The proteasome is a large multiprotein complex that degrades unwanted cellular proteins. The mechanisms that control this protein-eating machine are being uncovered Inside eukaryotic cells there is a massive protein complex called the proteasome whose raison d'être is to remove unnecessary proteins by breaking them down into short peptides. The proteasome is thus responsible for an important aspect of cellular regulation because the timely and controlled proteolysis of key cellular factors regulates numerous biological processes such as cell cycle, differentiation, stress response, neuronal morphogenesis, cell surface receptor modulation, secretion, DNA repair, transcriptional regulation, long-term memory, circadian rhythms, immune response, and biogenesis of organelles . With thWith so many proteins to target for degradation, the activity of the proteasome is subject to multiple levels of regulation. In the overwhelming majority of cases, selected proteins are first “labeled” by the addition of several copies of a small protein tag called ubiquitin and are thus targeted for degradation in the proteasome . The ubiInterestingly, ubiquitination is a reversible process. Even when a protein has been tagged with ubiquitin, its fate is not sealed—specific hydrolytic enzymes called deubiquitinases can remove the ubiquitin label intact . By deubThe proteasome itself is made up of a multiprotein core particle (CP) where proteolysis occurs and a separate multiprotein regulatory particle (RP) that recognizes and prepares substrates for degradation by the CP. A base subcomplex of the RP is pivotal in anchoring polyubiquitin chains during this process, either directly or via auxiliary ubiquitin-binding proteins . The basAn intriguing evolutionary and structural relationship between the proteasome lid and an independent complex, the COP9 signalosome (CSN), may shed light on their respective roles in regulated protein degradation. Both are made up of eight homologous protein subunits that contain similar structural and functional motifs. While a lot is still unknown, the CSN appears to mediate responses to signals  in a manner that is intimately linked to the ubiquitin–proteasome system. This is accomplished, for instance, by suppressing ubiquitin E3 ligase activity or interacting with various components of the pathway . In partSurprisingly, the proteasome itself harbors intrinsic deubiquitination activity . Moreove+/JAMM motif garnered great attention. The trademark of the MPN+/JAMM motif is a consensus sequence E—HxHx(7)Sx(2)D that bears some resemblance to the active site of zinc metalloproteases. Members of this family were predicted to be hydrolytic enzymes, some of which are specific for removal of ubiquitin or ubiquitin-like domains from their targets  and the auxiliary deubiquitinating enzymes (situated in the regulatory particle) is clear-cut: the latter cleave between ubiquitin domains, while the core proteolytic subunits process the target protein itself . HoweverOnce bound to the proteasome, a polyubiquitinated substrate can be unfolded by the RP and irreversibly translocated into the CP. It has been proposed that long polyubiquitin chains commit a substrate to unfolding and degradation by the proteasome, whereas short chains are poor substrates because they are edited by deubiquitinating enzymes, resulting in premature substrate release . ExtendeIdentifying Rpn11 and Csn5 as members of a novel class of metallohydrolases immediately elevates them into promising “drugable” candidates. Undoubtedly, the molecular structures deciphered by the groups of Deshaies  and Bycr


Proteins may be the workhorse of the cell, but when a cell can synthesize one protein in a matter of minutes, chances are some will become obsolete. Though many proteins put in years of productive service, others quickly outlive their usefulness and can even damage the cell. Proteins that help form bone and muscle, for example, function for years while regulators of mitosis and cell proliferation might finish their jobs in seconds. Such short-timers are soon tagged as superfluous by a chain of small proteins called ubiquitin, which marks the proteins for degradation in an enzyme called the proteasome. Once in the proteasome, these proteins are broken down and can then be recycled for more productive ventures.A massive structure by cellular standards, the proteasome consists of multiple subunits, including a cylindrical core particle called 20S, which catalyzes degradation, and regulatory complexes called 19S caps, which form lid and base structures at both ends of the core. While the structure and biomechanics of the 20S core have been well characterized, much less is known about the functional mechanics of the regulatory complexes. The lid--base complex recognizes only ubiquitin-tagged proteins, which are then unfolded so they can enter the proteasome. But first ubiquitin chains must be detached from the protein, a task performed by an enzyme in the proteasome called Rpn11 isopeptidase. How the lid–base complex removes the ubiquitin tag, unfolds the protein, and shuttles it into the proteasome's core is not clear. Now Raymond Deshaies and colleagues present the structure of a homolog of the 19S lid's isopeptidase enzymatic center and provide new insights into these questions.The proteasome Rpn11 subunit contains a key region called the JAMM motif, which Deshaies' lab has shown previously is required for the proteasome to remove ubiquitin tags. For the work discussed in this paper, the researchers set out to understand how the proteasome strips off ubiquitin tags from proteins about to be destroyed by determining the three-dimensional structure of the JAMM motif.Archaeoglobus fulgidus. After determining the structure of the JAMM protein , the researchers discovered that AfJAMM looks nothing like the well-known deubiquitinating enzymes. But the arrangement of a set of amino acids that binds a zinc ion and forms the proposed active site of AfJAMM does resemble that found in a well-known protein-degrading metalloprotease called thermolysin, even though in other respects AfJAMM and thermolysin have very different features. The researchers mutated amino acid residues in another JAMM protein called Csn5  and found that the residues are indeed important for Csn5 function. These results suggest that JAMM does indeed represent a novel family of metalloproteases.The researchers tested many genes to look for a JAMM-containing protein that would crystallize properly and found one in the heat-loving prokaryote As for the wider function of JAMM proteins, the researchers speculate that these proteins are likely to be involved in a variety of important regulatory systems since they appear in life forms that lack ubiquitin and ubiquitin-like proteins. The crystal structure reported in this paper will provide a valuable tool for investigations into the underlying structural and functional mechanisms of these enzymes. And it may have important therapeutic implications. Proteasome inhibitors are promising anticancer therapies—fighting cancer by blocking machinery required by rapidly dividing cells. In the hopes of developing more targeted therapies, scientists are trying to fine-tune their control of the ubiquitin system and the proteasome. Inhibiting the JAMM domain of enzymes like Csn5, which remove ubiquitin-like tags from proteins upstream of the proteasome, for example, might just do the trick.


