NS1-mediated upregulation of ZDHHC22 acyltransferase in influenza a virus infected cells.

Influenza A viruses contain two S-acylated proteins, the ion channel M2 and the glycoprotein hemagglutinin (HA). Acylation of the latter is essential for virus replication. Here we analysed the expression of each of the 23 members of the family of ZDHHC acyltransferases in human airway cells, the site of virus replication. RT-PCR revealed that every ZDHHC acyltransferase (except ZDHHC19) is expressed in A549 and Calu cells. Interestingly, expression of one ZDHHC, ZDHHC22, is upregulated in virus-infected cells; this effect is more pronounced after infection with an avian compared to a human virus strain. The viral protein NS1 triggers ZDHHC22 expression in transfected cells, whereas recombinant viruses lacking a functional NS1 gene did not cause ZDHHC22 upregulation. CRISPR/Cas9 technology was then used to knock-out the ZDHHC22 gene in A549 cells. However, acylation of M2 and HA was not reduced, as analysed for intracellular HA and M2 and the stoichiometry of S-acylation of HA incorporated into virus particles did not change according to MALDI-TOF mass spectrometry analysis. Comparative mass spectrometry of palmitoylated proteins in wt and ΔZDHHC22 cells identified 25 potential substrates of ZDHHC22 which might be involved in virus replication.

Human Hepatitis B Viral Infection Outcomes Are Linked to Naturally Occurring Variants of HLA-DOA That Have Altered Function.

HLA molecules of the MHC class II (MHCII) bind and present pathogen-derived peptides for CD4 T cell activation. Peptide loading of MHCII in the endosomes of cells is controlled by the interplay of the nonclassical MHCII molecules, HLA-DM (DM) and HLA-DO (DO). DM catalyzes peptide loading, whereas DO, an MHCII substrate mimic, prevents DM from interacting with MHCII, resulting in an altered MHCII-peptide repertoire and increased MHCII-CLIP. Although the two genes encoding DO (DOA and DOB) are considered nonpolymorphic, there are rare natural variants. Our previous work identified DOB variants that altered DO function. In this study, we show that natural variation in the DOA gene also impacts DO function. Using the 1000 Genomes Project database, we show that ∼98% of individuals express the canonical DOA*0101 allele, and the remaining individuals mostly express DOA*0102, which we found was a gain-of-function allele. Analysis of 25 natural occurring DOα variants, which included the common alleles, identified three null variants and one variant with reduced and nine with increased ability to modulate DM activity. Unexpectedly, several of the variants produced reduced DO protein levels yet efficiently inhibited DM activity. Finally, analysis of associated single-nucleotide polymorphisms genetically linked the DOA*0102 common allele, a gain-of-function variant, with human hepatitis B viral persistence. In contrast, we found that the DOα F114L null allele was linked with viral clearance. Collectively, these studies show that natural variation occurring in the human DOA gene impacts DO function and can be linked to specific outcomes of viral infections.

Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction.

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FADox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s(-1), in a step that is coupled to the rapid dissociation of NAD(+). Electrochemical and equilibrium-binding studies indicate that NSMOA binds FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein-protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD(+) after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.

CD4+ T Cell Epitope Identification from Complex Parasite Antigen Mixtures.

Antigen complexity represents a major challenge for scoring CD4+ T cell immunogenicity, a key hallmark of immunity and with great potential to improve vaccine development. In this chapter, we provide a comprehensive picture of a pipeline that can be applied to virtually any complex antigen to overcome different limitations. Antigens are characterized by Mass Spectrometry to determine the available protein sources and their abundances. A reconstituted in vitro antigen processing system is applied along with bioinformatics tools to prioritize the list of candidates. Finally, the immunogenicity of candidate peptides is validated ex vivo using PBMCs from HLA-typed individuals. This protocol compiles the essential information for executing the whole pipeline while focusing on the candidate epitope prioritizing scheme.

Preclinical Toxicological Assessment of A Novel siRNA, SLN360, Targeting Elevated Lipoprotein (a) in Cardiovascular Disease.

SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with <1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies.

Pre-clinical assessment of SLN360, a novel siRNA targeting LPA, developed to address elevated lipoprotein (a) in cardiovascular disease.

