Gene Therapy: Contest between Adeno-Associated Virus and Host Cells and the Impact of UFMylation.

Adeno-associated virus (AAV)-based gene therapy is currently limited by (1) decline in therapeutic gene expression over time, (2) immune cell activation and (3) neutralization by pre-existing antibodies. Hence, studying the interaction of AAV vectors with various cellular pathways during the production and transduction process is necessary to overcome such barriers. Post-translational modifications (PTM) of AAV vectors during the production and transduction process is known to limit its transduction efficiency and further evoke the immune response. Further, AAV vectors are known to trigger cellular stress, resulting in an upregulation of distinct arms of the unfolded protein response (UPR) pathway. Recognition of the AAV genome by Toll-like receptor-9 triggers the myeloid differentiation primary response signaling cascade for innate (IL-6, IFN-α, IFN-ß) and adaptive (CD8+ T-cell, B-cell) immune response against the viral capsid and the transgene product. Herein, we highlight a potential intersection of the UPR, PTMs, and intracellular trafficking pathways, which could be fine-tuned to augment the outcome of AAV-based gene delivery.

Extracellular vesicles and post-translational protein deimination signatures in haemolymph of the American lobster (Homarus americanus).

The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EVs in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.

Unconventional post-translational modifications in immunological signaling.

The activity of a cell is governed by the signals it receives from the extracellular milieu, which are 'translated' into the appropriate biological output, such as activation, survival, proliferation, migration or differentiation. Signaling pathways are responsible for converting environmental cues into discrete intracellular events. The alteration of existing proteins by post-translational modification (PTM) is a key feature of signal-transduction pathways that allows the modulation of protein function. Research into PTMs has long been dominated by the investigation of protein phosphorylation; other PTMs, such as methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, have received comparatively little attention. This Review aims to present an overview of these PTMs, with an emphasis on their role in cells of the immune system.

Posttranslational modification of differentially expressed mitochondrial proteins in the retina during early experimental autoimmune uveitis.

PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.

Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk.

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.

Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule.

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4(+) T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4(+) T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.

Tobacco reduces membrane HLA class I that is restored by transfection with transporter associated with antigen processing 1 cDNA.

HLA class I molecules are recognized by CTL that eliminate virally infected and malignantly transformed cells presenting foreign peptide-a process termed immunosurveillance. Many tumors have reduced levels of membrane HLA class I. Tumor cells with mutations that reduce HLA class I avoid immunosurveillance and continue to proliferate. As tobacco use can induce tumors, we examined the effect of tobacco extracts on membrane HLA class I. These studies show that culture of cells in media containing tobacco extracts reduces membrane HLA class I, but not other proteins, on primary keratinocytes and other cell types. Culture in tobacco extracts, but not extracts of other substances, reduces TAP1 protein, but does not reduce expression of HLA class I H chain, L chain, or the housekeeping protein beta-actin. The reduction of TAP1 protein occurs within 4 h and is dose-dependent. Culture in tobacco extracts reduces TAP1 protein abundance, but not steady-state mRNA abundance. Tobacco-treated cells show defects in HLA class I biosynthesis similar to those found in TAP1-deficient cell lines. Transfection with TAP1 cDNA restores TAP1 protein abundance, HLA class I biosynthesis, and cell surface expression. Combined, these data show that culture in tobacco extracts reduces TAP1 protein abundance and membrane HLA class I levels. Reduction in membrane HLA class I could permit subsequent malignant transformation of cells to be undetected by the immune system.

Antioxidants inhibit indoleamine 2,3-dioxygenase in IFN-gamma-activated human macrophages: posttranslational regulation by pyrrolidine dithiocarbamate.

Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-gamma is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-kappaB. Here we show that IFN-gamma treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor delta-aminolevulinic acid increased IDO activity. Also, incubation of IFN-gamma-activated hMDM with delta-[(14)C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-gamma-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.

The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.