Defective Protein Degradation Research Articles

Abnormalities of NBR1, a novel autophagy-associated protein, in muscle fibers of sporadic inclusion-body myositis

Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-β and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.

A Conserved F Box Regulatory Complex Controls Proteasome Activity in Drosophila

The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated by islet amyloid polypeptide-induced UCH-L1 deficiency.

OBJECTIVEThe islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum–associated degradation/ubiquitin/proteasome system.RESEARCH DESIGN AND METHODSWe used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated.RESULTSWe report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis.CONCLUSIONSOur results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability.

Ubiquitin Proteasome Dysfunction in Human Hypertrophic and Dilated Cardiomyopathies

The ubiquitin proteasome system maintains a dynamic equilibrium of proteins and prevents accumulation of damaged and misfolded proteins, yet its role in human cardiac dysfunction is not well understood. The present study evaluated ubiquitin proteasome system function in human heart failure and hypertrophic cardiomyopathy (HCM). Proteasome function was studied in human nonfailing donor hearts, explanted failing hearts, and myectomy samples from patients with HCM. Proteasome proteolytic activities were markedly reduced in failing and HCM hearts compared with nonfailing hearts (P<0.01). This activity was partially restored after mechanical unloading in failing hearts (P<0.01) and was significantly lower in HCM hearts with pathogenic sarcomere mutations than in those lacking these mutations (P<0.05). There were no changes in the protein content of ubiquitin proteasome system subunits (ie, 11S, 20S, and 19S) or in active-site labeling of the 20S proteolytic subunit beta-5 among groups to explain decreased ubiquitin proteasome system activity in HCM and failing hearts. Examination of protein oxidation revealed that total protein carbonyls, 4-hydroxynonenylated proteins, and oxidative modification to 19S ATPase subunit Rpt 5 were increased in failing compared with nonfailing hearts. Proteasome activity in HCM and failing human hearts is impaired in the absence of changes in proteasome protein content or availability of proteolytic active sites. These data provide strong evidence that posttranslational modifications to the proteasome may account for defective protein degradation in human cardiomyopathies.

Deletion of the BH3-only protein puma protects motoneurons from ER stress-induced apoptosis and delays motoneuron loss in ALS mice

BH3-only proteins couple diverse stress signals to the evolutionarily conserved mitochondrial apoptosis pathway. Previously, we reported that the activation of the BH3-only protein p53-up-regulated mediator of apoptosis (Puma) was necessary and sufficient for endoplasmic reticulum (ER) stress- and proteasome inhibition-induced apoptosis in neuroblastoma and other cancer cells. Defects in protein quality control have also been suggested to be a key event in ALS, a fatal neurodegenerative condition characterized by motoneuron degeneration. Using the SOD1(G93A) mouse model as well as human post mortem samples from ALS patients, we show evidence for increased ER stress and defects in protein degradation in motoneurons during disease progression. Before symptom onset, we detected a significant up-regulation of Puma in motoneurons of SOD1(G93A) mice. Genetic deletion of puma significantly improved motoneuron survival and delayed disease onset and motor dysfunction in SOD1(G93A) mice. However, it had no significant effect on lifespan, suggesting that other ER stress-related cell-death proteins or other factors, such as excitotoxicity, necrosis, or inflammatory injury, may contribute at later disease stages. Indeed, further experiments using cultured motoneurons revealed that genetic deletion of puma protected motoneurons against ER stress-induced apoptosis but showed no effect against excitotoxic injury. These findings demonstrate that a single BH3-only protein, the ER stress-associated protein Puma, plays an important role during the early stages of chronic neurodegeneration in vivo.

Lafora disease proteins malin and laforin are recruited to aggresomes in response to proteasomal impairment

Lafora disease (LD), an autosomal recessive neurodegenerative disorder, is characterized by the presence of cytoplasmic polyglucosan inclusions known as Lafora bodies in several tissues including the brain. Laforin, a protein phosphatase, and malin, an ubiquitin ligase, are two of the proteins that are known to be defective in LD. Malin interacts with laforin and promotes its polyubiquitination and degradation. Here we show that malin and laforin co-localize in endoplasmic reticulum (ER) and that they form centrosomal aggregates when treated with proteasomal inhibitors in both neuronal and non-neuronal cells. Laforin/malin aggregates co-localize with gamma-tubulin and cause redistribution of alpha-tubulin. These aggregates are also immunoreactive to ubiquitin, ubiquitin-conjugating enzyme, ER chaperone and proteasome subunits, demonstrating their aggresome-like properties. Furthermore, we show that the centrosomal aggregation of laforin and malin is dependent on the functional microtubule network. Laforin and malin form aggresome when expressed together or otherwise, suggesting that the two proteins are recruited to the centrosome independent of each other. Taken together, our results suggest that the centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin or laforin may thus lead to increased levels of misfolded and/or target proteins, which may eventually affect the physiological processes of the neuron. Thus, defects in protein degradation and clearance are likely to be the primary trigger in the physiopathology of LD.

