Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo

ABSTRACT Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C26S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C26S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.

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Substitutions in an Active Site Loop ofEscherichia coliIscS Result in Specific Defects in Fe-S Cluster and Thionucleoside Biosynthesisin Vivo

Journal of Biological Chemistry ◽

10.1074/jbc.m401261200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19551-19558 ◽

Cited By ~ 31

Author(s):

Charles T. Lauhon ◽

Elizabeth Skovran ◽

Hugo D. Urbina ◽

Diana M. Downs ◽

Larry E. Vickery

Keyword(s):

Active Site ◽

Cluster Formation ◽

Wild Type ◽

Cysteine Desulfurase ◽

Mutant Proteins ◽

Active Site Cysteine ◽

Sulfur Transfer ◽

Amino Acid Region

IscS catalyzes the fragmentation ofl-cysteine tol-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. InEscherichia coliIscS, the active site cysteine Cys328resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys328identified additional residues important for IscS functionin vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesisin vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s2C and ms2i6A) yet showed wild type levels of Fe-S-independent thionucleosides (s4U and mnm5s2U) that require persulfide formation and transfer.In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesisin vivowithout detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

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Operative ubiquitin-specific protease 22 deubiquitination confers a more invasive phenotype to cholangiocarcinoma

Cell Death and Disease ◽

10.1038/s41419-021-03940-0 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Yu Tian ◽

Bo Tang ◽

Chengye Wang ◽

Yan Wang ◽

Jiakai Mao ◽

...

Keyword(s):

Tumour Growth ◽

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Sirtuin 1 ◽

Mesenchymal Transition ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Paired Tumour

AbstractOncogenic ubiquitin-specific protease 22 (USP22) is implicated in a variety of tumours; however, evidence of its role and underlying molecular mechanisms in cholangiocarcinoma (CCA) development remains unknown. We collected paired tumour and adjacent non-tumour tissues from 57 intrahepatic CCA (iCCA) patients and evaluated levels of the USP22 gene and protein by qPCR and immunohistochemistry. Both the mRNA and protein were significantly upregulated, correlated with the malignant invasion and worse OS of iCCA. In cell cultures, USP22 overexpression increased CCA cell proliferation and mobility, and induced epithelial-to-mesenchymal transition (EMT). Upon an interaction, USP22 deubiquitinated and stabilized sirtuin-1 (SIRT1), in conjunction with Akt/ERK activation. In implantation xenografts, USP22 overexpression stimulated tumour growth and metastasis to the lungs of mice. Conversely, the knockdown by USP22 shRNA attenuated the tumour growth and invasiveness in vitro and in vivo. Furthermore, SIRT1 overexpression reversed the USP22 functional deficiency, while the knockdown acetylated TGF-β-activated kinase 1 (TAK1) and Akt. Our present study defines USP22 as a poor prognostic predictor in iCCA that cooperates with SIRT1 and facilitates tumour development.

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NLRP7 deubiquitination by USP10 promotes tumor progression and tumor-associated macrophage polarization in colorectal cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01920-y ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Bing Li ◽

Zhi-Peng Qi ◽

Dong-Li He ◽

Zhang-Han Chen ◽

Jing-Yi Liu ◽

...

Keyword(s):

Tumor Progression ◽

Acceptor Site ◽

Protein Levels ◽

Qrt Pcr ◽

Chemokine Ligand ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Chemokine Ligand 2

Abstract Background NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR, and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role in tumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulated in colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumor progression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism. Methods NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRC progression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitation and mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellular ubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning and mutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokines affected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry. Results NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expression was associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro and interacted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability and protein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in the ubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysine acceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κB nuclear translocation and activation of C-C motif chemokine ligand 2 transcription. Conclusions We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism by which NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serve as a biomarker and novel therapeutic target for the treatment of CRC.

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Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

Journal of Virology ◽

10.1128/jvi.75.21.10054-10064.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10054-10064 ◽

Cited By ~ 18

Author(s):

Jerg Schmidt ◽

Volker Gerdts ◽

Jörg Beyer ◽

Barbara G. Klupp ◽

Thomas C. Mettenleiter

Keyword(s):

