Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.

Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.

Phosphorylation is a post-translational protein modification regulating most known cellular processes. While protein kinases constitute a large family of highly conserved enzymes, identification of active kinases is challenging due to a low abundance of some of these signaling molecules. Although chicken is the first agricultural animal to have a sequenced genome, annotation of the kinome, i.e., a complement of all protein kinases in the genome is limited. We used chemical probes consisting of ATP and ADP derivatives binding to specific lysine (Lys) residues within the ATP-binding pocket of kinases, combined with proteomics, to identify 267 peptides labeled with the ATP and ADP acyl derivatives and 188 corresponding chicken kinases in chicken spleen and liver. Our description of active chicken kinases and ATP binding sites will support future studies focused on identifying the role of this important class of enzymes in chicken health and disease. SIGNIFICANCE: Advances made in understanding chicken enzymes are critical for the improved knowledge of the regulatory pathways controlling physiological processes in chicken. Since protein phosphorylation controls multiple aspects of cell fate, it is often linked to pathological conditions, and understanding of the kinase expression in chicken is essential for future therapeutic approaches. We coupled proteomics and labeling with active-site probes binding to Lys residues within the ATP-binding pocket of kinases to identify 188 kinases and corresponding 267 peptides labeled with the ATP and ADP acyl derivatives in chicken spleen and liver. Results of the present study describing catalytically active kinases is a starting point for chemoproteomic-based interrogation of kinases in chicken exposed to different conditions. Kinases identified in this study are available through the Chickspress genome browser that has previously published mRNA, miRNA, and shotgun proteomics data.

Social play is the most characteristic form of social interaction which is necessary for adolescents to develop proper cognitive, emotional, and social competency. The information available on neural substrates and the mechanism involved in social play is limited. This study characterized social play by proteomic and transcriptional profiling studies. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioral testing. The proteomic analysis led to the identification of 170 differentially expressed proteins (p ≤ 0.05) with 67 upregulated and 103 downregulated proteins. The transcriptomic analysis led to the identification of 188 genes (FDR ≤ 0.05) with 55 upregulated and 133 downregulated genes. DAVID analysis of gene/protein expression data revealed that social play altered GABAergic signaling, glutamatergic signaling, and G-protein coupled receptor (GPCR) signaling. These data suggest that the synaptic levels of GABA and glutamate increased during play. Ingenuity Pathway Analysis (IPA) confirmed these alterations. IPA also revealed that differentially expressed genes/proteins in our data had significant over representation of neurotransmitter signaling systems, including the opioid, serotonin, and dopamine systems, suggesting that play alters the systems involved in the regulation of reward. In addition, corticotropin-releasing hormone signaling was altered indicating that an increased level of stress occurs during play. Overall, our data suggest that increased inhibitory GPCR signaling in these neurotransmitter pathways occurs following social play as a physiological response to regulate the induced level of reward and stress and to maintain the excitatory-inhibitory balance in the neurotransmitter systems.

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa). We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family and their impact on numerous biological and pathological processes has now been widely appreciated. Many DUB functions have to be tightly controlled within the cell, and this can be achieved in several ways, such as substrate-induced conformational changes, binding to adaptor proteins, proteolytic cleavage, and post-translational modifications (PTMs). This review is focused on the role of PTMs including monoubiquitination, sumoylation, acetylation, and phosphorylation as characterized and putative regulative factors of DUB function. Although this aspect of DUB functionality has not been yet thoroughly studied, PTMs represent a versatile and reversible method of controlling the role of DUBs in biological processes. In several cases PTMs might constitute a feedback mechanism insuring proper functioning of the ubiquitin proteasome system and other DUB-related pathways.

APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium, which can invade and survive within macrophages. Pathogenic salmonellae induce the secretion of specific cytokines from these phagocytic cells and interfere with the host secretory pathways. In this study, we describe the extracellular proteome of human macrophages infected with S Typhimurium, followed by analysis of canonical pathways of proteins isolated from the extracellular milieu. We demonstrate that some of the proteins secreted by macrophages upon S Typhimurium infection are released via exosomes. Moreover, we show that infected macrophages produce CD63+ and CD9+ subpopulations of exosomes at 2 h postinfection. Exosomes derived from infected macrophages trigger the Toll-like receptor 4-dependent release of tumor necrosis factor alpha (TNF-α) from naive macrophages and dendritic cells, but they also stimulate secretion of such cytokines as RANTES, IL-1ra, MIP-2, CXCL1, MCP-1, sICAM-1, GM-CSF, and G-CSF. Proinflammatory effects of exosomes are partially attributed to lipopolysaccharide, which is encapsulated within exosomes. In summary, we show for the first time that proinflammatory exosomes are formed in the early phase of macrophage infection with S Typhimurium and that they can be used to transfer cargo to naive cells, thereby leading to their stimulation.

The central nucleus of the amygdala (CeA) is a striatum-like structure that contains mainly inhibitory circuits controlling a repertoire of (mal)adaptive behaviors related to pain, anxiety, motivation, and addiction. Neural activity in the CeA is also necessary for the expression of persistent and robust drug seeking, also termed 'incubation of drug craving.' However, neuroadaptations within this brain region supporting incubated drug craving have not been characterized. Here, we conducted a comprehensive analysis of protein expression in the CeA of male rats after prolonged (45-day) abstinence from extended-access cocaine self-administration using a quantitative proteomic approach. The proteomic analysis identified 228 unique proteins altered in cocaine rats relative to animals that received saline. Out of the identified proteins, 160 were downregulated, while 68 upregulated. Upregulation of tyrosine hydroxylase and downregulation of neural cell-adhesion protein contactin-1 were validated by immunoblotting. Follow-up analysis by the Ingenuity Pathway Analysis tool revealed alterations in protein networks associated with several neurobehavioral disorders, cellular function and morphology, as well as axogenesis, long-term potentiation, and receptor signaling pathways. This study suggests that chronic cocaine self-administration, followed by a prolonged abstinence results in reorganization of specific protein signaling networks within the CeA that may underlie incubated cocaine craving and identifies potential novel 'druggable' targets for the treatment of cocaine use disorder (CUD).

Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20-300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SIGNIFICANCE: This study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.

Although it is known that the composition of extracellular vesicles (EVs) is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs (LiEVs) containing parasite-derived molecules. In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages, is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that significantly increase numbers as the infection progresses. To evaluate the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10, and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha, and IFN-gamma and not M2 signature molecules. Finally, in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers, although they exhibit differences in their spatial distribution in infected tissues. In conclusion, EVs produced by macrophages during Leishmania infection lead to the gene expression consistent with M2 polarization. In contrast, the EVs produced during S. Typhimurium infection stimulated the transcription of genes associated with M1 polarization.

Small extracellular vesicles (sEVs) produced by antigen-presenting cells represent a novel mechanism of cell-to-cell communication. The sEVs have been shown to drive Th1-type adaptive immune responses against intracellular infections such as Salmonella. In this study, we have demonstrated that an administration of sEVs produced by Salmonella-infected macrophages to BALB/c mice that were then challenged with Salmonella infection decreased bacterial load in infected animals and led to protection against a lethal dose of Salmonella. Second, the same sEVs induced a robust production of IgA anti-Salmonella antibodies (Abs) in BALB/c mice, including IgA anti-OmpD Abs. These results show that the nanoscale sEVs stimulate adaptive immune responses against intracellular pathogens and that these sEVs can be used to provide animals with complete protection against lethal infection, such as the systemic bacterial infection in immunodeficient BALB/c mice.

Although protein ubiquitination has been shown to regulate multiple processes during host response to Salmonella enterica serovar Typhimurium infection, specific functions of host deubiquitinating enzymes remain unknown in this bacterial infection. By using chemical proteomics approach, in which deubiquitinating enzymes were labeled by an active-site probe and analyzed by quantitative proteomics, we identified novel deubiquitinases in chicken macrophages based on their reactivity with the probe. Also, we detected down-regulation of UCH-L3, and USP4 as well as up-regulation of USP5 and UCH-L5 deubiquitinating enzymes in macrophages infected with Salmonella Typhimurium. We showed that decrease in either UCH-L5 activity, or in UCH-L5 protein amount in chicken and human macrophages infected or stimulated with LPS/nigericin, led to decreased IL-1β release. These data point towards a putative role of UCH-L5 in inflammasome regulation during Salmonella infection. Because inflammasome activation is important in innate resistance to these bacteria, one would expect that naturally occurring or therapeutically induced alteration in UCH-L5 activation would influence disease outcome and could represent a target for new therapeutic approaches.

Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Polyubiquitination promotes proteasomal degradation, or signaling and localization, of targeted proteins. Here we show that the E3 ubiquitin ligase Hectd3 is necessary for pathogenic Th17 cell generation in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. Hectd3-deficient mice have lower EAE severity, reduced Th17 program and inefficient Th17 cell differentiation. However, Stat3, but not RORγt, has decreased polyubiquitination, as well as diminished tyrosine-705 activating phosphorylation. Additionally, non-degradative polyubiquitination of Malt1, critical for NF-κB activation and Th17 cell function, is reduced. Mechanistically, Hectd3 promotes K27-linked and K29-linked polyubiquitin chains on Malt1, and K27-linked polyubiquitin chains on Stat3. Moreover, Stat3 K180 and Malt1 K648 are targeted by Hectd3 for non-degradative polyubiquitination to mediate robust generation of RORγt+IL-17Ahi effector CD4+ T cells. Thus, our studies delineate a mechanism connecting signaling related polyubiquitination of Malt1 and Stat3, leading to NF-kB activation and RORγt expression, to pathogenic Th17 cell function in EAE.

Eicosanoids are cellular metabolites, which shape the immune response, including inflammatory processes in macrophages. The effects of these lipid mediators on inflammation and bacterial pathogenesis are not clearly understood. Certain eicosanoids are suspected to act as molecular sensors for the recruitment of neutrophils, while others regulate bacterial uptake. In this study, gene expression analyses indicated that genes involved in eicosanoid biosynthesis including COX-1, COX-2, DAGL, and PLA-2 are differentially regulated in THP-1 human macrophages infected with Salmonella enterica Typhimurium or Yersinia enterocolitica. By using targeted metabolomics approach, we found that the eicosanoid precursor, arachidonic acid (AA) as well as its derivatives, including prostaglandins (PGs) PGF2α or PGE2/PGD2, and thromboxane TxB2, are rapidly secreted from macrophages infected with these Gram-negative pathogenic bacteria. The magnitude of eicosanoid biosynthesis in infected host cells depends on the presence of virulence factors of Y. enterocolitica and S. Typhimurium strains, albeit in an opposite way in Y. enterocolitica compared to S. Typhimurium infection. Trials with combinations of EP2/EP4 PGE2 receptor agonists and antagonists suggest that PGE2 signaling in these infection models works primarily through the EP4 receptor. Downstream of EP4 activation, PGE2 enhances inflammasome activation and represses M2 macrophage polarization while inducing key M1-type markers. PGE2 also led to a decreased numbers of Y. enterocolitica within macrophages. To summarize, PGE2 is a potent autocrine/paracrine activator of inflammation during infection in Gram-negative bacteria, and it affects macrophage polarization, likely controlling bacterial clearance by macrophages.

This data article contains the proteomic and transcriptomic data of the amygdala of adolescent rats involved in social play compared to non-behavioural animals. Social play was performed on male Sprague Dawley rats on postnatal day 38 and protein and gene expression in the amygdala was determined following behavioural testing. The protein expression was measured by analysing trypsin digested protein samples using a LTQ Orbitrap Velos mass spectrometer equipped with an Advion nanomate ESI source. The obtained tandem mass spectra were extracted by Thermo Proteome Discoverer 1.3 and the data were displayed with Scaffold v 4.5.1. The transcriptomic data were generated by llumina HiSeq 4000 system. Cuffdiff (v2.2.1) program was used to calculate RNA-seq based gene expression levels. For further interpretation of data presented in this article, please see the research article 'Proteomic and Transcriptional Profiling of Rat Amygdala Following Social Play' (Alugubelly et al. 2019).