The polypeptide ubiquitin is used in many processes as different as endocytosis, multivesicular body formation, and regulation of gene transcription. Conjugation of a single ubiquitin moiety is typically used in these processes. A polymer of ubiquitin moieties is required for tagging proteins for proteasomal degradation. Besides its role in protein degradation, ubiquitin is also engaged as mono- or polymer in intracellular signalling and DNA repair. Since free ubiquitin is present in limiting amounts in cells, changes in the demands for ubiquitin in any of these processes is likely to indirectly affect other ubiquitin modifications. For example, proteotoxic stress strongly increases poly-ubiquitylated proteins at the cost of mono-ubiquitylated histones resulting in chromatin remodelling and altered transcription. Here we discuss the interconnection between ubiquitin-dependent processes and speculate on the functional significance of the ubiquitin equilibrium as a signalling route translating cellular stress into molecular responses. Ubiquitin is abundantly expressed in eukaryotes and can be found in all cell types and tissues with up to 10per cell -6; Figurper cell . A largeUbiquitin contains seven lysine residues, all of which can be used to form poly-ubiquitin chains . Poly-ubA cascade of different classes of enzymes is required for identification and ubiquitylation of proteins . TheSeveral groups have described the existence of various forms of ubiquitin in eukaryotic cells, including free ubiquitin molecules, mono- and poly-ubiquitylated proteins -34. ThesTo monitor the amount of free ubiquitin in living cells the nuclear pore was used as a molecular sieve in a FLIP (Fluorescence Loss In Photobleaching) protocol wherein the GFP fluorescence in either the complete nucleus or cytoplasm was bleached and the effect of GFP-Ub fluorescence in the other compartment was measured. The rationale behind this approach is that proteins up to approximately 50 kDa can passively diffuse through the nuclear pore, whereas larger species (like conjugated ubiquitin) are excluded . The FLIGiven the availability of a small pool of free ubiquitin, free ubiquitin has to be replenished continuously by DUBs. This ubiquitin cycle is essential to supply ubiquitin to substrates in a multitude of nuclear and cytosolic processes Figure . But whaIn principle, histone deubiquitylation could be the result of enhanced deubiquitylation activity following proteotoxic stress. This was assayed using photo-activated GFP-Ub in a protocol where the fate (i.e. the off-rate) of ubiquitin fluorescence was followed in one half of the nucleus. Proteotoxic stress did not affect the off-rate of fluorescent (photoactivated) GFP-Ub from histones indicating the DUBs were not activated by proteotoxic stress . AntibodUbiquitylation of histones is one of the major (and largest) modifications in chromatin. This modification is in mammalian cells mainly found on the core histone H2A. Approximately 5–15% of histone H2A is ubiquitylated, and this is associated with condensed DNA and gene silencing -44. H2B Regulated protein turnover by the ubiquitin-proteasome system (UPS) is essential for the survival of eukaryotic cells. This process is required for various cellular processes such as cell cycle control, signalling pathways, transcription and protein quality control. Alterations in the UPS are correlated with a variety of human pathologies, like cancer, immunological disorders, inflammation and neurodegenerative diseases . The exaMany neurological disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are caused by an accumulation of aberrant proteins leading to the formation of protein aggregates, inclusions and plaques. It is not completely clear why the UPS is failing to clear these aberrant proteins. For polyglutamine diseases like Huntington's disease it has been demonstrated that the UPS is unable to clear inclusions -52, and The flux of free ubiquitin between different cellular processes could be a passive mechanism in which unconjugated ubiquitin diffuses intracellular until it is utilized in ubiquitylation processes. However, a recent publication suggest that active factors could actually be involved in channelling ubiquitin from one ubiquitin-dependent process to another with temporally a higher priority . It has Ubiquitin seems more then just a signalling molecule involved in the regulation of various distinct processes in eukaryotic cells. The dynamic behaviour of ubiquitin modifications creates an equilibrium which allows crosstalk between different cellular processes that may allow cells to translate cellular stress to molecular responses by affecting the transcriptome.DUBs, deubiquitylation enzymes; uH2A, ubiquitylated H2A; UPS, ubiquitin proteasome system.The author(s) declare that they have no competing interests.FAS and TAMG conceived the manuscript. JN and NPD revised the manuscript. All authors approved the final version.


Lead concentration in whole blood (BPb) is the primary biomarker used to monitor exposure to this metallic element. The U.S. Centers for Disease Control and Prevention and the World Health Organization define a BPb of 10 μg/dL (0.48 μmol/L) as the threshold of concern in young children. However, recent studies have reported the possibility of adverse health effects, including intellectual impairment in young children, at BPb levels < 10 μg/dL, suggesting that there is no safe level of exposure. It appears impossible to differentiate between low-level chronic Pb exposure and a high-level short Pb exposure based on a single BPb measurement; therefore, serial BPb measurements offer a better estimation of possible health outcomes. The difficulty in assessing the exact nature of Pb exposure is dependent not so much on problems with current analytical methodologies, but rather on the complex toxicokinetics of Pb within various body compartments . If we are to differentiate more effectively between Pb stored in the body for years and Pb from recent exposure, information on other biomarkers of exposure may be needed. None of the current biomarkers of internal Pb dose have yet been accepted by the scientific community as a reliable substitute for a BPb measurement. This review focuses on the limitations of biomarkers of Pb exposure and the need to improve the accuracy of their measurement. We present here only the traditional analytical protocols in current use, and we attempt to assess the influence of confounding variables on BPb levels. Finally, we discuss the interpretation of BPb data with respect to both external and endogenous Pb exposure, past or recent exposure, as well as the significance of Pb determinations in human specimens including hair, nails, saliva, bone, blood , urine, feces, and exfoliated teeth. Over the last two decades, atmospheric concentrations of lead have decreased significantly around the globe as more and more nations have chosen to remove tetraethylead from gasoline . HoweverAfter Pb enters the body, it can travel along several pathways depending on its source and, by extension, its bioavailability. The fraction of Pb that is absorbed depends mainly on the physical and chemical form, particularly particle size and the solubility of the specific compound. Other important factors are specific to the exposed subject, such as age, sex, nutritional status and, possibly, genetic background . One of Like many other “bone-seeking” elements, Pb from blood is incorporated into calcified tissues such as bone and teeth, where it can remain for years . AccordiPhysiologic differences between children and adults account for much of the increased susceptibility of small children to the deleterious effects of Pb: whereas in adults 94% of Pb body burden is stored in bones and teeth, this proportion is only 70% in children . In addiBiomonitoring for human exposure to Pb reflects an individual’s current body burden, which is a function of recent and/or past exposure. Thus, the appropriate selection and measurement of biomarkers of Pb exposure is of critical importance for health care management purposes, public health decision making, and primary prevention activities.It is well known that Pb affects several enzymatic processes responsible for heme synthesis. Lead directly inhibits the activity of the cytoplasmic enzyme δ-aminolevulinic acid dehydratase (ALAD), resulting in a negative exponential relationship between ALAD and BPb. Pb depresses coproporphyrinogen oxidase, resulting in increased coproporphyrin activity. Pb also interferes with the normal functioning of the intramitochondrial enzyme ferrochelatase, which is responsible for the chelation of iron by protoporphyrin. Failure to insert Fe into the protoporphyrin ring results in depressed heme formation and an accumulation of protoporphyrin; this in turn chelates zinc in place of Fe, to form zinc protoporphyrin. These effects also result in modifications of some other metabolite concentrations in urine (ALA-U), blood, (ALA-B) and plasma (ALA-P), coproporphyrin in urine (CP). The activities of pyrimidine nucleotidase (P5’N) and nicotinamide adenine dinucleotide synthase (NADS) are also modified in blood after Pb exposure. Levels of these various metabolites in biological fluids have been used in the past to diagnose Pb poisoning when direct Pb levels were difficult to obtain in tissues or body fluids  or as inThroughout the last five decades, whole blood has been the primary biological fluid used for assessment of Pb exposure, both for screening and diagnostic purposes and for biomonitoring purposes in the long term. Although BPb measurements reflect recent exposure, they may also represent past exposures, as a result of Pb mobilization from bone back into blood . In thosData collected as part of the U.S. National Health and Examination Survey (NHANES) give the 95th percentile for BPb as 7.0 μg/dL for children 1–5 years of age and as 5.20 μg/dL for adults 20 years of age and older [U.S. Centers for Disease Control and Prevention . In lighMany studies have reported statistically significant associations between BPb levels and various health effect outcomes. Some, however, have been statistically weak, with the magnitude of the effect relatively small. According to Plasma-Pb likely represents a more relevant index of exposure to, distribution of, and health risks associated with Pb than does BPb. Indeed, from a physiologic point of view, we can assume that the toxic effects of Pb are primarily associated with plasma-Pb because this fraction is the most rapidly exchangeable one in the blood compartment. In recent years increased attention has been paid to monitoring the concentration of Pb in plasma (or serum). However, research on associations between plasma-Pb and toxicologic outcomes is still sparse, and a significant gap in knowledge remains.Plasma/serum Pb levels in nonexposed and exposed individuals reported in older publications range widely, from 0.02 to 14.5 μg/L . This isThe use of advanced analytical techniques is not the only essential requirement to ensure accurate and reliable plasma-Pb data. Contamination of the specimen may occur at the preanalytical phase, namely, during collection, manipulation, or storage. Use of Class-100 biosafety cabinets