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AbstractObjective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11’s role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs–human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

Abstract Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCFFBXO9, that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCFFBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCFFBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the latest variant in the coronavirus family, causing COVID-19, has resulted in global pandemic since early 2020 leading to severe public health concern. So far, the pandemic has caused more than 200 million infections and 4 million deaths worldwide. Most of the studies are focused on developing prevention, intervention, and therapeutic strategies. However, underlying pathophysiology of the disease is important as well, which needs further attention. Cell death is one of the major causative mechanisms that leads to severe inflammation, and it is also an a posteriori consequence of the hyperinflammatory storm that renders poor prognosis of the disease. Substantial cell death has been reported in biopsy samples from post mortem patients. Among the distinct cell death pathways, apoptosis, the regulated programmed cell death plays an important role in the pathogenesis of the disease. Understanding the role of SARS-CoV-2 infection in apoptosis is critical to linearize the pathogenesis of the virus as well as the resultant disease, that may uncover novel therapeutic targets in treatment of COVID-19 patients. Here, we review the current progress on the underlying molecular mechanism(s) of SARS-CoV-2-induced apoptosis, not only at the level of the virus but also at its individual proteins.

ABSTRACT The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become a severe global public health crisis. Therefore, understanding the molecular details of SARS-CoV-2 will be critical for fighting the virus’s spread and preventing future pandemics. In this study, we globally profiled the stability of SARS-CoV-2-encoded proteins, studied their degradation pathways, and determined their correlation with the antibody responses in patient plasma. We identified 18 proteins with unstable half-lives and 6 relatively stable proteins with longer half-lives. The labile SARS-CoV-2 proteins were degraded mainly by the ubiquitin-proteasome pathway. We also observed a significant correlation between antibody levels and protein half-lives, which indicated that a stable antigen of SARS-CoV-2 could be more effective for eliciting antibody responses. In addition, levels of antiviral antibodies targeting NSP10 were found to be negatively correlated with systemic levels of interleukin 6 (IL-6) in patients. These findings may facilitate the development of novel therapeutic or diagnostic approaches. IMPORTANCE SARS-CoV-2, the etiological cause of COVID-19, carries 29 genes in its genome. However, our knowledge of the viral proteins in biological and biochemical aspects is limited. In this study, we globally profiled the stability of the viral proteins in living lung epithelial cells. Importantly, the labile SARS-CoV-2-encoded proteins were mainly degraded through the ubiquitin-proteasome pathway. Stable proteins, including spike and nucleocapsid, of SARS-CoV-2 were more effective in eliciting antibody production. The levels of antiviral antibodies targeting NSP10 were negatively correlated with systemic levels of IL-6 in COVID-19 patients.

Abstract Background Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. Methods To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. Results T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Abstract Background Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. Results Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24 h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-κB signaling. Conclusions Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.

Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for the cause of acute lung injury and acute respiratory distress syndrome. P. aeruginosa isthe leading species isolated from patients with nosocomial infection and is detected in almost all the patients with long term ventilation in critical care units. P. aeruginosa infection is also the leading cause of deleterious chronic lung infections in patients suffering from cystic fibrosis as well as the major reason for morbidity in people with chronic obstructive pulmonary disease. P. aeruginosa infections are linked to diseases with high mortality rates and are challenging for treatment, for which no effective remedies have been developed. Massive lung epithelial cell death is a hallmark of severe acute lung injury and acute respiratory distress syndrome caused by P. aeruginosa infection. Lung epithelial cell death poses serious challenges to air barrier and structural integrity that may lead to edema, cytokine secretion, inflammatory infiltration, and hypoxia. Here we review different types of cell death caused by P. aeruginosa serving as a starting point for the diseases it is responsible for causing. We also review the different mechanisms of cell death and potential therapeutics in countering the serious challenges presented by this deadly bacterium.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are characterized by an inflammatory response, alveolar edema, and hypoxemia. ARDS occurs most often in the settings of pneumonia, sepsis, aspiration of gastric contents, or severe trauma. The prevalence of ARDS is approximately 10% in patients of intensive care. There is no effective remedy with mortality high at 30–40%. Most functional proteins are dynamic and stringently governed by ubiquitin proteasomal degradation. Protein ubiquitination is reversible, the covalently attached monoubiquitin or polyubiquitin moieties within the targeted protein can be removed by a group of enzymes called deubiquitinating enzymes (DUBs). Deubiquitination plays an important role in the pathobiology of ALI/ARDS as it regulates proteins critical in engagement of the alveolo-capillary barrier and in the inflammatory response. In this review, we provide an overview of how DUBs emerge in pathogen-induced pulmonary inflammation and related aspects in ALI/ARDS. Better understanding of deubiquitination-relatedsignaling may lead to novel therapeutic approaches by targeting specific elements of the deubiquitination pathways.

