Deubiquitinase Research Articles

PINK1/Parkin Pathway Activation for Mitochondrial Quality Control - Which Is the Best Molecular Target for Therapy?

There has been long-term interest in drugging the PINK1-Parkin pathway with therapeutics as a treatment for Parkinson’s disease (PD). Despite significant structural data on Parkin as well as the PINK1 kinase and the multiple conformational changes it undergoes, activation of these targets is non-trivial. This review highlights small molecule screening results that suggests that activation of Parkin biochemically does not necessarily translate to activation of Parkin within cells. There are also issues with activation of PINK1 with kinetin analogs, which do not appear to rescue rodent models of PD. The counter-measure of activating the mitophagy pathway with deubiquitinase (DUB) inhibitors such as USP30 inhibitors is progressing in the clinic for kidney disease and the proof of biology for this target will be tested in these trials. An alternative mechanism of activating Parkin in response to oxidative stress via Parkin phosphorylation by the AMPK-ULK1 pathway may be a simpler way to lower the energy barrier Parkin activation.

Deubiquitinases in cell death and inflammation.

Apoptosis, pyroptosis, and necroptosis are distinct forms of programmed cell death that eliminate infected, damaged, or obsolete cells. Many proteins that regulate or are a part of the cell death machinery undergo ubiquitination, a post-translational modification made by ubiquitin ligases that modulates protein abundance, localization, and/or activity. For example, some ubiquitin chains target proteins for degradation, while others function as scaffolds for the assembly of signaling complexes. Deubiquitinases (DUBs) are the proteases that counteract ubiquitin ligases by cleaving ubiquitin from their protein substrates. Here, we review the DUBs that have been found to suppress or promote apoptosis, pyroptosis, or necroptosis.

On the Study of Deubiquitinases: Using the Right Tools for the Job.

Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design.

The requirement of ubiquitin C-terminal hydrolase L1 in mouse ovarian development and fertility†.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Identification of Deubiquitinases (DUBs) that modulate PAR1‐mediated p38 MAPK inflammatory signaling in endothelial cells

Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. To address this gap in knowledge, we sought to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. Currently, I am examining the impact of USP34‐mediated modulation of thrombin‐p38 signaling on endothelial inflammatory responses. I will present new data examining the role of USP34 on thrombin‐induced endothelial barrier disruption and induction of cytokine expression. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. Here, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

An siRNA library screen for endothelial PAR1‐specific deubiquitinases regulating p38 MAPK inflammatory signaling

Endothelial dysfunction is induced by inflammatory mediators that signal through GPCRs and a hallmark of inflammation. Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. The objective of this study was to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. New data examining the function of CYLD on thrombin‐induced inflammatory responses will be presented. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. In this study, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

OTUB1: A Key Player in the Adaptive Cardiac Hypertrophy

Deubiquitinases (DUBs), a family enzymes that reverse the process of ubiquitination, participate in diverse cellular processes through regulating protein degradation, intracellular trafficking, cell signaling, and protein‐protein interaction. The functional importance of DUBs in cardiac pathophysiology remains poorly defined. Herein, we report a critical role of OTUB1, one of the most highly expressed DUBs in the heart, in adaptive cardiac remodeling. Analysis of RNA sequencing data revealed that OTUB1 is upregulated in the failing human hearts. OTUB1 is also upregulated and activated in cardiomyocytes (CMs) treated with adrenergic agonists. OTUB1‐deficient CMs were resistant to phenylephrine (PE) ‐ and isoproterenol‐induced CM hypertrophy as characterized by attenuated cell enlargement, decreased fetal gene activation, and diminished protein synthesis. In vivo, CM‐specific deletion of OTUB1 in the developing mouse heart caused dilated cardiomyopathy without a prior development of compensatory cardiac hypertrophy, eventually leading to heart failure and premature lethality of mice by 6 months of age. The severe cardiac phenotype is accompanied by lipid accumulation, ER dilatation, prevalent autophagic vesicles containing ribosomes, as well as dysregulated expression of metabolic and ribosome genes. Delivery of wild‐type OTUB1, but not the enzymatic dead mutant, via AAV9 to OTUB1‐deficient hearts attenuated cardiac dysfunction and prolonged the lifespan of mice. On the molecular level, OTUB1 deficiency blunted mTOR signaling in cultured CMs and mouse hearts. Our findings indicate that OTUB1 is indispensable for the development of adaptive cardiac hypertrophy, possibly through modulating mTOR signaling.