and clean rooms for specimen preparation and analysis is mandatory. Moreover, all analytical reagents used must be of the highest purity grade. These conditions are far more rigorous than are typically required for clinical BPb measurements performed in a commercial laboratory. After the blood specimen has been collected, the serum/plasma separation must be performed as soon as possible because there is high potential for Pb to move from the dominant BPb subcompartment repository, namely, the erythrocytes, into the plasma via hemolysis, leading to erroneously high results for plasma-Pb. Plasma hemolysis can be estimated by analyzing hemoglobin levels in the specimen because these levels are likely to become abnormally elevated with hemolysis . MateriaCommercial evacuated blood tubes, prepared specifically for BPb measurements, are available with < 5 μg/L Pb , but it There are many reports of plasma-Pb measurements where validation data are either weak or absent. For example, some simply cite successful participation of the analyzing laboratory in quality assurance (QA) programs for whole blood Pb operated by the CDC and the College of American Pathologists , whereasSaliva has been proposed as a diagnostic specimen for various purposes, as it is easily collected . HoweverSome data suggest an association between Pb levels in saliva and those in either plasma or blood . MoreoveUncontrolled variation in salivary flow rates, lack of standard or certified reference materials, and absence of reliable reference values for human populations are major factors that limit the utility of saliva Pb measurements. In addition the very low levels of Pb present in saliva limit the range of suitable analytical techniques, thereby further diminishing the utility and reliability of this biomarker for evaluating Pb exposure.Hair is a biological specimen that is easily and noninvasively collected, with minimal cost, and it is easily stored and transported to the laboratory for analysis. These attributes make hair an attractive biomonitoring substrate, at least superficially. Because Pb is excreted in hair, many have suggested it for assessing Pb exposure, particularly in developing countries where specialized laboratory services may be unavailable and resources are limited . However, an extensive debate is ongoing about the limitations of hair as a biomarker of metal exposure generally. Here we limit our discussion to Pb exposure, although many of the issues for Pb, such as preanalytical concerns for contamination control, sampling, and reference ranges, also apply to other metals.The ability to distinguish between Pb that is endogenous, namely, absorbed into the blood and incorporated into the hair matrix, and Pb that is exogenous, namely, derived from external contamination, is a major problem. During the washing step it is assumed that exogenous Pb is completely removed, whereas endogenous Pb is not. However, no consensus exists about how removal of exogenous Pb is best accomplished. Some publications that describe the use of hair for assessing Pb exposure reference a hair washing method proposed by the International Atomic Energy Agency (IAEA) in 1978. The approach entailed washing hair specimens with acetone/water/acetone . HoweverAnother issue is the significant variation in the Pb concentration profile among various subpopulations according to age, sex, hair color, and smoking status . MoreoveRecently, the ATSDR established an expert advisory panel to review current knowledge about the use of hair analysis for trace metals in biomonitoring . The genIn addition to the preanalytical issues and the absence of reliable reference ranges, the quality of analytical techniques used for determining Pb, as well as other trace metals, in hair has been questioned. In a recent interlaboratory study of commercial laboratories that specifically market the test for trace metals in hair, interlaboratory agreement was judged very poor, with wide discrepancies observed for Pb as well as for other elements .2EDTA. In unexposed subjects BPb levels remained stable even after 5 hr of CaNa2EDTA administration. However, plasma-Pb levels in the unexposed group decreased by as much as one half, while urine-Pb increased by a factor of 10. In the Pb-exposed group the same amount of chelation therapy resulted in plasma-Pb levels increasing by a factor of 2, while BPb levels decreased by a factor of 2, with a higher urine-Pb excretion. Thus, it seems that in nonexposed subjects a major contribution for urine-Pb is derived from the Pb fraction in soft tissues that is in equilibrium with that in plasma compartment. We could speculate that the larger the amount of erythrocyte-bound Pb, the weaker the binding forces, and that a significant fraction of Pb is released from red blood cell membranes into plasma and is then filtered by the kidney. Because the amount of Pb excreted is very high, the kidneys are unable to remove it rapidly from the blood stream; this may account for the temporal elevation of plasma-Pb levels.The determination of Pb in urine (urine-Pb) is considered to reflect Pb that has diffused from plasma and is excreted through the kidneys. Collection of urine for Pb measurements is noninvasive and is favored for long-term biomonitoring, especially for occupational exposures. However, a spot urine specimen is particularly unreliable because it is subject to large biological variations that necessitate a creatinine excretion correction. Urine-Pb originates from plasma-Pb that is filtered at the glomerular level; thus, according to some authors , urine-PThe availability of reliable urine quality-control materials and reference materials certified for Pb content and participation in external quality assessment schemes for urine-Pb are important factors in assuring the accuracy of analytical results. However, the tendency for urate salts to precipitate out of urine during transit and storage can be a complicating factor in the analysis. Moreover, because only a few studies have examined associations between urine-Pb and other biomarkers, the use of urine-Pb measurements is essentially limited to long-term occupational monitoring programs, monitoring patients during chelation-therapy, and, until very recently, to clinical evaluation of potential candidates for chelation therapy.Measurement of fecal-Pb content over several days is one possible approach to estimating the overall magnitude of childhood Pb intake. According to Like hair, nails have many superficial advantages as a biomarker for Pb exposure, especially because specimen collection is noninvasive and simple and because nail specimens are very stable after collection, not requiring special storage conditions. Nail-Pb is considered to reflect long-term exposure because this compartment remains isolated from other metabolic activities in the body . BecauseLead concentration in nails depends on the subject’s age , but it Because bone accounts for > 94% of the adult body burden of Pb (70% in children) , many re206Pb/204Pb isotopic ratios in immigrant Australian subjects, Australian-born subjects, and environmental samples. The immigrant population exhibited Pb isotopic ratios from 17.7 to 18.5, distinct from the ratio in Australian-born subjects (~ 17.0). This difference allowed a distinction to be drawn between current exposure acquired from Australian sources and older bone-stored Pb that was not acquired from Australian sources.As pointed by Differing bone types have differing bone-Pb mobilization characteristics. For example, the tibia principally consists of cortical bone, whereas the patella is largely trabecular bone. Pb in trabecular bone is more biologically active than Pb in cortical bone, and trabecular bone has a shorter turnover time. The endogenous contribution of Pb from bone stores is an important health consideration. The O’Flaherty kinetic model can be used to indicate the quantity of Pb delivered from bone as a function of bone turnover and Pb exchange . A recenin vivo X-ray fluorescence (XRF) methods have become increasingly accepted. The technique uses fluorescing photons to remove an inner-shell electron from a Pb atom, leaving it in an excited state. The result is emission of X-ray photons that are characteristic of Pb. Measurements are performed by using one of four kinds of XRF: two involve fluorescence of the K-shell electrons of Pb (K-XRF), and the other two involve fluorescence of the L-shell electrons (L-XRF)  dose (as compared to L-XRF) (Over the last decade bone-Pb measurements based on noninvasive  (L-XRF) . Severalo L-XRF) . The radCalibration is usually performed with Pb-doped plaster-of-Paris phantoms . Method Thus, an appropriate selection of the precise bone type to be analyzed for Pb content must be made before commencing. Moreover, further research on the relationship between various bone-Pb subcompartments and other Pb measures is warranted.Like bone, teeth accumulate Pb over the long term. However, there is some evidence that teeth are superior to bone as an indicator of cumulative Pb exposure because the losses from teeth are much slower . MoreoveChronic Pb exposure from mouthing activity in early childhood may be camouflaged by “dilution” effects during periods of rapid skeletal growth in young children and adolescents and may not be detected by a single BPb measurement. However, most published data on tooth-Pb have been based on whole tooth analysis, with no attempt to distinguish among tooth types (different teeth are formed at different ages) or to differentiate the Pb concentration in enamel from that in dentin . The influence of age and/or sex have also not been considered . Furtherin vivo enamel biopsy of children. In this approach superficial minerals are leached from teeth and Pb is determined by electrothermal atomic absorption spectrophotometry. One important drawback to this approach is that, because an accumulation gradient for Pb has not yet been established for enamel, only biopsies of a given depth can be compared to one another. Another issue related to tooth-Pb measurements is whether Pb that accumulates in the first few micrometers of the enamel surface was incorporated posteruptively  rather than during the period when the tooth was mineralized inside the bone.Recently, in utero and thus may provide information on prenatal exposure to Pb. This information could be valuable in improving our understanding of dose–effect relationships for embryonic anomalies, particularly neurotoxic dysfunction. The dentine of the primary teeth provides evidence of exposure during the early childhood years, when hand-to-mouth activity is usually an important contributor to Pb body burden .A critical question that might be asked with respect to an individual’s bone-Pb measurement is what does it mean in terms of health risk or, perhaps, clinical management? To answer this question, we may need to distinguish between bone-Pb measurements in children and pregnant women, namely, those with high bone turnover rate compared to (nonpregnant) adults. In children, bone-Pb may have little effect on BPb levels, but it may help us to estimate the extent to which BPb derives from endogenous sources and the possible contribution to the labile plasma-Pb pool.