Summary: Acute lung injury (ALI) is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1). Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme. : It is unknown why people with severe bacterial infections develop mitochondrial dysfunction with impaired cellular oxygenation. Here, Mallampalli and colleagues show that S. aureus lung infection in cells and mice induces an ubiquitin E3 ligase subunit to mediate degradation of a mitochondrial biosynthetic enzyme, CLS1, after its phosphorylation by PINK1. The data provide mechanistic insights into mitochondrial bioenergetics during pneumonia.

The participation of large-conductance Ca2+ activated K+ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca2+ sparks and caffeine-induced global Ca2+ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro.

Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

Background: One hallmark of sepsis is the reduced number of lymphocytes, termed lymphopenia, that occurs from decreased lymphocyte proliferation or increased cell death contributing to immune suppression. Histone modification enzymes regulate immunity by their epigenetic and non-epigenetic functions; however, the role of these enzymes in lymphopenia remains elusive.Methods: We used molecular biological approaches to investigate the high expression and function of a chromatin modulator protein arginine N-methyltransferase 4 (PRMT4)/coactivator-associated arginine methyltransferase 1 in human samples from septic patients and cellular and animal septic models.Results: We identified that PRMT4 is elevated systemically in septic patients and experimental sepsis. Gram-negative bacteria and their derived endotoxin lipopolysaccharide (LPS) increased PRMT4 in B and T lymphocytes and THP-1 monocytes. Single-cell RNA sequencing results indicate an increase of PRMT4 gene expression in activated T lymphocytes. Augmented PRMT4 is crucial for inducing lymphocyte apoptosis but not monocyte THP-1 cells. Ectopic expression of PRMT4 protein caused substantial lymphocyte death via caspase 3-mediated cell death signalling, and knockout of PRMT4 abolished LPS-mediated lymphocyte death. PRMT4 inhibition with a small molecule compound attenuated lymphocyte death in complementary models of sepsis.Conclusions: These findings demonstrate a previously uncharacterised role of a key chromatin modulator in lymphocyte survival that may shed light on devising therapeutic modalities to lessen the severity of septic immunosuppression. [ABSTRACT FROM AUTHOR]

FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here we show that TNFα, a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation as reflected by reduced expression of myogenin and impaired myotube formation. TNFa did not destabilize the Fbxl2 transcript (t 10 h), but inhibited SP1 transactivation of its core promoter, localized to 160/+42 base pairs within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect less pronounced during proliferation or after TNFα exposure. TNFα, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key pro-inflammatory myopathic cytokine. [ABSTRACT FROM AUTHOR]

Cigarette smoking increases susceptibility for microbial infection in respiratory system. However, the underlying molecular mechanism(s) is not fully elucidated. Here we report that cigarette smoking extract (CSE) increases bacterial load in lung epithelial cells via downregulation of the ubiquitin-specific protease 25 (USP25)/histone deacetylase 11 (HDAC11) axis. CSE treatment decreases HDAC11 at protein level in lung epithelial cells without significant changes of its transcription. Concomitantly, CSE treatment accelerates a ubiquitin-specific protease USP25 ubiquitination and degradation. Coimmunoprecipitation studies showed that USP25 associated with HDAC11. USP25 catalyzes deubiquitination of HDAC11, which regulates HDAC11 protein stability. CSE-mediated degradation of USP25 thereafter reduces HDAC11 at the protein level. Interestingly, CSE-downregulated USP25/HDAC11 axis increases the bacterial load of Pseudomonas aeruginosa in lung epithelial cells. These findings suggest that CSE-downregulated USP25 and HDAC11 may contribute to high susceptibility of bacterial infection in the cigarette smoking population. [ABSTRACT FROM AUTHOR]