Functional Dynamics of Deubiquitinase A

Deubiquitinase A (DUBA) is an isopeptidase that cleaves K63‐linked and K48‐linked ubiquitin chains from substrate proteins. DUBA belongs to the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs) and was initially identified as a negative regulator of the production of type I interferons. DUBA is also known to regulate the production of several other cytokines in both innate and adaptive immune cells. More recently, its role in regulation of the stability of chromatin regulators essential for human development has been revealed. DUBA is a model system for understanding how the activity and substrate specificity of DUBs are regulated by post‐translation modifications (PTMs) through modulation of the conformational ensemble that pre‐exists in the apo form of the enzyme. We have previously identified changes in the conformational ensemble of DUBA upon phosphorylation, which is essential for DUBA activity, using solution nuclear magnetic resonance (NMR) spectroscopy and activity assays. We have also characterized the conformational dynamics on the microsecond‐to‐millisecond timescales using NMR relaxation dispersion experiments. We will present NMR and activity data on several mutants of DUBA, which allowed us to identify the structural determinants of the previously observed conformational dynamics and to understand the functional roles of motions in DUBA. We will also present NMR data on the enzyme‐substrate complex, which shed light on the mechanism for the specificity of DUBA for the K63‐linked ubiquitin chain. Overall, our data suggest that the conformational dynamics of DUBA can be modulated by phosphorylation and are essential for the function of DUBA. Our study highlights the functional importance of conformational dynamics in the DUB family and the need to define dynamic properties of DUBs in order to fully understand how activity and substrate specificity can both be modulated by PTMs.

Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action.

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.

Structural basis for the linkage specificity of ubiquitin-binding domain and deubiquitinase.

Ubiquitination is a post-translational modification system essential for regulating a wide variety of biological processes in eukaryotes. Ubiquitin (Ub) itself undergoes post-translational modifications, including ubiquitination. All seven lysine residues and one N-terminal amino group of Ub can act as acceptors for further ubiquitination, producing eight types of Ub chains. Ub chains of different linkage types have different cellular functions and are referred to as the 'ubiquitin code'. Decoder molecules that contain linkage-specific Ub-binding domains (UBDs) recognize the Ub chains to regulate different cellular functions. On the other hand, deubiquitinases (DUBs) cleave Ub chains to reverse ubiquitin signals. This review discusses the molecular mechanisms of linkage-specific recognitions of Ub chains by UBDs and DUBs, which have been revealed by structural studies.

Loss of ubiquitin-specific peptidase 18 destabilizes 14-3-3ζ protein and represses lung cancer metastasis

ABSTRACT Cancer metastasis is a major cause of cancer-related mortality. Strategies to reduce metastases are needed especially in lung cancer, the most common cause of cancer mortality. We previously reported increased ubiquitin-specific peptidase 18 (USP18) expression in lung and other cancers. Engineered reduction of USP18 expression repressed lung cancer growth and promoted apoptosis. This deubiquitinase (DUB) stabilized targeted proteins by removing the complex interferon-stimulated gene 15 (ISG15). This study explores if the loss of USP18 reduced lung cancer metastasis. USP18 knock-down in lung cancer cells was independently achieved using small hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs). USP18 knock-down reduced lung cancer growth, wound-healing, migration, and invasion versus controls (P < .001) and markedly decreased murine lung cancer metastases (P < .001). Reverse Phase Protein Arrays (RPPAs) in shRNA knock-down lung cancer cells showed that 14-3-3ζ protein was regulated by loss of USP18. ISG15 complexed with 14-3-3ζ protein reducing its stability. Survival in lung adenocarcinomas (P < .0015) and other cancers was linked to elevated 14-3-3ζ expression as assessed by The Cancer Genome Atlas (TCGA). The findings were confirmed and extended using 14-3-3ζ immunohistochemical assays of human lung cancer arrays and syngeneic murine lung cancer metastasis models. A direct 14-3-3ζ role in controlling lung cancer metastasis came from engineered 14-3-3ζ knock-down in lung cancer cell lines and 14-3-3ζ rescue experiments that reversed migration and invasion inhibition. Findings presented here revealed that USP18 controlled metastasis by regulating 14-3-3ζ expression. These data provide a strong rationale for developing a USP18 inhibitor to combat metastases.