The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation.The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics.We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process. Ageing is due to the gradual accumulation of unrepaired random molecular faults, which leads to an increased fraction of damaged cells and eventually to the functional impairment of older tissues and organs [reviewed in ]. CellulTerman mainly applied his theory to the lysosomal pathway of degradation. He suggested that lysosomes gradually accumulate lipofuscin and that the lipofuscin granules attract lysosomal enzymes which, however, fail to degrade the pigment. This leads to a shortage in lysosomal enzymes, resulting in a decrease in degradation by this pathway. However, the major pathway for the rapid degradation of proteins is the ubiquitin-proteasome pathway. Proteins need to be degraded either because they are short-lived e.g. key regulatory proteins or because they have become damaged and partially unfolded. Partially unfolded proteins have exposed hydrophobic surfaces and are liable to form aggregates. Molecular chaperones bind to the exposed surfaces and an attempt is made to refold the protein. If refolding is unsuccessful then chaperones assist in the removal of these proteins and so prevent their aggregation .Interestingly, proteasomes themselves are degraded by the lysosomal system and there is evidence for cross-talk between the two degradation pathways ,9. It haIt has been reported that aggregated protein directly impairs the function of the ubiquitin-proteasome system ,20. AnotMany neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome . It has In order to be recognised and degraded by the 26S proteasome, proteins must first be tagged with chains of four or more ubiquitin molecules. Ubiquitin is a small protein found in all eukaryotic organisms. Ubiquitination occurs as a result of the sequential activity of three classes of enzymes, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin protein ligase). In the first step, ubiquitin is activated through ATP hydrolysis mediated by an E1 enzyme. The activated ubiquitin is then transferred to an E2 (ubiquitin conjugate) enzyme. There are approximately 50 different E2 proteins in mammalian cells  that havA large number of de-ubiquitinating enzymes (DUBs) are also found within all eukaryotic cells . In addiA model of protein turnover and the role of the molecular chaperone Hsp90 in maintaining protein homeostasis has previously been developed . This moThe aim of this paper is to show that the model previously used to simulate chaperone function can be extended to model ubiquitin-mediated proteolysis, incorporating molecular details of this degradation system. We have also sought to incorporate details of the aggregation process. One approach would be to take the computer code for the chaperone model and then extend the code. However, a better approach is to build a new and separate model that can be tested individually and then linked with the chaperone model. A network diagram of the ubiquitin-proteasome model is given in Figure The model was simulated with the initial values and parameter values given in Tables k1, k2, k67, k68, and k69. Varying the parameter for protein synthesis, k1, affects the levels of native protein but only an increase in k1 has any affect on any of the other species. Increasing protein synthesis leads to a depletion in monomeric ubiquitin pools due to the increase pool of protein requiring degradation. An increase in the parameter for misfolding, k2, leads to an increase in misfolded protein, a decrease in native protein, all the ubiquitin ending up in conjugates and an increase in degradation. Conversely, a decrease in k2, leads to higher levels of native protein, more monomeric ubiquitin, less ubiquitin conjugates and less degradation. Increasing the parameter for binding of polyubiquitinated misfolded protein to the proteasome, k67, has no effect on the model output, but a decrease in k67 leads to a depletion in monomeric ubiquitin pools, an increase in unbound polyubiquitinated misfolded protein and less degradation. Increasing the parameter for the chain shortening of polyubiquitinated misfolded protein, k68, has the same effect as decreasing k67. However, decreasing k68 has little effect on model output except that nearly all the degradation reactions are via the substrates with the longest ubiquitin chain. Increasing the parameter for proteasome activity, k69, leads to more degradation and less ubiquitin conjugates, whereas decreasing k69 has the opposite effect and also results in depletion of monomeric ubiquitin due to less recycling of ubiquitin.We carried out a sensitivity analysis to see how changing each of the model parameters affects the simulation output. The only parameters which have a significant effect on the results when increased or decreased by an order of magnitude were in silico experiment of inhibiting the proteasome by setting the parameter k69 = 0. The model predicts that there is initially a build up of ubiquitinated protein and that the pool of monomeric ubiquitin is depleted. The depletion of ubiquitin is followed by a steady rise in the level of misfolded protein , the only parameters which affect aggregation are k1, k2, k3 and k61, Either an increase in protein synthesis (k1) or in the misfolding rate (k2) leads to an increase in aggregated protein which in turn leads to both an increase in sequestered aggregated protein (SeqAggP) and binding of aggregated protein to the proteasome (AggP_Proteasome). This is due to the increase level of misfolded protein which can not be degraded. On the other hand an increase in refolding (k3) leads to a reduction in aggregation. Increased refolding could occur if there was an upregulation in molecular chaperones. Increasing the rate of binding of E3 to misfolded protein (k61) also reduces the level of aggregation suggesting that, if a particular protein was involved in aggregation, then overexpressing the relevant E3 for a misfolded protein could help to prevent the accumulation of aggregates. However, aggregates are usually composed of a variety of proteins, so this approach may not be feasible. As expected, increasing the rate of aggregation by either increasing k71 or k72 leads to more aggregation, and varying the parameters k73 or k74 changes the ratio of SeqAggP to AggP_Proteasome.We also varied each of the parameters in turn to see which parameters affect the kinetics of aggregation. We found that apart from the parameters actually involved in the aggregation steps  is most sensitive with a ten-fold increase leading to a ten-fold decrease in protein half-life, and a ten-fold decrease leading to a ten-fold increase in half-life  only has an effect when decreased, with a ten-fold decrease leading to a four-fold increase in protein half-life , leads to a slight increase (× 1.4) in protein half-life . These results suggest that the availability of chaperones and E3 enzymes are important in maintaining protein homeostasis.We also examined which parameters affect protein half-life. The most important parameter in this respect was the parameter for misfolding with a ten-fold increase/decrease leading to a ten-fold decrease/increase in protein half-life. Since the misfolding reaction can be driven by the level of ROS in the cell, our model would predict a similar outcome by changing ROS levels, with an increase in ROS leading to higher misfolding and an increase in degradation, consistent with current thought linking protein oxidation and ageing . Our modThere are also many other modifications that we could make to the model. For example, we have assumed that E3 binds to misfolded protein in one simple step; however it is likely that when a native protein misfolds, the first step would be for a molecular chaperone (e.g. Hsp90) to bind to the exposed hydrophobic surface to prevent inappropriate interactions. We could easily add this detail by including the following steps:MisP + Hsp90 ↔ Hsp90_MisP (reversible reaction)Hsp90_MisP + E3 → E3_MisP + Hsp90Similarly, we could include the possibility that chaperones aid in delivery of ubiquitinated proteins to the proteasome.Our model includes a HECT E3, but it would be easy to adapt the model for other types of E3 enzymes. For example if E3 is a RING ligase, then it has to be in complex with E2 before it can bind to the misfolded protein. This could be represented by the following reactions:E3 + E2_Ub ↔ E2_E3_Ub (reversible reaction)MisP + E2_E3_Ub → E2_E3_MisP_UbE2_E3_MisP_Ub → MisP_Ub + E2 + E3Our model only includes one pool of long-lived proteins and so far ignores the fact that the majority of proteasome activity is involved in the turnover of short-lived regulatory proteins and also the elimination of incorrect newly-synthesised proteins. It would make the model more realistic to include an additional pool of short-lived proteins and this work is currently in progress.We have so far restricted our models of protein turnover to the ubiquitin-proteasome system; however work is currently in progress to develop other models of protein turnover. It will be important to incorporate models of lysosomal pathways since studies suggest that protein aggregates such as mutant huntingtin are removed by autophagy [reviewed in ].We have included the possibility of aggregated protein inhibiting proteasomes. Another possible cause of proteasome inhibition is via unaggregated damaged protein which could also sequester proteasomes. It would be fairly straight forward to add this extra detail to our current model. By running simulations over long time periods, the models can then be used to see how proteasome activity is affected by damaged and/or aggregated protein. This would be more realistic, than simply setting the parameter for proteasome activity to zero.Our model predicted that levels of native protein did not change when misfolded protein levels rise, but in reality we would not expect total protein levels to continually rise. This prediction was due to our assumption of a constant protein synthesis rate. We could later include detail of feedback mechanisms such as the unfolded protein response which downregulates translation when unfolded protein accumulates in the endoplasmic reticulum.We have encoded our model using SBML which not only allows for easy portability but also enables the model to be modified and extended in a very straight-forward way. The model is available on the BASIS website and can be freely accessed from the public repository, so that the interested reader can run simulations, make their own modifications, or download the SBML code. The BASIS website has a stochastic simulator and a database for storing models and simulation results.Computer models of other molecular mechanisms involved in ageing are being developed at Newcastle University, for example the role of mitochondria, the antioxidant system, and DNA damage signalling pathways. Methods to link models in an automated way are also under development which will greatly facilitate the development of integrated models. For example, our model of the ubiquitin-proteasome system could be linked to a model of the mitochondria. This would be very valuable since neurodegeneration is not only associated with an increase in aggregated protein but also an increase in damaged mitochondrial DNA .We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. We have used SBML to encode the model to ensure that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process. The model and its extensions will be an invaluable aid to further our understanding of the cellular mechanisms involved in maintaining protein homeostasis and how it is disturbed during ageing.2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of a 2:1 mixture of fetal bovine and donor bovine serum. ts20 cells harbouring a temperature sensitive E1 ubiquitin activating enzyme  were maintained in 5% CO2 at 34°C in DMEM as above. Inactivation of E1 was achieved by incubating the cells at 42°C for the indicated time. When proteasome inhibitor  was used, it was added to the culture medium 3 or 4 hours prior to harvesting the cells for western analysis at a final concentration of 50 μM.U87MG human glioblastoma cells, NIH-3T3 mouse fibroblasts and IMR90 human fibroblasts were maintained in 5% COCells were harvested in protein lysis buffer , 0.5 mM EDTA, 5% glycerol) containing the following protease and phosphates inhibitors: phenylmethylsulfonyl fluoride, leupeptin , aprotinin , sodium fluoride (NaF), sodium pyrophosphate (NaPPi) and N-ethylmaleimide. The cell extracts were sonicated and centrifuged at 14,000 rpm for 20 minutes at 4°C to pellet cellular debris. The soluble fractions were recovered and the protein concentration was determined using the Bradford protein assay . 40 μg of cytoplasmic extracts were then resolved on a two-phase SDS-polyacrylamide gel (15 and 8%) and electroblotted onto a hybond C nitrocellulose membrane . The membranes were autoclaved on a liquid cycle for 45 minutes to enhance the detection of poly-ubiquitinated proteins, stained with Ponceau S  and analyzed by western blotting with the indicated antibody. The proteins were visualized by a horseradish peroxidase method using the ECL kit from Kirkegaard and Perry Laboratories Inc., Gaithersburg, Maryland, USA.The rabbit polyclonal antibody used to detect ubiquitin was purchased from Dako. The mouse monoclonal antibodies recognizing actin and GAPDH were from Sigma  and Stressgen Bioreagents  respectively.The model was built by first drawing a diagram of the system , ubiquitin conjugating enzyme (E2), ubiquitin ligase (E3), de-ubiquitinating enzymes (DUB), proteasomes, ATP, ADP, AMP, reactive oxygen species (ROS), and complexes to represent binding of elements e.g. ubiquitinated protein. A full list of the species with their names, database terms and their initial amounts is shown in Table , ubiquitIn our previous model of the chaperone system , we inclwhere MisP represents the pool of misfolded protein.We now replace this single step with a more detailed representation of the ubiquitin-proteasome system.The first step in the degradation pathway is that the substrate (in this case MisP) is bound to its specific ubiquitin ligase (E3). The binding of E3 to MisP produces a complex E3_MisP. This reaction is reversible and so we have two reactions as follows:k61<#E3><#MisP>, where # denotes the number of molecules. The disassociation reaction is a first-order reaction and is given by kr61<#E3_MisP>. The values for k61 and kr 61can be estimated from knowledge of the protein half-life and the steady state level of protein in its misfolded state.We use mass action stochastic kinetics for the rate laws . The binBefore a substrate can be ubiquitinated, ubiquitin must be activated by the ubiquitin-activating enzyme (E1). This reaction requires one molecule of ATP. The binding of E1 to ubiquitin (Ub) produces a complex which we denote by E1_Ub:The activated ubiquitin is then transferred to the ubiquitin-conjugating enzyme (E2) which releases E1 making it available for further ubiquitin-activating reactions:Although it is possible that this reaction is reversible, it seems unlikely that E2 would return the Ub to E1. So we assume that the reverse reaction is negligible and do not include it in the model. Ubiquitin bound to E2 is then transferred to the misfolded protein bound to E3, releasing E2 and E3:Further ubiquitin is then attached to the monoubiquitinated protein via the action of E2. Since each ubiquitin molecule must be activated by E1 before being transferred to E2, each step in the formation of polyubiquitin chain uses one molecule of ATP. The extension of the ubiquitin chain is modelled by the following set of reactions:Each of the above reactions is reversible, with the reversible reactions requiring the activity of a deubiquitinating enzyme (DUB). We assume that the shortening of ubiquitin chains by DUBs is a step-wise process, although we could later modify the model to allow whole chains to be removed if it becomes of interest to model this. The step-wise removal of ubiquitin is modelled by the following reactions:We assume that ubiquitinated protein first binds to the proteasome and waits for degradation and that it can remain bound so long as at least 4 ubiquitin sub-units are attached. This can be modelled by the following reactions:where MisP_Ub4_Proteasome etc. represents an ubiquitinated protein bound to the proteasome.Bound ubiquitinated protein can also be de-ubiquitinated in a step-wise process, so that proteins with longer chains stay longer at the proteasome. This is modelled by the following set of reactions:When a bound protein has a chain shorter than four, then it dissociates from the proteasome. This is shown in the following reaction:Any protein which has a chain of four or more ubiquitin molecules may be degraded by the proteasome in an ATP dependent reaction:This model can either be combined with the chaperone model  or can bThe rate of misfolding depends on the level of reactive oxygen species (ROS) within