Nodular fasciitis of the breast: Report of two cases illustrating the diagnostic implications for USP6 gene rearrangement and brief review of the literature.

Nodular fasciitis is a benign, self-limited, pseudosarcomatous neoplasm that is cytogenetically characterized by recurrent USP6 gene rearrangement. Involvement of the breast by nodular fasciitis is very rare with only a few documented cases. It can clinically, radiologically and histologically mimic a malignancy, posing significant diagnostic challenges to clinicians, radiologists, and pathologists. In this study, we report 2 cases of nodular fasciitis occurring in the female breast, reviewing the literature and emphasizing the application of fluorescence in situ hybridization analysis of USP6 gene rearrangement in its diagnosis and differential diagnosis.

USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS.

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

Expression, purification and characterization of the second DUSP domain of deubiquitinase USP20/VDU2.

Deubiquitinase USP20/VDU2 (VHL-interacting Deubiquitinating Enzyme 2) has been proved to play vital roles in multiple cellular processes by controlling the life-span of substrate proteins including hypoxia-inducible factor HIF1α, ß2-adrenergic receptor, and type 2 iodothyronine deiodinase etc. USP20 contains four distinct structural domains, which include the N-terminal zinc-finger ubiquitin binding domain (ZnF-UBP), the catalytic domain (USP domain), and two tandem DUSP domains (DUSP1 and DUSP2). Here in this study, we report the setting up of the production approach for USP20 DUSP2, and the NMR characterization of the produced target protein. With the assistance of GB1 tag and glycerol, both the solubility and stability of USP20 DUSP2 are significantly enhanced. And by using the optimized protein production procedure, monomeric and stable 15N, 13C-labeled USP20 DUSP2 sample for NMR data acquisition was obtained. The secondary structural elements of USP20 DUSP2 were then revealed by the analysis of recorded NMR spectra, and USP20 DUSP2 forms an AB3 fold in solution. The production protocol and NMR characterization results reported in this manuscript could be utilized in the extended structural and functional studies of USP20 DUSP2.

Ubiquitin-specific protease as the underlying gene biomarker for aortic stenosis.

BACKGROUND: Aortic stenosis is a common heart valvular disease whose pathological processes include an inflammatory reaction and lipid accumulation. However, its detailed pathogenesis is yet to be completely elucidated. Therefore, it is of great significance to further explore the molecular mechanisms of aortic stenosis. METHODS: Four datasets were downloaded from the Gene Expression Omnibus (GEO) database. Firstly, the differently expressed genes (DEGs) were screened between control and aortic stenosis samples. Secondly, weighted gene co-expression network analysis (WGCNA) was performed to find the highly relevant gene modules. Enrichment analysis and protein-protein interaction (PPI) networking were also performed, then Cytoscape was used to identify hub genes. Finally, the six participants (3 control participants and 3 patients with aortic stenosis) were recruited at the Tianjin Chest Hospital. In order to verify the expression level of USP14, several molecular experiments were performed, including hematoxylin-eosin (HE) staining, immunohistochemistry, immunofluorescence technology, real time-quantitative polymerase chain reaction (RT-qPCR), and western blotting. RESULTS: A total of 9636 DEGs were found between the control and aortic stenosis samples. The DEGs were mainly enriched in the autophagy-animal, cellular lipid catabolic process, apoptosis, and glycoside metabolic process categories. Eleven hub genes were identified via four different algorithms. Following verification of the patient samples, Ubiquitin-specific protease 14 (USP14) was found to be displayed at higher levels in the aortic stenosis samples. CONCLUSION: USP14 might be involved in the occurrence and development of aortic stenosis, so it would be a molecular target for early diagnosis and specific treatment of aortic stenosis. There is a significant association between the high expression of USP14 and aortic stenosis, indicating that this gene may be a genetic risk factor for aortic stenosis.

Single-step functionalization of poly-catecholamine nanofilms for ultra-sensitive immunosensing of ubiquitin carboxyl terminal hydrolase-L1 (UCHL-1) in spinal cord injury.

Rapid, selective, and ultra-sensitive detection of brain and spinal cord injury markers in bodily fluids is an unmet clinical need. In this work, Polycatecholamine as a rich source of amine moieties was used for single-step fabrication of ultrasensitive immunosensors for the detection of Ubiquitin carboxyl-terminal hydrolase (UCHL-1) biomarker of brain and spinal cord injuries and address the clinical need. The surface of graphene electrodes was modified by electropolymerizing aqueous solution of dopamine (DA) and norepinephrine (NE) monomers for generating polycatecholamines nanofilms on the surface of graphene screen printed electrodes (GSPE) in a single functionalization step. Amine moieties of the polymer allowed immobilization of UCHL-1 antibody on the electrode. The single-step modification of GSPE offered a simple, ultrasensitive, and stable production of immunosensors for the detection of UCHL-1. The operational range of the UCHL-1 immunosensor developed with Polynorepinephrine pNE-modified is 0.1 pg mL-1 - 105 pg mL-1 (LOD: 1.91 pg mL-1), and 1 pg mL-1 - 105 pg mL-1 (LOD: 0.70 pg mL-1) with Polydopamine (pDA) modification, satisfying the clinical range. Both pNE and pDA modified immunosensors, detected UCHL-1 spiked in phosphate buffer saline, artificial cerebrospinal fluid, and serum. Along with the sensitive detections, selective performances were recorded in the above matrices in the presence of interfering neurotransmitters GABA and Glutamate as well as glial fibrillary acidic protein (GFAP). Upon testing clinical samples of spinal cord injury patients and healthy controls, both pNE and pDA immunosensors, delivered a comparable response for UCHL-1, thereby, making immunosensors useful for clinical settings.

Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47.

In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ∼146 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

Deubiquitinases and their emerging roles in ß-arrestin-mediated signaling.

The two homologous mammalian proteins called ß-arrestin1 (also known as arrestin2) and ß-arrestin2 (also called arrestin3) are now widely accepted as endocytic and signaling adaptors for G protein-coupled receptors (GPCRs), growth factor receptors, and ion channels. The sustained interactions of ß-arrestins with activated GPCRs have been shown to correlate with the agonist-induced ubiquitination on distinct domains in the ß-arrestin molecule. Additionally, ubiquitination of ß-arrestin promotes its interaction with proteins that mediate endocytosis (e.g., clathrin) and signaling (e.g., c-RAF). Recent studies have demonstrated that deubiquitination of ß-arrestin by specific deubiquitinating enzymes (DUBs) acts as an important regulatory mechanism, which determines the stability of ß-arrestin-GPCR binding and fine-tunes ß-arrestin-dependent signaling to downstream kinases. Accordingly, ubiquitination/deubiquitination of ß-arrestin can serve as an on/off switch for its signaling and endocytic functions. Moreover, by regulating the stability and localization of signalosomes, deubiquitination of ß-arrestins by DUBs imparts spatial and temporal resolution in GPCR signaling.

USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing.

Post-translational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of ubiquitin-specific peptidase 49 (USP49) as a histone H2B-specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. USP49 forms a complex with RuvB-like1 (RVB1) and SUG1 and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression but affects the abundance of >9000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) levels at these exons as well as upstream 3' and downstream 5' intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B-specific deubiquitinase and uncovers a critical role for H2B deubiquitination in cotranscriptional pre-mRNA processing events.

Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

UAF1 is a subunit of multiple deubiquitinating enzyme complexes.

A balance between ubiquitination and deubiquitination regulates numerous cellular processes and pathways, and specific deubiquitinating enzymes often play the decisive role of controlling this balance. We recently reported that the USP1 deubiquitinating enzyme, which regulates the Fanconi anemia pathway by deubiquitinating the central player of the pathway, FANCD2, is activated by the WD40-repeat containing UAF1 protein through formation of a stable USP1/UAF1 protein complex. Here we present the isolation of two novel multisubunit deubiquitinating enzyme complexes containing USP12 and USP46, respectively. Both complexes contain the UAF1 protein as a bona fide subunit. Interestingly, UAF1 regulates the enzymatic activity of both enzyme complexes, suggesting that this activator protein may regulate a subclass of human deubiquitinating enzymes. We postulate that additional WD40-containing proteins may also form complexes with other human deubiquitinating enzymes and thereby regulate their activity and substrate specificity.

Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

Integral protein microarrays for the identification of lung cancer antigens in sera that induce a humoral immune response.

The identification of biomarkers (both molecules and profiles) in patient sera offers enormous interest for the diagnosis of cancers. In this context, the detection of antibodies to tumor cell autologous antigens possesses great potential. The humoral immune response represents a form of biological amplification of signals that are otherwise weak because of very low concentrations of antigen, especially in the early stages of cancers. Herein we present the use of integral microarrays spotted with tumor-derived proteins to investigate the antibody repertoire in the sera of lung cancer patients and controls. The use of two-dimensional liquid chromatography allowed us to separate proteins from the lung adenocarcinoma cell line A549 into 1760 fractions, which were printed in duplicate, along with various controls, onto nitrocellulose coated slides. The sensitivity and specificity of the microarrays to detect singular antibodies in fluids were first validated through the recognition of fractions containing a lung marker antigen by antibody probing. Twenty fractions were initially selected as highly reactive against the anti-PGP9.5 antibody, and subsequent mass spectrometry analyses confirmed the identity of PGP9.5 protein in four of them. As a result, the importance of neighboring fractions in microarray detection was revealed due to the spreading of proteins during the separation process. Next, the microarrays were individually incubated with 14 serum samples from patients with lung cancer patients, 14 sera from colon cancer patients, and 14 control sera from normal subjects. The reactivity of the selected fractions was analyzed, and the level of immunoglobulin bound to each fraction by each serum sample was quantified. Eight of the 20 fractions offered p values < 0.01 and were recognized by an average of four reacting patients, whereas no serum from normal individuals was positive for those fractions. Protein microarrays from tumor-derived fractions hold the diagnostic potential of uncovering antigens that induce an immune response in patients with certain types of cancers.

Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles.

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.

Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering.

Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.

Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.