USP13: Multiple Functions and Target Inhibition.

As a deubiquitination (DUB) enzyme, ubiquitin-specific protease 13 (USP13) is involved in a myriad of cellular processes, such as mitochondrial energy metabolism, autophagy, DNA damage response, and endoplasmic reticulum-associated degradation (ERAD), by regulating the deubiquitination of diverse key substrate proteins. Thus, dysregulation of USP13 can give rise to the occurrence and development of plenty of diseases, in particular malignant tumors. Given its implications in the stabilization of disease-related proteins and oncology targets, considerable efforts have been committed to the discovery of inhibitors targeting USP13. Here, we summarize an overview of the recent advances of the structure, function of USP13, and its relations to diseases, as well as discovery and development of inhibitors, aiming to provide the theoretical basis for investigation of the molecular mechanism of USP13 action and further development of more potent druggable inhibitors.

USP20 regulates the stability of EMT transcription factor SOX4 and influences colorectal cancer metastasis

BackgroundColorectal cancer (CRC) is a familiar malignancy accompanied by higher morbidity and mortality. The deubiquitination enzyme USP20 has been discovered to be one key factor in several cancers progression. SOX4 is a critical transcription factor to regulate the expression of various genes, and participates into the occurrence and progression of cancers. In this study, it was aimed to illustrate the role of USP20 and the regulatory relationship between USP20 and SOX4 in CRC. MethodsThe protein expressions of USP20, SOX4, E-cadherin, N-cadherin, Snail and slug were tested through western blot. The cell proliferation ability was verified through CCK-8 assay. The migration and invasion abilities were detected through Transwell assay. The mRNA expression of SOX4 was confirmed through RT-qPCR. The interaction between USP20 and SOX4 was notarized through Co-IP assay. ResultOur study demonstrated that USP20 displayed higher expression, and facilitated CRC progression through regulating cell proliferation, migration, invasion and EMT process markers. USP20 was found to modulate SOX4 protein expression. Next, it was verified that USP20 regulated SOX4 degradation through deubiquitination. Finally, through rescue assays, we revealed that USP20 mediated SOX4 expression to accelerate CRC progression. ConclusionsIn this study, USP20 regulated the stability of EMT transcription factor SOX4 and aggravated colorectal cancer metastasis. This finding might highlight the function of USP20 in the treatment of CRC.

DUB to the rescue

Henning etal. (2022) report development of a novel class of agents, bivalent deubiquitinase (DUB)-targeting chimeras (DUBTACs), that can selectively stabilize protein targets. These findings encourage further pursuit of targeted protein stabilization as a new paradigm in chemical biology and drug discovery.

C-MYC-USP49-BAG2 axis promotes proliferation and chemoresistance of colorectal cancer cells in vitro

Deubiquitinases (DUBs) play critical roles in tumorigenesis and are emerging as potential therapeutic targets. However, it remains less clear which DUBs may play important roles and represent a realistic vulnerability for a particular type of tumor. Here we revealed that Ubiquitin Specific Peptidase 49 (USP49) is transcriptionally activated by c-MYC in colorectal cancer (CRC), and CRC patients with elevated USP49 levels exhibited significantly shorter survival. Knockdown of USP49 markedly inhibited CRC cell proliferation, colony formation, and chemotherapy resistance in vitro. Investigation of mechanisms unravels that USP49 deubiquitinates and stabilizes Bcl-2-Associated Athanogene 2 (BAG2), a well-known protein that antagonizes apoptosis and enables adaptive response of CRC cells. This study identified a novel mechanism by which USP49 promotes CRC cell survival by stabilizing BAG2 through the c-MYC-USP49-BAG2 axis, indicating that USP49 may become a potential therapeutic target for CRC.