E3 ubiquitin ligase recognizes its protein substrates via specific molecular signatures for ubiquitin proteasomal degradation. However, the role of acetylation/deacetylation in the process of E3 ubiquitin ligase recognizing its protein substrates is not fully studied. Here, we report that a tandem IK motif in protein arginine methyltransferase 1 (PRMT1) forms an acetyldegron to recruit the F-box/LRR-repeat protein 17 (FBXL17), a component of the SKP1--CUL1--F-box protein (SCF)-type E3 ubiquitin ligase complex. PRMT1 is polyubiquitylated for proteasome degradation with a half-life of approximately 4 h in lung epithelial cells. SCFFbxl17 mediates PRMT1 polyubiquitylation at K117. SCFFbxl17 specifically binds PRMT1 via a unique motif IKxxxIK. Strikingly, the acetylation/deacetylation status of the lysine residues within the motif determines Fbxl17 binding. Deacetylation on both K200 and K205 by Sirtuin 1 (Sirt1) and acetylation of p300 (EP300) on K205 collaboratively prepare the motif for SCFFbxl17 binding thereby triggering PRMT1 protein degradation. Pathogen-derived lipopolysaccharide (LPS) downregulates Sirt1 and p300 to protect PRMT1 from degradation. This study demonstrates that LPS promotes PRMT1 stability by blockade of PRMT1 and SCFFbxl17 binding via an acetylation/deacetylation-modified acetyldegron; and LPS-elevated levels of PRMT1 lead to bronchial epithelial cell overgrowth in pulmonary inflammatory diseases. [ABSTRACT FROM AUTHOR]

Oxidative stress impacts normal cellular function leading to the pathogenesis of various diseases including pulmonary illnesses. Protein arginine methyltransferase 4 (PRMT4) is critical for normal lung alveolar epithelial cell development; however, the regulation of PRMT4 within such pulmonary diseases has yet to be elucidated. Using biochemical approaches, we uncovered that peroxide (H2O2) treatment decreases PRMT4 protein stability in murine lung epithelial (MLE12) cells to impede cell migration. Protein kinase glycogen synthase kinase 3β (GSK-3β) interacts with PRMT4 and catalyzes PRMT4 T132 phosphorylation that protects PRMT4 from ubiquitin proteasomal degradation. H2O2 downregulates GSK-3β to reduce PRMT4 at protein level. PRMT4 promotes cell migration and H2O2 degrades PRMT4 to inhibit lung epithelial cell migration. These observations demonstrate that oxidative stress destabilizes PRMT4 via GSK-3β signaling to impede lung epithelial cell migration that may hinder the lung repair and regeneration process. [ABSTRACT FROM AUTHOR]

Cardiolipin (also known as PDL6) is an indispensable lipid required for mitochondrial respiration that is generated through de novo synthesis and remodeling. Here, the cardiolipin remodeling enzyme, acyl-CoA:lysocardiolipin-acyltransferase-1 (Alcat1; SwissProt ID, Q6UWP7) is destabilized in epithelia by lipopolysaccharide (LPS) impairing mitochondrial function. Exposure to LPS selectively decreased levels of carbon 20 (C20)-containing cardiolipin molecular species, whereas the content of C18 or C16 species was not significantly altered, consistent with decreased levels of Alcat1. Alcat1 is a labile protein that is lysosomally degraded by the ubiquitin E3 ligase Skp--Cullin--F-box containing the Fbxo28 subunit (SCF-Fbxo28) that targets Alcat1 for monoubiquitylation at residue K183. Interestingly, K183 is also an acetylation-acceptor site, and acetylation conferred stability to the enzyme. Histone deacetylase 2 (HDAC2) interacted with Alcat1, and expression of a plasmid encoding HDAC2 or treatment of cells with LPS deacetylated and destabilized Alcat1, whereas treatment of cells with a pan-HDAC inhibitor increased Alcat1 levels. Alcat1 degradation was partially abrogated in LPS-treated cells that had been silenced for HDAC2 or treated with MLN4924, an inhibitor of Cullin--RING E3 ubiquitin ligases. Thus, LPS increases HDAC2-mediated Alcat1 deacetylation and facilitates SCF-Fbxo28-mediated disposal of Alcat1, thus impairing mitochondrial integrity. [ABSTRACT FROM AUTHOR]

