Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability.

Spastin is one of the proteins which lead to hereditary spastic paraplegia (HSP), whose dysfunction towards microtubule severing and membrane transporting is critically important. The present study is to elucidate the mechanisms of the protein stability regulation of spastin. The ubiquitin encoding plasmids are transfected into COS-7 cells with different fusion tags including Green Fluorescent Protein (GFP), mCherry and Flag. The expression level of spastin was detected, microtubule severing activity and neurite outgrowth were quantified. The data showed that ubiquitin overexpression significantly induced the decreased expression of spastin, suppressed the activity of microtubule severing in COS-7 cells and inhibited the promoting effect on neurite outgrowth in cultured hippocampal neurons. Furthermore, when modulating the overexpression experiments of ubiquitin, it was found that relatively small tag like Flag, but not large tags such as GFP or mCherry fused with ubiquitin, retained the activity on spastin stability. The present study investigated the effects of small/large tags addition to ubiquitin and the novel mechanisms of post-transcriptional modifications of spastin on regulating neurite outgrowth, in the attempt to experimentally elucidate the mechanisms that control the level or stability of spastin in hereditary spastic paraplegia.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

Dysregulations of Expression of Genes of the Ubiquitin/SUMO Pathways in an In Vitro Model of Amyotrophic Lateral Sclerosis Combining Oxidative Stress and SOD1 Gene Mutation.

Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.

Facile Semisynthesis of Ubiquitylated Peptides with the Ligation Auxiliary 2-Aminooxyethanethiol.

The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.

Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa.

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64-65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55-68 kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with an anti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

Assembly of branched ubiquitin oligomers by click chemistry.

Ubiquitin monomers functionalized with an azide or multiple alkynes were utilized for the assembly of branched ubiquitin oligomers (K6/K11, K11/K48, K11/K63, K6/K11/K48) by click chemistry. The oligomers resist deubiquitylase-catalysed hydrolysis and exhibit stability in eukaryotic cell lysates.

Protein Degradation and the Pathologic Basis of Disease.

The abundance of any protein is determined by the balance of protein synthesis and protein degradation. Regulated protein degradation has emerged as a powerful means of precisely controlling individual protein abundance within cells and is largely mediated by the ubiquitin-proteasome system (UPS). By controlling the levels of key regulatory proteins, the UPS contributes to nearly every aspect of cellular function. The UPS also functions in protein quality control, rapidly identifying and destroying misfolded or otherwise aberrant proteins that may be toxic to cells. Increasingly, we understand that dysregulation of protein degradation pathways is critical for many human diseases. Conversely, the versatility and scope of the UPS provides opportunities for therapeutic intervention. In this review, we will discuss the basic mechanisms of protein degradation by the UPS. We will then consider some paradigms of human disease related to protein degradation using selected examples. Finally, we will highlight several established and emerging therapeutic strategies based on altering pathways of protein degradation.

Dietary supplementation with ketoacids protects against CKD-induced oxidative damage and mitochondrial dysfunction in skeletal muscle of 5/6 nephrectomised rats.

BACKGROUND: A low-protein diet supplemented with ketoacids (LPD + KA) maintains the nutritional status of patients with chronic kidney disease (CKD). Oxidative damage and mitochondrial dysfunction associated with the upregulation of p66SHC and FoxO3a have been shown to contribute to muscle atrophy. This study aimed to determine whether LPD + KA improves muscle atrophy and attenuates the oxidative stress and mitochondrial damage observed in CKD rats. METHODS: 5/6 nephrectomy rats were randomly divided into three groups and fed with either 22% protein (normal-protein diet; NPD), 6% protein (low-protein diets; LPD) or 5% protein plus 1% ketoacids (LPD + KA) for 24 weeks. Sham-operated rats with NPD intake were used as the control. RESULTS: KA supplementation improved muscle atrophy and function in CKD + LPD rats. It also reduced the upregulation of genes related to the ubiquitin-proteasome system and 26S proteasome activity, as well as protein and mitochondrial oxidative damage in the muscles of CKD + LPD rats. Moreover, KA supplementation prevented the drastic decrease in activities of mitochondrial electron transport chain complexes, mitochondrial respiration, and content in the muscles of CKD + LPD rats. Furthermore, KA supplementation reversed the elevation in p66Shc and FoxO3a expression in the muscles of CKD + LPD rats. CONCLUSIONS: Our results showed that KA supplementation to be beneficial to muscle atrophy in CKD + LPD, which might be associated with improvement of oxidative damage and mitochondrial dysfunction through suppression of p66Shc and FoxO3a.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

SHORT-COMMUNICATION Evaluation of perfused bovine udder for gene expression studies in dairy cows.