Deubiquitinase Vulnerabilities Identified through Activity-Based Protein Profiling in Non-Small Cell Lung Cancer.

To aid in the prioritization of deubiquitinases (DUBs) as anticancer targets, we developed an approach combining activity-based protein profiling (ABPP) with mass spectrometry in both non-small cell lung cancer (NSCLC) tumor tissues and cell lines along with analysis of available RNA interference and CRISPR screens. We identified 67 DUBs in NSCLC tissues, 17 of which were overexpressed in adenocarcinoma or squamous cell histologies and 12 of which scored as affecting lung cancer cell viability in RNAi or CRISPR screens. We used the CSN5 inhibitor, which targets COPS5/CSN5, as a tool to understand the biological significance of one of these 12 DUBs, COPS6, in lung cancer. Our study provides a powerful resource to interrogate the role of DUB signaling biology and nominates druggable targets for the treatment of lung cancer subtypes.

Impacts of Cancer-associated Mutations on the Structure–Activity Relationship of BAP1

BRAC1 associated protein-1 (BAP1) is a major tumor suppressor involved in many cancers. The deubiquitinase (DUB) activity of BAP1 is essential for its nuclear localization, histone remodeling and proteostasis associated with mitochondrial calcium flux. Loss of the DUB activity due to catalytic mutations within the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) directly contributes to oncogenesis. Nevertheless, it is non-trivial to rationalize how the other high-frequency but non-catalytic mutations within the BAP1-UCH lead to malignancies. Here we used multiplex spectroscopic, thermodynamic and biophysical analyses to investigate the impacts of eleven high-occurrence mutations within BAP1-UCH on the structure, folding and function. Several mutations significantly destabilize BAP1-UCH and increase its aggregation propensity. Hydrogen-deuterium exchange mass spectrometry data revealed allosteric destabilizations caused by mutations distant from the catalytic site. Our findings gave a comprehensive and multiscale account of the molecular basis of how these non-catalytic mutations within BAP1-UCH may be implicated in oncogenesis.

Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis.

MYC as a transcriptional factor plays a crucial role in breast cancer progression. However, the mechanisms underlying MYC deubiquitination in breast cancer are not well defined. Here, we report that OTUB1 is responsible for MYC deubiquitination. OTUB1 could directly deubiquitinate MYC at K323 site, which blocks MYC protein degradation. Moreover, OTUB1 mediated MYC protein stability is also confirmed in OTUB1-knockout mice. Stabilized MYC by OTUB1 promotes its transcriptional activity and induces HK2 expression, which leads to enhance aerobic glycolysis. Therefore, OTUB1 promotes breast tumorigenesis in vivo and in vitro via blocking MYC protein degradation. Taken together, our data identify OTUB1 as a new deubiquitination enzyme for MYC protein degradation, which provides a potential target for breast cancer treatment.