The article discusses a study focusing on the mediation of epithelial cell death in a mouse model of pneumonia by mortality factor 4 like 1 (Morf4l1) protein. Topics discussed include the involvement of Morf4l1 in chromatin remodeling, the effect of exposure to pseudomonas aeruginosa or lipopolysaccharide on Morf4l1 in humans, and the lung epithelial cell death due to overexpression of Morf4l1 in mice.

Influenza A virus (IAV) has been raising public health and safety concerns worldwide. Cyanovirin-N (CVN) is a prominent anti-IAV candidate, but both cytotoxicity and immunogenicity have hindered the development of this protein as a viable therapy. In this article, linker- CVN (LCVN) with a flexible and hydrophilic polypeptide at the N-terminus was efficiently produced from the cytoplasm of Escherichia coli at a >15-l scale. PEGylation at the N-terminal α-amine of LCVN was also reformed as 20 kDa PEGylated linkered Cyanovirin-N (PEG20k-LCVN). The 50% effective concentrations of PEG20k-LCVN were 0.43 ± 0.11 µM for influenza A/HK/8/68 (H3N2) and 0.04 ± 0.02 µM for A/Swan/Hokkaido/51/96 (H5N3), dramatically lower than that of the positive control, Ribavirin (2.88 ± 0.66 × 10³ µM and 1.79 ± 0.62 × 10³ µM, respectively). A total of 12.5 µM PEG20k-LCVN effectively inactivate the propagation of H3N2 in chicken embryos. About 2.0 mg/kg/day PEG20k-LCVN increased double the survival rate (66.67%, P=0.0378) of H3N2 infected mice, prolonged the median survival period, downregulated the mRNA level of viral nuclear protein and decreased (attenuated) the pathology lesion in mice lung. A novel PEGylated CVN derivative, PEG20k-LCVN, exhibited potent and strain-dependent anti-IAV activity in nanomolar concentrations in vitro, as well as in micromolar concentration in vivo. [ABSTRACT FROM AUTHOR]

The Skp-Cul-F box (SCF) ubiquitin E3 ligase machinery recognizes predominantly phosphodegrons or, less commonly, an (I/L)Q molecular signature within substrates to facilitate their recruitment in mediating protein ubiquitination and degradation. Here, we examined the molecular signals that determine the turnover of the multifunctional enzyme nucleoside diphosphate kinase A (NDPK-A) that controls cell proliferation. NDPK-A protein exhibits a half-life of ~6 h in HeLa cells and is targeted for ubiquitylation through actions of the F-box protein FBXO24. SCF-FBXO24 polyubiquitinates NDPK-A at K85, and two NH2-terminal residues, L55 and K56, were identified as important molecular sites for FBXO24 interaction. Importantly, K56 acetylation impairs its interaction with FBXO24, and replacing K56 with Q56, an acetylation mimic, reduces NDPK-A FBXO24 binding capacity. The acetyltransferase GCN5 catalyzes K56 acetylation within NDPK-A, thereby stabilizing NDPK-A, whereas GCN5 depletion in cells accelerates NDPK-A degradation. Cellular expression of an NDPK-A acetylation mimic or FBXO24 silencing increases NDPK-A life span which, in turn, impairs cell migration and wound healing. We propose that lysine acetylation when presented in the appropriate context may be recognized by some F-box proteins as a unique inhibitory molecular signal for their recruitment to restrict substrate degradation. [ABSTRACT FROM AUTHOR]