the cell. We have omitted details of the chaperone activity, where a misfolded protein would bind to Hsp90 before refolding can take place. However, when we combine the models, this simple reaction will be replaced with the more detailed reactions given in Proctor et al., . In thisIf a misfolded protein is not removed immediately by refolding or degradation, then there is a chance that it will interact with another misfolded protein to form a small aggregate, or it may interact with an existing aggregate to form a larger aggregate. (Here we will not be concerned with the size of aggregates – a more detailed model is in preparation). An aggregate may be sequestered to prevent it interfering with the cellular machinery or it may bind to the proteasome. We include enough detail to be able to examine whether an increase in misfolding (for example by an increase in levels of ROS) leads to an increase in aggregation and inhibition of the proteasome which in turn leads to an even greater level of aggregated protein.The following set of reactions show how protein aggregation can be simply modelled:where AggP represents the pool of aggregates. The aggregates may be sequestered (SeqAggP) which keeps them out of harms way, or they may bind to the proteasome (AggP_Proteasome) and so inhibit its function. Ubiquitinated misfolded protein may also form aggregates but this will not normally occur unless the proteasome is inhibited. As there are many ways in which misfolded proteins with ubiquitin chains of different lengths can interact, we only list a subset of the reactions:6 cells and 61.1 pmol/106 cells respectively. From this we can calculate that there are approximately 107 ubiquitin molecules per cell . Since we are only modelling part of the total cellular protein, we have scaled down the level of ubiquitin to 500 molecules per cell. The total level of proteasomes per cell has been estimated to be 8 × 105 per cell in L929 cells [Before the model can be simulated, it is necessary to specify the initial amounts of each species and the parameter values. There is experimental data on levels of ubiquitin and proteasomes in human cells. For example, Haas et al.  measured29 cells  which giThe level of E1 has been measured in IMR90 cells by western analysis of cell lysate compared to recombinant protein  and is estimated to be 1 million molecules per cell. This is one order less than the level of ubiquitin and so we chose an initial amount of 100 for E1. We do not have data for E2 or E3 enzymes but for the sake of building the model assume that for specific substrates these would be present at roughly the same abundance as E1. We do not have any experimental data for the abundance of DUBs within cells but assume that these are less abundant than ubiquitin and set the initial amount to 200.The level of native protein, NatP, was set at 500 and at this level the ratio of native protein to ubiquitin and proteasomes enabled efficient degradation to take place. If we increased the level of NatP to 5000, then it was also necessary to increase the pool of ubiquitin and proteasomes to prevent depletion of monomeric ubiquitin under normal conditions. The levels of ATP, ADP and AMP were set at a fairly arbitrary level of 10000, 1000 and 1000 respectively and the levels were kept constant by imposing a boundary condition to these species. We chose to do this instead of allowing the levels to fluctuate as cells possess mechanisms to maintain ATP levels at fairly constant levels and it is unlikely that the ubiquitin-proteasome system would cause major changes to levels of these molecules. ROS levels were set at an arbitrary value of 10 and the level remained constant as there are no mechanisms in this model that would cause ROS levels to change. The initial amounts of all the species are shown in Table The default values for the model parameters are shown in Tables k1, k2 and k3 were set so that the half-life of the pool of protein equals about 10 hours, and under normal conditions, there is only a very low proportion of misfolded protein [k61 for E3/MisP binding is set so that the average time for one E3 to bind to MisP is about 10 minutes. The parameter kr 61is set so that the reverse reaction (dissociation of E3 from MisP) happens once every 100 minutes. So the forward reaction is 10 times stronger than the reverse reaction. The parameter k62 for E1/Ub binding is set so that about 40 ubiquitin molecules are activated every minute and the parameter k63 for E2/Ub binding is set to give about 6 reactions per minute. The parameter k64 for monoubiquitination is set so that once MisP is bound by E3 it takes about 10 seconds to receive the first Ub molecule. The parameter k65 for polyubiquitination is set so that it took about one second for each additional Ub molecule to be added to the chain. So each polyubiquitination step is about 10 times faster than the first ubiquitination reaction. The parameter k66 for the de-ubiquitination reactions is set so that it takes about 8 minutes for each reaction. It is necessary that this reaction is very slow compared to the ubiquitination reactions so that the model output fits the experimental data when E1 activity is shut down (see section 4.4). The parameter k67 for the binding of ubiquitinated proteins (with chains of 4 or more ubiquitin molecules) to the proteasome is set so that under normal conditions when there are about 200 ubiquitin conjugates present in the cell, on average about 5 reactions per second are occurring. The parameter k68 for the de-ubiquitination of proteasome-bound ubiquitinated proteins is set to be equal to k66. We assume that this reaction is slow so that proteins with long chains are highly likely to reside long enough at the proteasome for degradation to take place, whereas proteins with short chains may be shortened to below the required threshold and would then dissociate from the proteasome. The parameter k69 for proteasome activity is set so that it takes about 15 seconds to degrade each proteasome-bound ubiquitinated protein. We initially set k71 and k72 to be equal to 10-7 as in the model of Proctor et al. [k71 and k72 set equal to 10-8, The parameter k73 for sequestering aggregates is set so that if there is a low level of aggregates in the cell (1–10), then on average there are 0.005 reactions per second, so each reaction takes about 3 minutes for each reaction.We now describe each of the parameters in turn and give an interpretation of their values in terms of reaction kinetics. The parameters  protein . The parr et al. . Howeverk74 is set so that there is an equal probability of an aggregated protein being sequestered or binding to the proteasome when all the proteasomes are available for binding.The parameter in silico experiment of inhibiting the proteasome by setting the parameter k69 = 0. We also varied each of the other parameters in turn to see which parameters affect the aggregation kinetics.We carried out an k1, to zero we can use our model to check the half-life of the protein pool. We also varied each of the other parameters in turn to see which parameters affect the half-life. For each parameter we increased and decreased its value ten-fold, re-ran the simulations and plotted the model output.By setting the parameter for protein synthesis, in silico model by adding an event to the SBML code so that at a certain time-point the parameter for the E1/Ub binding reaction is set to zero. This event is equivalent to putting the cells at the non-permissive temperature in the wet lab experiment. We chose 30 minutes as the time-point at which this event occurred to allow time for ubiquitin conjugates to form. The details of the event are given in Table It is very straight forward to mimic this experiment with our Statistical analysis of repeated simulations was carried using the R programming language.CJP constructed and coded the models, ran simulations, plotted results and drafted the manuscript. MT designed and performed laboratory experiments and wrote sections of the manuscript. DAG designed the experiments, advised on the model construction and contributed sections of the manuscript. All authors read and approved the final manuscript.Proctor2007_UPSProteinDegradation.xml. SBML code for the ubiquitin-proteasome modelClick here for file