Characterisation of the nascent polypeptide-associated complex in fission yeast

The nascent polypeptide-associated complex (NAC) is an abundant and phylogenetically conserved protein complex. It is composed of two subunits and interacts with nascent polypeptide chains emerging from the ribosome. It has been proposed to protect the nascent chains from premature interaction with other cell proteins, but has also been found to associate with DNA junctions, and to be involved in other processes including transcription regulation and mitochondrial protein import.Here, we characterize NAC in fission yeast. We find that NAC is associated with ribosomes, while a significant fraction remains in a free form. The NAC alpha subunit contains a ubiquitin-associated (UBA) domain, which is found in several proteins involved in the ubiquitin-proteasome pathway for protein degradation. However, NAC does not associate with ubiquitin chains and mutants lacking NAC did not exhibit any obvious defects in protein degradation. Accordingly, we find that the NAC UBA domain belongs to an ancient and distinct subgroup of the UBA family. In contrast to the situation with budding yeast, fission yeast cells devoid of NAC were not temperature sensitive. However, they displayed resistance to the amino acid analogue canavanine, in accordance with the idea that NAC is involved in protein quality control.

A dominant-negative form of the E3 ubiquitin ligaseCullin-1disrupts the correct allocation of cell fate in the neural crest lineage

Selective protein degradation is an efficient and rapid way of terminating protein activity. Defects in protein degradation are associated with a number of human diseases, including potentially DiGeorge syndrome, which is characterised by abnormal development of the neural crest lineage during embryogenesis. We describe the identification of Xenopus Cullin-1, an E3 ubiquitin ligase, and show that blocking the function of endogenous Cullin-1 leads to pleiotropic defects in development. Notably, there is an increased allocation of cells to a neural crest fate and within this lineage, an increase in melanocytes at the expense of cranial ganglia neurons. Most of the observed effects can be attributed to stabilisation ofβ-catenin, a known target of Cullin-1-mediated degradation from other systems. Indeed, we show that blocking the function of Cullin-1leads to a decrease in ubiquitinated β-catenin and an increase in totalβ-catenin. Our results show that Cullin-1-mediated protein degradation plays an essential role in the correct allocation of neural crest fates during embryogenesis.

A synthetic defect in protein degradation caused by loss of Ufd4 and Rad23

The UFD (ubiquitin fusion degradation) pathway is responsible for multiubiquitination of the fusion proteins that bear a “non-removable” N-terminal ubiquitin moiety. Previous reports have shown that the UFD pathway is conserved from yeast to human. The essential elements of the UFD pathway have also been identified in Saccharomyces cerevisiae. These studies, however, are limited to use of engineered UFD substrates. The biological significance of the UFD pathway remains unknown. Here we demonstrate that Ufd4, the E3 component of the UFD pathway, is involved in controlling the degradation of Rad4, a nucleotide excision repair protein. Moreover, simultaneous loss of Ufd4 and Rad23 exhibits a synthetic inhibitory effect on Rad4 degradation, presenting the first example that a UBA/UBL-domain protein functionally overlaps with a ubiquitin ligase in determining the turnover rate of a protein substrate. The current work also provides a direction for further investigation of the physiological functions of the UFD pathway.

Functional Analysis of Rpn6p, a Lid Component of the 26 S Proteasome, Using Temperature-sensitive rpn6 Mutants of the Yeast Saccharomyces cerevisiae

Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.

Neurobiology of alpha-synuclein.

Alpha-synuclein is an abundant neuronal protein that has been linked both to normal synaptic function and to neurodegeneration. Most significantly, mutations in the gene encoding for alpha-synuclein are responsible for Parkinson's disease (PD) in rare familial cases, and the aggregated protein is a major component of Lewy bodies found in sporadic PD. Here we review recent data regarding the structure, the regulation at the transcriptional and posttranslational level, and the physiologic and aberrant functions of alpha-synuclein. We focus in particular on the fibrilization potential of alpha-synuclein and on its link with defects in protein degradation.

Proteolysis, the ubiquitin-proteasome system, and renal diseases.

Protein degradation is a critical process for the growth and function of cells. Proteolysis eliminates abnormal proteins, controls many cellular regulatory processes, and supplies amino acids for cellular remodeling. When substrates of proteolytic pathways are poorly recognized or there is mistiming of proteolysis, profound changes in cell function can occur. Based on these potential problems, it is not surprising that alterations in proteolytic enzymes/cofactors or in the structure of protein substrates that render them more or less susceptible to degradation are responsible for disorders associated with kidney cell malfunctions. Multiple pathways exist for protein degradation. The best-described proteolytic system is the ubiquitin-proteasome pathway, which requires ATP and degrades the bulk of cellular and some membrane proteins. This review will survey examples of renal abnormalities that are associated with defective protein degradation involving the ubiquitin-proteasome pathway. Loss of muscle mass associated with chronic renal failure, von Hippel-Lindau disease, Liddle syndrome, and ischemic acute renal failure will be discussed. These examples are indicative of the diverse roles of the ubiquitin-proteasome system in renal-associated pathological conditions.