Intramammary infections are one of the main causes of productivity loss in dairy cows. To better understand the immune system response and to avoid the use of live animals, we validated the use of isolated bovine udder as an ex situ model. Six mammary glands were collected from cows ready for culling. Three udders were perfused with Tyrode's solution and three were not-perfused. During six hours, we collected perfusate samples for biochemical analysis. We also collected alveolar and teat canal tissue to evaluate gene expression. The biochemical parameters indicated that the perfused udders remained viable for the entire period of the experiment. A real-time polymerase chain reaction showed an increase in 18S rRNA gene expression in the alveolar tissue at 3 and 4 h after perfusion. There was also an increase in the Ubiquitin gene in the teat canal from not-perfused udders at 1, 3, and 4 h after slaughter. In general, gene expression was stable during the experiment. Our results indicated that the isolated perfused bovine udder model is appropriate for genetic studies, opening a new perspective in animal experimentation methods.

Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains.

Ubiquitination of a subset of proteins by ubiquitin chain elongation factors (E4), represented by Ufd2p in Saccharomyces cerevisiae, is a pivotal regulator for many biological processes. However, the mechanism of Ufd2p-mediated ubiquitination is largely unclear. Here, we show that Ufd2p catalyses K48-linked multi-monoubiquitination on K29-linked ubiquitin chains assembled by the ubiquitin ligase (Ufd4p), resulting in branched ubiquitin chains. This reaction depends on the interaction of K29-linked ubiquitin chains with two N-terminal loops of Ufd2p. Only following the addition of K48-linked ubiquitin to substrates modified with K29-linked ubiquitin chains, can the substrates be escorted to the proteasome for degradation. We demonstrate that this ubiquitin chain linkage switching reaction is essential for ERAD, oleic acid and acid pH resistance in yeast. Thus, our results suggest that Ufd2p functions by switching ubiquitin chain linkages to allow the degradation of proteins modified with a ubiquitin linkage, which is normally not targeted to the proteasome.

Erythrocyte heat shock protein responses to chronic (in vivo) and acute (in vitro) temperature challenge in diploid and triploid salmonids.

This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions.

A chemical biology route to site-specific authentic protein modifications.

Many essential biological processes are controlled by posttranslational protein modifications. The inability to synthetically attain the diversity enabled by these modifications limits functional studies of many proteins. We designed a three-step approach for installing authentic posttranslational modifications in recombinant proteins. We first use the established O-phosphoserine (Sep) orthogonal translation system to create a Sep-containing recombinant protein. The Sep residue is then dephosphorylated to dehydroalanine (Dha). Last, conjugate addition of alkyl iodides to Dha, promoted by zinc and copper, enables chemoselective carbon-carbon bond formation. To validate our approach, we produced histone H3, ubiquitin, and green fluorescent protein variants with site-specific modifications, including different methylations of H3K79. The methylated histones stimulate transcription through histone acetylation. This approach offers a powerful tool to engineer diverse designer proteins.

Perturbed proteostasis in autism spectrum disorders.

Dynamic changes in synaptic strength rely on de novo protein synthesis and protein degradation by the ubiquitin proteasome system (UPS). Disruption of either of these cellular processes will result in significant impairments in synaptic plasticity and memory formation. Mutations in several genes encoding regulators of mRNA translation and members of the UPS have been associated with an increased risk for the development of autism spectrum disorders. It is possible that these mutations result in a similar imbalance in protein homeostasis (proteostasis) at the synapse. This review will summarize recent work investigating the role of the UPS in synaptic plasticity at glutamatergic synapses, and propose that dysfunctional proteostasis is a common consequence of several genetic mutations linked to autism spectrum disorders. Dynamic changes in synaptic strength rely on de novo protein synthesis and protein degradation by the ubiquitin proteasome system (UPS). Disruption of either of these cellular processes will result in significant impairments in synaptic plasticity and memory formation. Mutations in several genes encoding regulators of mRNA translation (i.e. FMR1) and protein degradation (i.e. UBE3A) have been associated with an increased risk for autism spectrum disorders and intellectual disability (ASD/ID). These mutations similarly disrupt protein homeostasis (proteostasis). Compensatory changes that reset the rate of proteostasis may contribute to the neurological symptoms of ASD/ID. This review summarizes recent work investigating the role of the UPS in synaptic plasticity at glutamatergic synapses, and proposes that dysfunctional proteostasis is a common consequence of several genetic mutations linked to ASD. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Fluorescent Reporters for Ubiquitin-Dependent Proteolysis in Plants.