The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. Posttranslational modification by ubiquitin regulates processes such as proteasomal degradation, intracellular trafficking, and transcription. Ubiquitin is attached to substrates in covalent isopeptide linkage or as an N-terminal fusion Ubiquitin-like molecules show sequence and structural similarity to ubiquitin. Unlike ubiquitination, modification by UbLs usually does not target proteins for destruction by the proteasome. A notable exception may be FAT10, a modifier that serves as a ubiquitin-independent signal for proteasomal degradation A closely related homolog of ubiquitin in vertebrates is the UbL polypeptide ISG15, an interferon-inducible gene product that is strongly upregulated following viral or bacterial infection The C-terminal six amino acids of ubiquitin and ISG15 are identical. This tail region is required for specific recognition of ubiquitin by conjugating enzymes, and also for recognition of ubiquitin adducts by isopeptidases in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SUMO1VME and ISG15VS, respectively bona fide ISG15-isopeptidase USP18 displayed reactivity only towards ISG15VS , validating the choice of such C-terminal modifications analysis . Whereasthe cell , similar pattern . Consist nucleus . To demous  is contrasted by the functional differences and localization of these two proteases. These enzymes provide a unique opportunity to investigate the structural features that may contribute to ubiquitin versus ubiquitin-like specificity. A characteristic of USP5 and USP13 is the tandem occurrence of a UBA domain, which has been implicated in the binding of ubiquitin The ubiquitin gene is prone to duplications and insertions, leading to the formation of new fusion proteins USP2 and USP18 cDNAs were cloned from a human kidney cDNA library . The cDNAs encoding the other human DUBs were obtained from ATCC. All cDNAs encoding full-length DUBs were subcloned into pcDNA3.1. Point mutations were generated with the Stratagene “Quik Change II” kit. The Genbank Identification numbers of the cloned constructs are: USP2 (GI:21361712), USP3 (GI:10720340), USP5 (GI:4507855), USP7 (GI:4507857), USP10 (GI:24307889), USP12 (GI:32698815), USP13 (GI:4507849), USP14 (GI:4827050), USP17/DUB3 (GI:37778800), USP18 (GI:32313610), USP22 (GI:113426698), USP24 (GI:113408685), USP25 (GI:6941888), USP35 (GI:89034166), USP36 (GI:35250686), USP37 (GI:32698744), Bap1 (GI:4757836), CylD (GI:14165258), CGI-77 (GI:40254879), OTU1 (GI:109148508), OTU2 (GI:12962939), UCH-L1 (GI:93279272).http://www.psc.edu/biomed/genedoc/) by K.B. Nicholas & HB Nicholas Jr., using the putative active-site cysteine as an alignment anchor. The phylogram represents a consensus tree based on 100 bootstrap iterations, calculated by the Minimum Evolution method under default parameters The protein sequences of human DUBs were obtained from the National Library of Medicine and the core domains were aligned with the CLUSTALW algorithm (EBI server) IVT was performed using the “TNT-T7 Quick Reticulocyte Lysate System” kit (Promega) for 30 to 45 min (0.25–1 µg DNA per reaction). Then, aliquots of the reaction mix were treated with RNase B  for 10 min and incubated with the probes as described below. SDS-PAGE followed by fluorography was performed as described E. coli-expressed human recombinant USP14 for the experiments shown in 2>0.99):The synthesis of human ubiquitin and UbL probes has been described 2, 20 mM DTT, 40 mM ATP) was added ISG15  or GST-SUMO1  and biotinylated peptide 7-mer . The solution was mixed thoroughly and to this was added ISG15 activating enzyme  and UbcH8 , or SUMO activating enzyme  and UbcH9 . The solution was mixed and incubated for 15 hours at 37°C. The reaction mixture was transferred to a microcentrifuge membrane filter , diluted to 600 µL total volume with 50 mM Tris, pH 7.4 and concentrated at 4°C to 50 µL. This dilution/concentration procedure was repeated six times. The products were transferred to a clean tube and diluted with 50 mM Tris, pH 7.4 to a final volume of 100 µL. The SUMO1- and ISG15-linked biotinylated isopeptide was detected after transfer to a PVDF membrane (Perkin Elmer) with Streptavidin-HRP (Amersham).To 50 µL of conjugation buffer  and ISG15 was manually modeled onto USP2 and USP14 with the SPDB-Viewer


It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton.The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways.in vivo.We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), β-catenin, AP-1, and nuclear factor-kappa B (NF-κB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-β but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/β-catenin pathway activity, it induced NFκB signaling and inhibited AP-1 signaling.Pb-exposed MSCs showed enhanced basal and TGF-β/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-β, BMP, AP-1, and NFκB.The  In spite of effort to reduce human exposure, lead toxicity continues to be a major environmental health concern in the United States and in other industrial countries. Although recent research has focused on understanding its toxic effects in the developing peripheral and central nervous system, Pb also impacts other developmental processes, including those that occur in the hematologic and skeletal systems . In factin vitro experiments to elucidate the underlying mechanism of toxicity during chondrogenesis and chondrocyte differentiation. We employed micromass cultures of mesenchymal stem cells, a culturing technique that has been used previously as a model to study the early events of embryonic limb development  signaling in neurologic tissues, and interfere with long-term learning and other functions  with a DME/F12 mixture (40%/60%) supplemented with 10% fetal bovine serum  as previously described  by enzymatic digestion of limb bud tissue as previously described . Brieflyescribed . One mic2O, photomicrographs of the stained cultures were obtained. Quantification of alcian blue staining was performed using NIH Image . The threshold was standardized to detect nodule area, and the area of the staining was converted from pixels to square millimeters. The area and intensity values were recorded and used to determine staining intensity.Three days after seeding of micromass cultures and treatment with various Pb concentrations and/or factors of interest, wells were washed once with phosphate-buffered saline  and then fixed for 20 min with 1 mL 10% neutral buffered formalin . The cultures were then washed three times with sterile distilled, deionized water, with the last wash being left on the cells for 15 min. Subsequently, 0.5 mL 3% alcian blue (Sigma) in glacial acetic acid was added to each well for 24 hr at room temperature. After two washes with 70% ethanol and three washes with sterile distilled, deionized H, reactions were carried out using the Rotor Gene Real-Time DNA Amplification System  according to the manufacturers instructions. cDNA samples were treated with RNase A , and 50-ng aliquots were combined in a 20-μL final volume with 0.5 mM primers and the SYBR Green PCR Master Mix . The PCR protocol began with 94°C denaturation (5 min) and then followed by 35 cycles of the following: 94°C denaturation (20 sec); 50–60°C annealing ; and 68°C extension (30 sec). Dilutions of the cDNA pool were used for each primer set to generate a standard curve, which was utilized by RotorGene software  to perform quantitative analyses. Data were normalized to the β-actin expression level for each sample. All samples were run in triplicate and repeated four times (n = 4).Cultures were harvested at various time points, and mRNA was prepared using Trizol reagent  as directed by the manufacturer. cDNA was generated using the Advantage RT-for-PCR kit , and real-time quantitative RT-PCR was performed as described previously , 2004. BUse of mice in this experiment was reviewed and approved by the University of Rochester Institutional Animal Care and Use Committee (IACUC). Mice were treated humanely with regard for alleviation of suffering. At 10 weeks of age, female C57/B6 mice were divided into three groups of 35 mice. Groups were exposed to 0, 55, or 230 ppm Pb (in the form of Pb acetate) in drinking water for 6 weeks before initiation of the ectopic bone formation experiment. Our group previously used an atomic absorption method to establish that these doses of Pb exposure correspond to environmentally relevant whole-blood Pb concentrations of 15 and 40 μg/dL for 55 and 230 ppm, respectively .l-glutamine, and 1 μg/mL doxycycline to repress BMP-2 expression . Nine days before implantation, doxycycline was removed from the medium to allow BMP-2 expression, which was confirmed using an ELISA kit . As a negative control, wild type C3H10T1/2 cells were cultured in basal medium Eagle (BME) with 10% FBS, 