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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.

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-17A hi 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. Ubiquitination may control protein stability or function. Here the authors show that an ubiquitination enzyme, Hectd3, ubiquitinates Stat3 and Malt1 to modulate their function but not degradation in T cells, and thereby promoting the differentiation of pathogenic Th17 cells and susceptibility to a mouse model of multiple sclerosis.

Extracellular vesicles (EVs) have garnered significant interest in recent years due to their contributions to cell-to-cell communication and disease processes. EVs are composed of a complex profile of bioactive molecules, which include lipids, nucleic acids, metabolites, and proteins. Although the biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied, there is incomplete knowledge about how infection influences EV biogenesis. EVs from infected cells contain specific molecules of both host and pathogen origin that may contribute to pathogenesis and the elicitation of the host immune response. Intracellular pathogens exhibit diverse lifestyles that undoubtedly dictate the mechanisms by which their molecules enter the cell’s exosome biogenesis schemes. We will discuss the current understanding of the mechanisms used during infection to traffic molecules from their vacuolar niche to host EVs by selected intravacuolar pathogens. We initially review general exosome biogenesis schemes and then discuss what is known about EV biogenesis in Mycobacterium, Plasmodium, Toxoplasma , and Leishmania infections, which are pathogens that reside within membrane delimited compartments in phagocytes at some time in their life cycle within mammalian hosts. The review includes discussion of the need for further studies into the biogenesis of EVs to better understand the contributions of these vesicles to host-pathogen interactions, and to uncover potential therapeutic targets to control these pathogens.

Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Typhimurium and remains unknown. To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during infection. Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during infection.

Inflammation is a protective response of the organism to tissue injury or infection. It occurs when the immune system recognizes Pathogen-Associated Molecular Patterns (PAMPs) or Damage-Associated Molecular Pattern (DAMPs) through the activation of Pattern Recognition Receptors. This initiates a variety of signalling events that conclude in the upregulation of proinflammatory molecules, which initiate an appropriate immune response. This response is tightly regulated since any aberrant activation of immune responses would have severe pathological consequences such as sepsis or chronic inflammatory and autoimmune diseases. Accumulative evidence shows that the ubiquitin system, and in particular ubiquitin-specific isopeptidases also known as deubiquitinases (DUBs), plays crucial roles in the control of these immune pathways. In this review we will give an up-to-date overview on the role of DUBs in the NF-κB pathway and inflammasome activation, two intrinsically related events triggered by activation of the membrane TLRs as well as the cytosolic NOD and NLR receptors. Modulation of DUB activity by small molecules has been proposed as a way to control dysregulation or overactivation of these key players of the inflammatory response. We will also discuss the advances and challenges of a potential use of DUBs as therapeutic targets in inflammatory pathologies.

Human norovirus is the primary cause of non-bacterial gastroenteritis globally and is the second leading cause of diarrheal deaths in children in developing countries. However, effective therapeutics which prevent or clear norovirus infection are not yet available due to a lack of understanding regarding norovirus pathogenesis. Evidence shows that noroviruses can bind to the surface of commensal bacteria, and the presence of these bacteria alters both acute and persistent murine norovirus infection through the modulation of host immune responses. Interestingly, norovirus-bacterial interactions also affect the bacteria by inducing bacterial stress responses and increasing the production of bacterial extracellular vesicles. Given the established ability of these vesicles to easily cross the intestinal barriers, enter the lamina propria, and modulate host responses, we hypothesized that bacterial extracellular vesicles influence murine norovirus infection through modulation of the antiviral immune response. In this study, we show that murine norovirus can attach to purified bacterial vesicles, facilitating co-inoculation of target cells with both virus and vesicle. Furthermore, we have found that when murine noroviruses and vesicles are used to co-inoculate macrophages, viral infection is reduced compared to virus infection alone. Specifically, co-inoculation with bacterial vesicles results in higher production and release of pro-inflammatory cytokines in response to viral infection. Ultimately, given that murine norovirus infection increases bacterial vesicle production in vivo , these data indicate that bacterial vesicles may serve as a mechanism by which murine norovirus infection is ultimately controlled and limited to a short-term disease.

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.

Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome‐wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus‐infected mice compared to mock‐infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection.

Recent advances in the development and discovery of pharmacological interventions within the ubiquitin–proteasome system (UPS) have uncovered an enormous potential for possible novel treatments of neurodegenerative disease, cancer, immunological disorder and microbial infection. Interference with proteasome activity, although initially considered unlikely to be exploitable clinically, has already proved to be very effective against haematological malignancies, and more specific derivatives that target subsets of proteasomes are emerging. Recent small-molecule screens have revealed inhibitors against ubiquitin-conjugating and -deconjugating enzymes, many of which have been evaluated for their potential use as therapeutics, either as single agents or in synergy with other drugs. Here, we discuss recent advances in the characterisation of novel UPS modulators (in particular, inhibitors of ubiquitin-conjugating and -deconjugating enzymes) and how they pave the way towards new therapeutic approaches for the treatment of proteotoxic disease, cancer and microbial infection.

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.