Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling

Shi, Junyan; Wong, Jerry; Piesik, Paulina; Gabriel, Franck; Zhang, Jingchun; Jagdeo, Julienne; Li, Xiaotao; Jan, Eric; Luo, Honglin

doi:10.4161/auto.26059

Cited by 103 publications

(102 citation statements)

References 38 publications

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“…Similar to our previous observation, 11 Triton-insoluble ubiquitin conjugates were accumulated in cells overexpressing SQSTM1-WT ( Figures 4b and c). Interestingly, we found that SQSTM1-WT-mediated insoluble aggregate formation was blocked in the presence of SQSTM1-C (Figures 4b and c).…”

supporting

confidence: 77%

“…We have previously demonstrated that SQSTM1 is cleaved during coxsackievirus infection, resulting in the disruption of the function of SQSTM1. 11 As NBR1 may have a compensatory role for the loss of SQSTM1, we examined NBR1 expression after CVB3 infection. We found that NBR1 was also potentially cleaved after CVB3 infection, generating at least two cleavage fragments (B100 and B50 kDa, respectively) using an anti-N-terminal NBR1 antibody (Figure 1a).…”

Section: Resultsmentioning

confidence: 99%

“…11 The cleavage of SQSTM1 results in the dissociation of the N-terminal PB1 domain from the C-terminal UBA and LIR domains, and subsequently the loss-of-function of full-length SQSTM1 in mediating selective autophagy.…”

mentioning

confidence: 99%

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Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

et al. 2014

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Coxsackievirus infection induces an abnormal accumulation of ubiquitin aggregates that are generally believed to be noxious to the cells and have a key role in viral pathogenesis. Selective autophagy mediated by autophagy adaptor proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), are an important pathway for disposing of misfolded/ubiquitin conjugates. We have recently demonstrated that SQSTM1 is cleaved after coxsackievirus infection, resulting in the disruption of SQSTM1 function in selective autophagy. NBR1 is a functional homolog of SQSTM1. In this study, we propose to test whether NBR1 can compensate for the compromise of SQSTM1 after viral infection. Of interest, we found that NBR1 was also cleaved after coxsackievirus infection. This cleavage took place at two sites mediated by virus-encoded protease 2A pro and 3C pro , respectively. In addition to the loss-of-function, we further investigated whether cleavage of SQSTM1/NBR1 leads to the generation of toxic gain-of-function mutants. We showed that the C-terminal fragments of SQSTM1 and NBR1 exhibited a dominant-negative effect against native SQSTM1/NBR1, probably by competing for LC3 and ubiquitin chain binding. Finally, we demonstrated a positive, mutual regulatory relationship between SQSTM1 and NBR1 during viral infection. We showed that knockdown of SQSTM1 resulted in reduced expression of NBR1, whereas overexpression of SQSTM1 led to increased level of NBR1, and vice versa, further excluding the possible compensation of NBR1 for the loss of SQSTM1. Taken together, the findings in this study suggest a novel mechanism through which coxsackievirus infection induces increased accumulation of ubiquitin conjugates and subsequent viral damage.

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supporting

confidence: 77%

Section: Resultsmentioning

confidence: 99%

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Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

et al. 2014

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“…Indirect immunofluorescence staining was performed as previously described. 51 In brief, after fixation and permeabilization, cells were incubated with primary antibody (anti-HA at a dilution of 1 : 200 or anti-dsRNA at a dilution of 1 : 400) at 4°C overnight, and then with secondary antibody for 1 h. After washing with PBS, coverslips were mounted using Vectashield mounting medium containing 4' 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, #H1200). Images were taken with a Leica SP2 AOBS confocal fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

“…54,55 HeLa cell lysates (50 μg) were incubated with various concentrations of purified CVB3 2A, 3C, or 3C mutant as indicated in a cleavage reaction buffer (20 mM HEPES (pH 7.4), 150 mM KOAc and 1 mM DTT) at 37˚C for different times as previously described. 20,51 The reaction was stopped by addition of 6 × SDS-PAGE sample buffer and TDP-43 cleavage was assessed by western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

et al. 2015

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We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at aminoacid 327 between glutamine and alanine, resulting in the generation of an N-and C-terminal cleavage fragment of~35 and~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. Coxsackievirus B3 (CVB3) is a small, positive-stranded RNA enterovirus. 1 The single open reading frame of CVB3 is translated into a viral polypeptide that is subsequently cleaved by two virus-encoded proteases 2A and 3C to generate structural and non-structural proteins. 2 In addition to processing viral polyprotein, 2A and 3C target host proteins important for maintenance of protein translation and transcription, antiviral activity, and cellular architecture and signaling, contributing to virus-induced pathogenesis. [3][4][5] Although enteroviral replication takes place exclusively in the cytoplasm, viral infection has been demonstrated to lead to cytoplasmic translocation of nuclear proteins. 6 For example, heterogeneous ribonucleoprotein D (hnRNP D) has been shown to translocate from the nucleus to the cytoplasm during enteroviral infection. 5,7,8 Moreover, hnRNP D is cleaved by 3C and has an antiviral function against enteroviral infection. 5,7,8 Cytoplasmic translocation after enteroviral infection has also been demonstrated for several other hnRNPs (A1, C, and K)...

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Novel quantitative immunohistochemistry method using histone H3, family 3B as the internal reference standard for measuring human epidermal growth factor receptor 2 expression in breast cancer

Wang,

Xia,

Tian

et al. 2024

Cancer

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BackgroundImmunohistochemistry (IHC) is an essential technique in surgical and clinical pathology for detecting diagnostic, prognostic, and predictive biomarkers for personalized cancer therapy. However, the lack of standardization and reference controls results in poor reproducibility, and a reliable tool for IHC quantification is urgently required. The objective of this study was to describe a novel approach in which H3F3B (histone H3, family 3B) can be used as an internal reference standard to quantify protein expression levels using IHC.MethodsThe authors enrolled 89 patients who had human epidermal growth factor receptor 2 (HER2)‐positive breast cancer (BC). They used a novel IHC‐based assay to measure protein expression using H3F3B as the internal reference standard. H3F3B was uniformly expressed at the protein level in all tumor regions in cancer tissues. HER2 expression levels were measured with the H‐score using HALO software.ResultsKaplan–Meier analysis indicated that, among patients who had HER2‐positive BC in The Cancer Genome Atlas data set and the authors' data set, the subgroup with low HER2 expression had a significantly better prognosis than the subgroup with high HER2 expression. Furthermore, the authors observed that HER2 expression levels were precisely evaluated using the proposed method, which can classify patients who are at higher risk of HER2‐positive BC to receive trastuzumab‐based adjuvant therapy. Dual‐color IHC with H3F3B is an excellent tool for internal and external quality control of HER2 expression assays.ConclusionsThe proposed IHC‐based quantification method accurately assesses HER2 expression levels and provides insights for predicting clinical prognosis in patients with HER2‐positive BC who receive trastuzumab‐based adjuvant therapy.

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Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling

Cited by 103 publications

References 38 publications

Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Novel quantitative immunohistochemistry method using histone H3, family 3B as the internal reference standard for measuring human epidermal growth factor receptor 2 expression in breast cancer

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