General aspects of neurodegeneration.

Neurodegenerative diseases are morphologically featured by progressive cell loss in specific vulnerable neuronal populations of the central nervous system, often associated with cytoskeletal protein aggregates forming intracytoplasmic and/or intranuclear inclusions in neurons and/or glial cells. Most neurodegenerative disorders are now classified either according to the hitherto known genetic mechanisms or to the major components of their cellular protein inclusions. The major basic processes inducing neurodegeneration are considered multifactorial ones caused by genetic, environmental, and endogenous factors. They include abnormal protein dynamics with defective protein degradation and aggregation, many of them related to the ubiquitin-proteasomal system, oxidative stress and free radical formation, impaired bioenergetics and mitochondrial dysfunctions, and "neuroinflammatory" processes. These mechanisms that are usually interrelated in complex vitious circles finally leading to programmed cell death cascades are briefly discussed with reference to their pathogenetic role in many, albeit diverse neurodegenerative diseases, like Alzheimer disease, synucleinopathies, tauopathies, and polyglutamine disorders. The impact of protein inclusions on cell dysfunction, activation or prevention of cell death cascades are discussed, but the molecular basis for the underlying disease mechanisms remains to be elucidated.

Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson’s disease

We have detected granular and filamentous inclusions that are alpha-synuclein- and ubiquitin-immunoreactive in the cytoplasm of dopaminergic and cortical neurons of C57/black mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid. The immunoreactive aggregates only become evident several weeks after large-scale dopaminergic cell death and a downregulation of α-synuclein gene expression. Numerous lipofuscin granules accumulate α-synuclein in the nigral and limbic cortical neurons of treated mice. These data provide evidence that insoluble proteins, such as α-synuclein, build up as granular and filamentous inclusions in dopaminergic neurons that survive the initial toxic MPTP insult. They further suggest that defective protein degradation rather than altered gene expression underlies deposition of α-synuclein and that abundant lysosomal compartments are present to seal off the potentially toxic material.

Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation.

Srp1p (importin alpha) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. The srp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11 and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of the srp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressing srp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-beta-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not in srp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 in srp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins.

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.

Mutations in the Yeast Proteasome β-Type Subunit Pre3 Uncover Position-dependent Effects on Proteasomal Peptidase Activity and in Vivo Function

Proteasomes are highly complex proteases responsible for selective protein degradation in the eukaryotic cell. 26 S proteasomes consist of two regulatory 19 S cap complexes and the 20 S proteasome, which acts as the proteolytic core module. We isolated six mutants of the yeast Saccharomyces cerevisiae containing mutations in the 20 S proteasome beta-type subunit Pre3. Three mutations (pre3-2, pre3-3, and pre3-5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype. However, the pre3-2 mutation strengthened phenotypes induced by other 20 S proteasomal mutations, indicating that the peptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions. The other three mutations (pre3-1, pre3-4, and pre3-6) are located at diverse sites of the Pre3 protein and caused multiple defects in proteasomal peptide cleaving activities. pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g. fructose-1,6-bisphosphatase. In addition, pre3-1 and pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.

Yeast cycloheximide-resistant crl mutants are proteasome mutants defective in protein degradation.

In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3-2, had been found to be suppressed by a mutation, SCL1-1, which resided in an alpha-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3-2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1-1 suppressor mutation causes partial restoration of crl3-2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1-1, pre2-1, pre3-1, pre4-1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.

Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces cerevisiae.

Acidification inside vacuoles has been shown to play a key role in a number of physiologically important cellular events. We studied the role of vacuolar membrane H(+)-ATPase in the autophagic process of Saccharomyces cerevisiae. Mutants lacking VMA genes which encode their subunits of the vacuolar H(+)-ATPase accumulated autophagic bodies in vacuoles on starvation. vma mutants also had a defect in protein degradation induced by starvation. In vma mutants, the activities of vacuolar proteases were remarkably lower than those of the wild-type. Overexpression of vacuolar proteases did not overcome the defect in the disintegration of autophagic bodies in vma mutant, even the overexpressed proteinase A and proteinase B being substantially localized to the vacuolar compartment and undergoing proper proteolytic maturation. Our results showed that the acidification of vacuoles is not required for the formation and delivery of autophagosomes to vacuoles, but is essential for the disintegration of autophagic bodies.