Rac1, a member of the Rho family of GTPases, regulates diverse cellular functions, including cytoskeleton reorganization and cell migration. F-box proteins are major subunits within the Skp1-Cul1-F-box (SCF) E3 ubiquitin ligases that recognize specific substrates for ubiquitination. The role of F-box proteins in regulating Rac1 stability has not been studied. Mouse lung epithelial (MLE12) cells were used to investigate Rac1 stability and cell migration. Screening of an F-box protein library and in vitro ubiquitination assays identified FBXL19, a relatively new member of the F-box protein family that targets Rac1 for its polyubiquitination and proteasomal degradation. Overexpression of FBXL19 decreased both Rac1 active and inactive forms and significantly reduced cellular migration. Protein kinase AKT-mediated phosphorylation of Rac1 at serine71 was essential for FBXL19-mediated Rac1 ubiquitination and depletion. Lysine166 within Rac1 was identified as a polyubiquitination acceptor site. Rac1S71A and Rac1K166R mutant proteins were resistant to FBXL19-mediated ubiquitination and degradation. Further, ectopically expressed FBXL19 reduced cell migration in Rac1-overexpressing cells (P<0.01, Rac1 cells vs. FBXL19+Rac1 cells), but not in Rac1 lysine166 mutant-overexpressing cells. FBXL19 diminished formation of the migratory leading edge. Thus, SCFFBXL19 targets Rac1 for its disposal, a process regulated by AKT. These findings provide the first evidence of an F-box protein targeting a small G protein for ubiquitination and degradation to modulate cell migration. [ABSTRACT FROM AUTHOR]

Histone acetyltransferase binding to origin recognition complex (HBO1) plays a crucial role in DNA replication licensing and cell proliferation, yet its molecular regulation in cells is relatively unknown. Here an uncharacterized protein, Fbxw15, directly interacts with HBO1, a labile protein (t1/2 = ∼3 h), to mediate its ubiquitination (Lys338) and degradation in the cytoplasm. Fbxw15-mediated HBO1 depletion required mitogenactivated protein kinase 1 (Mek1), which was sufficient to trigger HBO1 phosphorylation and degradation in cells. Mek1 ability to produce HBO1 degradation was blocked by Fbxw15 silencing. Lipopolysaccharide induced HBO1 degradation, an effect abrogated by Fbxw15 or Mek1 cellular depletion. Modulation of Fbxw15 levels was able to differentially regulate histone H3K14 acetylation and cellular proliferation by altering HBO1 levels. These studies authenticate Fbxw15 as a ubiquitin E3 ligase subunit that mediates endotoxin-induced HBO1 depletion in cells, thereby controlling cell replicative capacity. [ABSTRACT FROM AUTHOR]

The enzyme acyl-CoA:lysophosphatidylcholine acyltransferase (Lpcat1) is a critical cytosolic enzyme needed for lung surfactant synthesis that catalyzes an acyltransferase reaction by adding a palmitate to the sn-2 position of lysophospholipids. Here we report that histone H4 protein is subject to palmitoylation catalyzed by Lpcat1 in a calcium-regulated manner. Cytosolic Lpcat1 was observed to shift into the nucleus in lung epithelia in response to exogenous Ca2+. Nuclear Lpcat1 colocalizes with and binds to histone H4, where it catalyzes histone H4 palmitoylation. Mutagenesis studies demonstrated that Ser47 within histone H4 serves as a putative acceptor site, indicative of Lpcat1-mediated O-palmitoylation. Lpcat1 knockdown or expression of a histone H4 Ser47A mutant protein in cells decreased cellular mRNA synthesis. These findings provide the first evidence of a protein substrate for Lpcat1 and reveal that histone lipidation may occur through its O-palmitoylation as a novel post-translational modification. This epigenetic modification regulates global gene transcriptional activity. [ABSTRACT FROM AUTHOR]

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser178 within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys221), as substitution of Lys221 with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβTrCP E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis. [ABSTRACT FROM AUTHOR]

Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor–mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met α-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential. [ABSTRACT FROM AUTHOR]