Ubiquitin is a small protein commonly used as a signal molecule which upon attachment to the proteins affects their function and their fate in the cells. For example, it can be used as a degradation marker by the cell. Ubiquitin plays a significant role in regulation of numerous cellular processes. Therefore, monitoring of ubiquitin-dependent proteolysis can provide important information. Here, we describe construction of YFP-based proteasome substrates containing modified ubiquitin and the protocol for their transient expression in plant cells for functional analysis of the ubiquitin/proteasome system. To facilitate further subcloning all plasmids generated by us are based on the Gateway(®) Cloning Technology and are compatible with the Gateway(®) destination vectors.

The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors.

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co- and post-translational processing. Using an unbiased biochemical approach we found that UCHL3, USP9X, USP7, USP5 and Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway.

Nanoparticles Exacerbate Both Ubiquitin and Heat Shock Protein Expressions in Spinal Cord Injury: Neuroprotective Effects of the Proteasome Inhibitor Carfilzomib and the Antioxidant Compound H-290/51.

Increased levels of ubiquitin and heat shock protein (HSP) 72 kD are often seen in spinal cord injury (SCI). However, their roles in cell injury or survival are not well known. Thus, we have investigated the possible relationship between ubiquitin and HSP expressions in relation to cell injury in healthy animals, or following nanoparticle (NP) intoxication in SCI animals. A focal SCI was inflicted on the T10-11 segments over the right dorsal horn; animals were allowed to survive from 5 to 8 h after trauma. Separate groups of rats were exposed to SiO2, Ag, or Cu NPs for 7 days and subjected to SCI on the eighth day. A marked increase in ubiquitin or HSP immunoreactive cells occurred in the T9 to T12 segments 5 h after the injury, which further extended to the T4 and L5 after 8 h of survival. At this time, a marked increase in blood-spinal cord barrier (BSCB) permeability to Evans blue and radioiodine, accompanied by an intense edema formation, was observed. Changes were further exacerbated in NP-treated traumatized rats. The most marked expressions of ubiquitin and HSP in SCI were seen in rats treated with SiO2, followed by Ag, and Cu NPs. Treatment with H-290/51 (50 mg/kg p.o., 30 to 60 min after injury) or carfilzomib (1 mg/kg, i.v., 30 to 60 min after trauma) significantly reduced the ubiquitin or HSP expressions, as well as the BSCB breakdown, the edema formation, and the cell injury in the cord both 5 and 8 h after the injury, in normal animals. However, a double dose of H-290/51 (100 mg/kg) or carfilzomib (2 mg/kg) is needed to reduce cord pathology or ubiquitin and HSP expressions in traumatized animals treated with NPs. H-290/51 showed superior beneficial effects in reducing cord pathology in SCI than carfilzomib. These observations are the first to demonstrate that (i) NP-treated traumatized animals induce a widespread BSCB leakage, edema formation, and cord pathology as well as an overexpression of ubiquitin and HSP, (ii) high doses of antioxidant compounds or proteasome inhibitors are required for neuroprotection in the NP-exposed traumatized group, and (iii) ubiquitin and HSP expressions play a key role in neuronal injury in SCI, not reported earlier.

Cardiac Arrest Alters Regional Ubiquitin Levels in Association with the Blood-Brain Barrier Breakdown and Neuronal Damages in the Porcine Brain.

