiquitin chain co-localized with KLHL7, which increased upon proteasome inhibition suggesting that KLHL7 mediates protein degradation via UPS. An adRP-causative missense mutation in the BACK domain of KLHL7 attenuated only the Cul3 interaction but not dimerization. Nevertheless, the incorporation of the mutant as a heterodimer in the Cul3-KLHL7 complex diminished the E3 ligase activity. Together, our results suggest that KLHL7 constitutes a Cul3-based E3 and that the disease-causing mutation inhibits ligase activity in a dominant negative manner, which may lead to the inappropriate accumulation of the substrates targeted for proteasomal degradation.     

Ubiquitin-proteasome system components are upregulated during intestinal regeneration

The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence suggests that the UPS plays a regulatory role in regeneration processes. Here, we explore the possibility that the UPS is involved during intestinal regeneration of the sea cucumber Holothuria glaberrima. These organisms can regenerate most of their digestive tract following a process of evisceration. Initially, we identified components of H. glaberrima UPS, including sequences for Rpn10, β3, and ubiquitin-RPL40. Predicted proteins from the mRNA sequences showed high degree of conservation that ranged from 60% (Rpn10) to 98% (Ub-RPL40). Microarrays and RT-PCR experiments showed that these genes were upregulated during intestinal regeneration. In addition, we demonstrated expression of alpha 20S proteasome subunits and ubiquitinated proteins during intestinal regeneration and detected them in the epithelium and connective tissue of the regenerating intestine. Finally, the intestinal regeneration was altered in animals treated with MG132, a proteasome inhibitor. These findings support our contention that proteasomes are playing an important role during intestinal regeneration.     

The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.     

The Ubiquitin–Proteasome System in Retinal Health and Disease

The ubiquitin–proteasome system (UPS) is the main intracellular pathway for modulated protein turnover, playing an important role in the maintenance of cellular homeostasis. It also exerts a protein quality control through degradation of oxidized, mutant, denatured, or misfolded proteins and is involved in many biological processes where protein level regulation is necessary. This system allows the cell to modulate its protein expression pattern in response to changing physiological conditions and provides a critical protective role in health and disease. Impairments of UPS function in the central nervous system (CNS) underlie an increasing number of genetic and idiopathic diseases, many of which affect the retina. Current knowledge on the UPS composition and function in this tissue, however, is scarce and dispersed. This review focuses on UPS elements reported in the retina, including ubiquitinating and deubiquitinating enzymes (DUBs), and alternative proteasome assemblies. Known and inferred roles of protein ubiquitination, and of the related, SUMO conjugation (SUMOylation) process, in normal retinal development and adult homeostasis are addressed, including modulation of the visual cycle and response to retinal stress and injury. Additionally, the relationship between UPS dysfunction and human neurodegenerative disorders affecting the retina, including Alzheimer's, Parkinson's, and Huntington's diseases, are dealt with, together with numerous instances of retina-specific illnesses with UPS involvement, such as retinitis pigmentosa, macular degenerations, glaucoma, diabetic retinopathy (DR), and aging-related impairments. This information, though still basic and limited, constitutes a suitable framework to be expanded in incoming years and should prove orientative toward future therapy design targeting sight-affecting diseases with a UPS underlying basis.     

Ubiquitin Signaling Regulates RNA Biogenesis,Processing, and Metabolism

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.     

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin–proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.