Host Range ◽

Pseudorabies Virus ◽

Natural Host ◽

Target Cells ◽

Glycoprotein D ◽

Wild Type ◽

Cellular Receptors ◽

Receptor Usage

ABSTRACT Infection of cells by herpesviruses is initiated by the interaction of viral envelope glycoproteins with cellular receptors. In the alphaherpesvirus pseudorabies virus (PrV), the causative agent of Aujeszky's disease in pigs, the essential glycoprotein D (gD) mediates secondary attachment of virions to target cells by binding to newly identified cellular receptors (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998). However, in the presence of compensatory mutations, infection can also occur in the absence of gD, as evidenced by the isolation in cell culture of an infectious gD-negative PrV mutant (PrV-gD− Pass) (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). PrV-gD− Pass is replication competent with an only moderate reduction in specific infectivity but appears to bind to receptors different from those recognized by wild-type PrV (A. Karger, J. Schmidt, and T. C. Mettenleiter, J. Virol. 72:7341–7348, 1998). To analyze whether this alteration in receptor usage in vitro influences infection in vivo, the model host mouse and the natural host pig were intranasally infected with PrV-gD− Pass and were compared to animals infected by wild-type PrV. For mice, a comparable progress of disease was observed, and all animals infected with mutant virus died, although they exhibited a slight delay in the onset of symptoms and, correspondingly, a longer time to death. In contrast, whereas wild-type PrV-infected pigs showed clinical signs and histological and histopathological findings typical of PrV infection, no signs of disease were observed after infection with PrV-gD− Pass. Moreover, in these animals, virus-infected cells were not detectable by immunohistochemical staining of different organ samples and no virus could be isolated from nasal swabs. Mutations in glycoproteins B and H were found to correlate with, and probably contribute to, gD-independent infectivity. In conclusion, although PrV-gD− Pass is virulent in mice, it is apparently unable to infect the natural host, the pig. This altered host range in vivo correlates with a difference of receptor usage in vitro and demonstrates for the first time the importance of gD receptors in alphaherpesvirus infection of an animal host.

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Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.7.3353-3365.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3353-3365 ◽

Cited By ~ 183

Author(s):

Chi-Long Lin ◽

Che-Sheng Chung ◽

Hans G. Heine ◽

Wen Chang

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

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Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽

10.1128/mbio.02675-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Autumn T. LaPointe ◽

V Douglas Landers ◽

Claire E. Westcott ◽

Kevin J. Sokoloski

Keyword(s):

Sindbis Virus ◽

Virus Disease ◽

Severe Disease ◽

Wild Type Virus ◽

Wild Type ◽

Genomic Rnas ◽

Mortality And Morbidity ◽

The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

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Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances the Kinetics of Retrograde, Transneuronal Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.02102-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1173-1183 ◽

Cited By ~ 28

Author(s):

D. Curanović ◽

M. G. Lyman ◽

C. Bou-Abboud ◽

J. P. Card ◽

L. W. Enquist

Keyword(s):

Pseudorabies Virus ◽

Particle Production ◽

Neural Circuit ◽

Neuronal Cultures ◽

Wild Type ◽

Infectious Particle ◽

Viral Spread ◽

Kinetics Of

ABSTRACT The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the UL21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha UL21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.

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Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3

Biochemical Journal ◽

10.1042/bj20101152 ◽

2010 ◽

Vol 431 (3) ◽

pp. 391-402 ◽

Cited By ~ 13

Author(s):

Boon Shang Chew ◽

Wee Leng Siew ◽

Benjamin Xiao ◽

Norbert Lehming

Keyword(s):

Mutant Strain ◽

Transcriptional Activation ◽

Glutathione Transferase ◽

Binding Protein ◽

Tata Binding Protein ◽

Mutant Protein ◽

Specific Protease ◽

Ubiquitin Specific Protease

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)–Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

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USP38 critically promotes asthmatic pathogenesis by stabilizing JunB protein

Journal of Experimental Medicine ◽

10.1084/jem.20172026 ◽

2018 ◽

Vol 215 (11) ◽

pp. 2850-2867 ◽

Cited By ~ 4

Author(s):

Siyuan Chen ◽

Fenglin Yun ◽

Yikun Yao ◽

Mengtao Cao ◽

Yifan Zhang ◽

...

Keyword(s):

Allergic Asthma ◽

Molecular Mechanisms ◽

Protein Stabilization ◽

Th2 Immune Response ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Th2 Immunity ◽

Th2 Development

Th2 immune response is critical for allergic asthma pathogenesis. Molecular mechanisms for regulating Th2 immunity are still not well understood. Here we report that the ubiquitin-specific protease USP38 is crucial for Th2-mediated allergic asthma. TCR stimulation up-regulated the USP38 level, and USP38 in turn mediated the protein stabilization of JunB, a transcription factor specific for Th2 development. Consequently, USP38 was specifically required for TCR-induced production of Th2 cytokines and Th2 development both in vitro and in vivo, and USP38-deficient mice were resistant to asthma pathogenesis induced by OVA or HDM. Mechanistically, USP38 directly associated with JunB, deubiquitinated Lys-48–linked poly-ubiquitination of JunB, and consequently blocked TCR-induced JunB turnover. USP38 represents the first identified deubiquitinase specifically for Th2 immunity and the associated asthma.

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Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010301117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 27989-27996

Author(s):

Yasushi Daimon ◽

Shin-ichiro Narita ◽

Ryoji Miyazaki ◽

Yohei Hizukuri ◽

Hiroyuki Mori ◽

...

Keyword(s):

Active Site ◽

Protease Activity ◽

Underlying Mechanism ◽

Zinc Ion ◽

Wild Type ◽

Protease Domain ◽

Loop Region ◽

Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

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