SAGA Complex Subunits in Candida albicans Differentially Regulate Filamentation, Invasiveness, and Biofilm Formation.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a highly conserved, multiprotein co-activator complex that consists of five distinct modules. It has two enzymatic functions, a histone acetyltransferase (HAT) and a deubiquitinase (DUB) and plays a central role in processes such as transcription initiation, elongation, protein stability, and telomere maintenance. We analyzed conditional and null mutants of the SAGA complex module components in the fungal pathogen Candida albicans; Ngg1, (the HAT module); Ubp8, (the DUB module); Tra1, (the recruitment module), Spt7, (the architecture module) and Spt8, (the TBP interaction unit), and assessed their roles in a variety of cellular processes. We observed that spt7Δ/Δ and spt8Δ/Δ strains have a filamentous phenotype, and both are highly invasive in yeast growing conditions as compared to the wild type, while ngg1Δ/Δ and ubp8Δ/Δ are in yeast-locked state and non-invasive in both YPD media and filamentous induced conditions compared to wild type. RNA-sequencing-based transcriptional profiling of SAGA mutants reveals upregulation of hyphal specific genes in spt7Δ/Δ and spt8Δ/Δ strains and downregulation of ergosterol metabolism pathway. As well, spt7Δ/Δ and spt8Δ/Δ confer susceptibility to antifungal drugs, to acidic and alkaline pH, to high temperature, and to osmotic, oxidative, cell wall, and DNA damage stresses, indicating that these proteins are important for genotoxic and cellular stress responses. Despite having similar morphological phenotypes (constitutively filamentous and invasive) spt7 and spt8 mutants displayed variation in nuclear distribution where spt7Δ/Δ cells were frequently binucleate and spt8Δ/Δ cells were consistently mononucleate. We also observed that spt7Δ/Δ and spt8Δ/Δ mutants were quickly engulfed by macrophages compared to ngg1Δ/Δ and ubp8Δ/Δ strains. All these findings suggest that the SAGA complex modules can have contrasting functions where loss of Spt7 or Spt8 enhances filamentation and invasiveness while loss of Ngg1 or Ubp8 blocks these processes.

MiR-103a-3p Promotes Zika Virus Replication by Targeting OTU Deubiquitinase 4 to Activate p38 Mitogen-Activated Protein Kinase Signaling Pathway.

BackgroundMicroRNAs (miRNAs) play critical roles in regulating virus infection and replication. However, the mechanism by which miRNA regulates Zika virus (ZIKV) replication remains elusive. We aim to explore how the differentially expressed miR-103a-3p regulates ZIKV replication and to clarify the underlying molecular mechanism.MethodsSmall RNA sequencing (RNA-Seq) was performed to identify differentially expressed miRNAs in A549 cells with or without ZIKV infection and some of the dysregulated miRNAs were validated by quantitative real time PCR (qRT-PCR). The effect of miR-103a-3p on ZIKV replication was examined by transfecting miR-103a-3p mimic or negative control (NC) into A549 cells with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and expression levels of ZIKV NS5 mRNA and NS1 protein were detected by qRT-PCR and Western blot, respectively. The potential target genes for miR-103a-3p were predicted by four algorithms and further validated by mutation analysis through luciferase reporter assay. The predicated target gene OTU deubiquitinase (DUB) 4 (OTUD4) was over-expressed by plasmid transfection or silenced by siRNA transfection into cells prior to ZIKV infection. Activation status of p38 MAPK signaling pathway was revealed by looking at the phosphorylation levels of p38 (p-p38) and HSP27 (p-HSP27) by Western blot.ResultsThirty-five differentially expressed miRNAs in ZIKV-infected A549 cells were identified by RNA-Seq analysis. Five upregulated and five downregulated miRNAs were further validated by qRT-PCR. One of the validated upregulated miRNAs, miR-103a-3p significantly stimulated ZIKV replication both at mRNA (NS5) and protein (NS1) levels. We found p38 MAPK signaling was activated following ZIKV infection, as demonstrated by the increased expression of the phosphorylation of p38 MAPK and HSP27. Blocking p38 MAPK signaling pathway using SB203580 inhibited ZIKV replication and attenuated the stimulating effect of miR-103a-3p on ZIKV replication. We further identified OTUD4 as a direct target gene of miR-103a-3p. MiR-103a-3p over-expression or OTUD4 silencing activated p38 MAPK signaling and enhanced ZIKV replication. In contrast, OTUD4 over-expression inhibited p38 MAPK activation and decreased ZIKV replication. In addition, OTUD4 over-expression attenuated the stimulating effect of miR-103a-3p on ZIKV replication and activation of p38 MAPK signaling.ConclusionZika virus infection induced the expression of miR-103a-3p, which subsequently activated p38 MAPK signaling pathway by targeting OTUD4 to facilitate ZIKV replication.