The possibility that ubiquitin expression is altered in cardiac arrest-associated neuropathology was examined in a porcine model using immunohistochemical and biochemical methods. Our observations show that cardiac arrest induces progressive increase in ubiquitin expression in the cortex and hippocampus in a selective and specific manner as compared to corresponding control brains using enzyme-linked immunoassay technique (enzyme-linked immunosorbent assay (ELISA)). Furthermore, immunohistochemical studies showed ubiquitin expression in the neurons exhibiting immunoreaction in the cytoplasm and karyoplasm of distorted or damaged cells. Separate Nissl and ubiquitin staining showed damaged and distorted neurons and in the same cortical region ubiquitin expression indicating that ubiquitin expression after cardiac arrest represents dying neurons. The finding that methylene blue treatment markedly induced neuroprotection following identical cardiac arrest and reduced ubiquitin expression strengthens this view. Taken together, our observations are the first to show that cardiac arrest enhanced ubiquitin expression in the brain that is related to the magnitude of neuronal injury and the finding that methylene blue reduced ubiquitin expression points to its role in cell damage, not reported earlier.

Downregulation of ubiquitin inhibits the proliferation and radioresistance of non-small cell lung cancer cells in vitro and in vivo.

Radioresistance has been an important factor in restricting efficacy of radiotherapy for non-small cell lung cancer (NSCLC) patients and new approaches to inhibit cancer growth and sensitize irradiation were warranted. Despite the important role of ubiquitin/proteasome system (UPS) during cancer progression and treatment, the expression and biological role of ubiquitin (Ub) in human NSCLC has not been characterized. In this study, we found that ubiquitin was significantly overexpressed in 75 NSCLC tissues, compared to their respective benign tissues by immunohistochemistry (P < 0.0001). Knock-down of ubiquitin by mixed shRNAs targeting its coding genes ubiquitin B (UBB) and ubiquitin C (UBC) suppressed the growth and increased the radiosensitivity in NSCLC H1299 cells. Apoptosis and γ H2AX foci induced by X-ray irradiation were enhanced by knock-down of ubiquitin. Western blot and immunostaining showed that knock-down of ubiquitin decreased the expression and translocation of NF-κB to the nucleus by reduced phospho-IκBα after irradiation. Suppression of ubiquitin decreased the proliferation and radioresistance of H1299 transplanted xenografts in nude mice by promoting apoptosis. Taken together, our results demonstrate the critical role of ubiquitin in NSCLC proliferation and radiosensitivity. Targeting ubiquitin may serve as a potentially important and novel approach for NSCLC prevention and therapy.

Plasma asymmetric and symmetric dimethylarginine in a rat model of endothelial dysfunction induced by acute hyperhomocysteinemia.

Hyperhomocysteinemia induces vascular endothelial dysfunction, an early hallmark of atherogenesis. While higher levels of circulating asymmetric dimethylarginine (ADMA) and symmetric dimethyl arginine (SDMA), endogenous inhibitors of nitric oxide synthesis, have been associated with increased cardiovascular risk, the role that ADMA and SDMA play in the initiation of hyperhomocysteinemia-induced endothelial dysfunction remains still controversial. In the present study, we studied the changes of circulating ADMA and SDMA in a rat model of acutely hyperhomocysteinemia-induced endothelial dysfunction. In healthy rats, endothelium-related vascular reactivity (measured as acetylcholine-induced transient decrease in mean arterial blood pressure), plasma ADMA and SDMA, total plasma homocysteine (tHcy), cysteine and glutathione were measured before and 2, 4 and 6 h after methionine loading or vehicle. mRNA expression of hepatic dimethylarginine dimethylaminohydrolase-1 (DDAH1), a key protein responsible for ADMA metabolism, was measured 6 h after the methionine loading or the vehicle. Expectedly, methionine load induced a sustained increase in tHcy (up to 54.9 ± 1.9 µM) and a 30 % decrease in vascular reactivity compared to the baseline values. Plasma ADMA and SDMA decreased transiently after the methionine load. Hepatic mRNA expression of DDAH1, cathepsin D, and ubiquitin were significantly lower 6 h after the methionine load than after the vehicle. The absence of an elevation of circulating ADMA and SDMA in this model suggests that endothelial dysfunction induced by acute hyperhomocysteinemia cannot be explained by an up-regulation of protein arginine methyltransferases or a down-regulation of DDAH1. In experimental endothelial dysfunction induced by acute hyperhomocysteinemia, down-regulation of the proteasome is likely to dampen the release of ADMA and SDMA in the circulation.