1% penicillin/streptomycin, and 2 mM l-glutamine. Cells from both lines were washed, trypsinized, counted, and resuspended in medium at a concentration of 106 cells/100 μL. Surgifoam  was cut into 2 mm × 2 mm × 2 mm segments and soaked in medium, then excess fluid was removed. Aliquots (100 μL) of the cell suspension were micropipetted into each section of gel foam and incubated for 2 hr to allow cells to adhere.Ectopic bone formation was induced in mice by the intramuscular implantation of a gel foam substrate impregnated with cells expressing BMP-2. Specifically, C9 cells derived from murine C3H10T1/2 mesenchymal stem cells, which express BMP-2 under control of a doxycycline-repressible promoter, were cultured in DMEM with 10% FBS, 1% penicillin/streptomycin, 2 mM n = 15 for statistical purposes).Using an IACUC-approved surgical protocol, we placed C9 cell-impregnated gel foam segments into a muscle pouch in the right quadricep of each mouse; C3H10T1/2-impregnated gel foam was placed in the contralateral limb as a negative control. Ectopic nodules were allowed to develop for 3–28 days after implantation. At each time point, tissue was harvested, fixed in 10% formalin for 3 days, embedded in paraffin, and cut into 5-μm sections. Three sections 30 μm apart were stained with alcian blue and counterstained with alcian blue/hematoxylin. Three representative sections from each sample at day 5 and day 7 were analyzed using Osteometrics software . Cartilage cross-sectional area was measured for each slide and included in statistical analysis , AP1, and cyclic adenosine monophosphate response element binding protein (CREB). Reporter and control plasmids for each of these pathways was transfected into MSCs plated as a monolayer, and then cells were trypsinized and replated as a micromass as described above. Thus, MSCs were initially plated in monolayer in 6-cm dishes at a density of 8 × 105 cells per dish. Twelve hours after plating, cells were transfected with either P3TP-luciferase (Luc), 12 × Smad binding element (SBE)-Luc, NF-kB-Luc, TOPFLASH, AP1-Luc, or cAMP-responsive element (CRE)-Luc. These transfections were performed using Superfect  according to the manufacturers instructions. The SV40 Renilla-Luc plasmid was co-transfected to facilitate determination of transfection efficiency on a well-by-well basis, as previously described . Aliquots of protein extract (25 μg) were separated by SDS-PAGE (10% polyacrylamide) and then transferred to a polyvinylidene fluoride membrane . The blots were probed overnight at 4°C with the following rabbit anti-mouse polyclonal antibodies: phospho-Smad3 , phospho-Smad1/5/8 , or mouse anti-β-actin monoclonal antibody . All primary antibodies were applied at a 1:1,000 dilution. Blots were further incubated for 1 hr at 20°C in the presence of horseradish peroxidase–conjugated secondary antibodies against rabbit or mouse  at a dilution of 1:3,000. The immune complexes were detected using ECL-Femto  and visualized via exposure of X-OMAT AR film .After rinsing the cell layer with PBS, protein was extracted from micromass cultures using Golden lysis buffer containing protease inhibitor cocktail tablets (Roche Applied Science) as previously described . The lysMSCs were placed in micromass culture and were treated with Pb in the presence and absence of BMP-2 (50 ng/mL) and TGF-β (5 ng/mL). After 3 days in culture, treatment with Pb resulted in a stimulation of chondrogenesis as measured by the presence of alcian blue nodules . The effSox9, col2, and aggrecan were determined via real time RT-PCR. Consistent with the alcian blue staining shown in Sox9, a 5-fold increase in col2, and a 2.5-fold increase in aggrecan compared with untreated cultures  for 6 weeks. In control C3H10T1/2 cell implanted limbs, we observed proliferation of host mesenchymal cells at the periphery of the implant, but no cartilage tissue formed  induced a 2.5-fold stimulation . HoweverMSCs were transfected with the BMP-responsive reporter 12 × SBE-Luc and treated with Pb in the presence and absence of BMP-2 (100 ng/mL). Pb itself did not alter reporter activity, but BMP-2 stimulated a 2.4-fold induction. Interestingly, Pb induced a dose-dependent inhibition of BMP-2–induced signaling on the 12 × SBE-Luc promoter, with a maximal 65% inhibition occurring at a concentration of 10 μM Pb . ConsistBecause Pb has no effect on basal TGF-β–Smad and BMP-Smad signaling, and has opposite effects on these signaling pathways in the presence of their respective growth factors, it is unlikely that the induction of chondrogenesis by Pb is related to direct regulation of Smad signaling. To determine the possible involvement of other pathways known to be important in the process of chondrogenesis, MSCs were transfected with AP1-Luc, NFκB-Luc, CRE-Luc, and TOPFLASH reporters, each containing multiple repeats of the respective consensus binding sites. Although there was no effect of Pb on CRE or TOPFLASH activity , Pb causThere is growing awareness of the pleiotropic effects of Pb, with targets of toxicity including multiple tissues that include the skeleton. In fact, at concentrations considered subtoxic, Pb is known to affect skeletal growth. The two most recent National Health and Nutrition Examination Surveys (NHANES II and NHANES III) have demonstrated that Pb exposure reduces skeletal growth independent of factors such as nutritional status, social/ economic class, and co-morbid diseases . Moreovea) block calcium signaling and inhibit Ca2+/phospholipid-dependent PKC signaling (b) induce ERK1/2 and p38 (MAPK) phosphorylation and activation (c) activate AP-1 signaling  indutivation ; and c) ignaling . We havecolX, Interestingly, this study also showed that inhibition of colX by TGF-β was completely reversed by Pb. Furthermore, although neither Pb nor TGF-β alone affected the expression of BMP-6, in combination they induced its expression 3-fold (Previous work has established that Pb regulates BMP and TGF-β signaling pathways. Oral administration of Pb chloride has been shown to cause a reduction in the expression of TGF-β in intestinal tissues in diabetes-prone NOD mice . In an en 3-fold . AlthougIn the present study, we found that BMP signaling is also regulated by Pb. BMP receptor signaling occurs in a manner analagous to the TGF-β pathway, with Smad1/5/8 binding to the type I BMP receptor, followed by phosphorylation of these factors upon ligand activation . Using ain vivo appears to stimulate osteogenesis over chondrogenesis (The complex regulation of the Smad signaling molecules makes it unlikely that the induction of chondrogenesis by Pb is cause by direct alteration of these pathways. Although BMP and TGF-β signaling pathways have antagonistic effects on some cells, including growth plate chondrocytes where TGF-β inhibits and BMP-2 stimulates maturation, both signals enhance chondrogenesis in mesenchymal stem cell populations . An earlogenesis , in humaogenesis . Similarogenesis . Overallin vitro Pb exposure in rats results in activation of AP-1 and NFκB levels in multiple regions of the brain and in astrocytes in culture (In vivo Pb exposure results in increased NFκB signaling in renal tubular cells and results in nephritis in rats (To extend these signaling results, we also examined the effect of Pb on other signaling pathways. Although no effects were observed on CRE and TOPFLASH activation using the respective luciferase-based reporters, the experiments showed that Pb inhibited basal AP-1-Luc reporter activity. AP-1 activation has been shown to inhibit chondrogenesis, so it is possible that the inhibition of AP-1 could be involved in the induction of chondrogenesis by Pb . The inh culture . In vivo in rats . Similar in rats , it is uin vivo murine model of fracture healing (The induction of chondrogenesis by Pb in the current study is consistent with findings observed in an  healing . Mice wi healing . OverallIn conclusion, the present study establishes that in addition to affecting chondrocyte maturation, Pb accelerates the differentiation of mesenchymal precursors into chondrocytes. Although Pb alters BMP and TGF-β signaling, the mechanism through which Pb regulates mesenchymal cell fate determination is complex and likely involves modulation and integration of multiple signaling pathways. Increased understanding of the mechanisms through which Pb regulates stem cell fate and subsequently affects tissue repair is critical, given the sensitivity of these tissues and the ubiquitous presence of Pb in our society and in other industrialized and developing nations.