The HGF gene is transcriptionally silenced in normal differentiated breast epithelial cells, but its repression fails to occur in mammary carcinoma tissues and cell lines. The molecular mechanisms underpinning aberrant HGF expression in breast cancer cells are unknown. Here we report the discovery of a DNA element located 750 bp upstream from the transcription start site in the human HGF promoter that acts as a transcriptional repressor and is a target of deletion mutagenesis in human breast cancer cells and tissues. This HGF promoter element consists of a mononucleotide repeat of 30 deoxyadenosines (30As), which we have termed "deoxyadenosine tract element" (DATE). Functional studies revealed that truncation mutations within DATE have profound local and global effects on the HGF promoter region by modulating chromatin structure and DNA-protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines. We found that 51% of African Americans and 15% of individuals of mixed European descent with breast cancer harbor a truncated DATE variant (25As or fewer) in their breast tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression. Notably, breast cancer patients with the truncated DATE variant are substantially younger than those with a wild-type genotype. We also suggest that DATE may be used as a potential genetic marker to identify individuals with a higher risk of developing breast cancer. [ABSTRACT FROM AUTHOR]

The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1β and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1β in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1β and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention. [ABSTRACT FROM AUTHOR]

Histone acetyltransferase mortality factor 4-like 1 (MORF4L1) is a relatively new histone acetyltransferase component that exists as a homodimer to exert its epigenetic function. The mechanism of MORF4L1 self-assembly is unknown. Here we report that Lys-148 deacetylation is indispensable for facilitating MORF4L1 self-assembly into a homodimeric unit. Among a stretch of ~10 amino acids in the NH2 terminus between the chromodomain and MORF4-related gene (MRG) domain within MORF4L1, Lys-148 is normally acetylated. Substitution of Lys-148 with arginine augments MORF4L1 self-assembly. However, acetylation mimics of MORF4L1, including K148L and K148Q, abolished its self-assembly of the histone acetyltransferase component. HDAC2, a deacetylase, interacts with and keeps MORF4L1 in a deacetylation status at Lys148 that triggers MORF4L1 self-assembly. Knockdown of HDAC2 reduces MORF4L1 self-assembly. HDAC2-dependent deacetylation of MORF4L1 enhances MORF4L1 homodimerization, thus facilitating the functionality of complex formation to repress cell proliferation. [ABSTRACT FROM AUTHOR]

Cytokine-driven inflammation underlies the pathobiology of a wide array of infectious and immune-related disorders. The TNFR-associated factor (TRAF) proteins have a vital role in innate immunity by conveying signals from cell surface receptors to elicit transcriptional activation of genes encoding proinflammatory cytokines. We discovered that a ubiquitin E3 ligase F box component, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2. Analysis of the Fbxo3 C-terminal structure revealed that the bacterial-like ApaG molecular signature was indispensible for mediating Fbxl2 disposal and stimulating cytokine secretion. By targeting this ApaG motif, we developed a highly unique, selective genus of small-molecule Fbxo3 inhibitors that by reducing TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells. The Fbxo3 inhibitors effectively lessened the severity of viral pneumonia, septic shock, colitis, and cytokine-driven inflammation systemically in murine models. Thus, pharmacological targeting of Fbxo3 might be a promising strategy for immune-related disorders characterized by a heightened host inflammatory response. [ABSTRACT FROM AUTHOR]

Cortactin, an actin-binding protein, is essential for cell growth and motility. We have shown that cortactin is regulated by reversible phosphorylation, but little is known regarding cortactin protein stability. Here, we show that lipopolysaccharide (LPS)-induced cortactin degradation is mediated by extracellular regulated signal kinase (ERK). LPS induces cortactin serine phosphorylation, ubiquitination, and degradation in mouse lung epithelia, an effect abrogated by ERK inhibition. Serine phosphorylation sites mutant, cortactinS405A/S418A, enhances its protein stability. Cortactin is polyubiquitinated and degraded within the proteasome, whereas a cortactinK79R mutant exhibited proteolytic stability during cyclohexamide (CHX) or LPS treatment. The E3 ligase subunit β-Trcp interacts with cortactin, and its overexpression reduced cortactin protein levels, an effect attenuated by ERK inhibition. Overexpression of β-Trcp was sufficient to reduce the protective effects of exogenous cortactin on epithelial cell barrier integrity, an effect not observed after expression of a cortactinK79R mutant. These results provide evidence that LPS modulation of cortactin stability is coordinately regulated by stress kinases and the ubiquitin-proteasomal network. [ABSTRACT FROM AUTHOR]