BACKGROUND AND AIMS: The LPA gene encodes apolipoprotein (a), a key component of Lp(a), a potent risk factor for cardiovascular disease with no specific pharmacotherapy. Here we describe the pharmacological data for SLN360, a GalNAc-conjugated siRNA targeting LPA, designed to address this unmet medical need. METHODS: SLN360 was tested in vitro for LPA knockdown in primary hepatocytes. Healthy cynomolgus monkeys received single or multiple subcutaneous doses of the SLN360 sequence ranging from 0.1 to 9.0 mg/kg to determine the pharmacokinetic and pharmacodynamic effects. Liver mRNA and serum biomarker analyses were performed. RESULTS: In vitro, the SLN360 sequence potently reduces LPA mRNA in primary cynomolgus and human hepatocytes, while no effect was observed on the expression of APOB or PLG. In vivo, SLN360 exposure peaks 2 h after subcutaneous injection with near full elimination by 24 h. Specific LPA mRNA reduction (up to 91% 2 weeks after dosing) was observed with only the 3 mg/kg group showing appreciable return to baseline (40%). No consistent dose- or time-dependent effect on the expression of APOB, PLG or a panel of sensitive markers of liver lipid accumulation was observed. Potent (up to 95%) and long lasting (≥9 weeks) serum Lp(a) reduction was observed, peaking in all active groups at day 21. The minimally effective dose was determined to be 0.3 mg/kg with an ED50 of 0.6 mg/kg. CONCLUSIONS: SLN360 induces a sustained reduction in serum Lp(a) levels in cynomolgus monkeys following subcutaneous dosing. SLN360 has potential to address the unmet need of Lp(a) reduction in cardiovascular diseases.

Splicing-accessible coding 3'UTRs control protein stability and interaction networks.

BACKGROUND: 3'-Untranslated regions (3'UTRs) play crucial roles in mRNA metabolism, such as by controlling mRNA stability, translation efficiency, and localization. Intriguingly, in some genes the 3'UTR is longer than their coding regions, pointing to additional, unknown functions. Here, we describe a protein-coding function of 3'UTRs upon frameshift-inducing alternative splicing in more than 10% of human and mouse protein-coding genes. RESULTS: 3'UTR-encoded amino acid sequences show an enrichment of PxxP motifs and lead to interactome rewiring. Furthermore, an elevated proline content increases protein disorder and reduces protein stability, thus allowing splicing-controlled regulation of protein half-life. This could also act as a surveillance mechanism for erroneous skipping of penultimate exons resulting in transcripts that escape nonsense mediated decay. The impact of frameshift-inducing alternative splicing on disease development is emphasized by a retinitis pigmentosa-causing mutation leading to translation of a 3'UTR-encoded, proline-rich, destabilized frameshift-protein with altered protein-protein interactions. CONCLUSIONS: We describe a widespread, evolutionarily conserved mechanism that enriches the mammalian proteome, controls protein expression and protein-protein interactions, and has important implications for the discovery of novel, potentially disease-relevant protein variants.

CD4+ Th immunogenicity of the Ascaris spp. secreted products.

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation.

Distinct editing functions of natural HLA-DM allotypes impact antigen presentation and CD4+ T cell activation.

Classical human leukocyte antigen (HLA) molecules of the major histocompatibility class II (MHCII) complex present peptides for the development, surveillance and activation of CD4+ T cells. The nonclassical MHCII-like protein HLA-DM (DM) catalyzes the exchange and loading of peptides onto MHCII molecules, thereby shaping MHCII immunopeptidomes. Natural variations of DM in both chains of the protein (DMA and DMB) have been hypothesized to impact peptide presentation, but no evidence for altered function has been reported. Here we define the presence of DM allotypes in human populations covered by the 1000 Genomes Project and probe their activity. The functional properties of several allotypes are investigated and show strong enhancement of peptide-induced T cell activation for a particular combination of DMA and DMB. Biochemical evidence suggests a broader pH activity profile for the new variant relative to that of the most commonly expressed DM allotype. Immunopeptidome analysis indicates that the compartmental activity of the new DM heterodimer extends beyond the late endosome and suggests that the natural variation of DM has profound effects on adaptive immunity when antigens bypass the canonical processing pathway.

Dynamic palmitoylation events following T-cell receptor signaling.

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation.

Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility.

The major histocompatibility complex of class II (MHCII) immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU)] allowing the identification and label-free quantification (LFQ) of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAtEAU algorithm in facilitating robust and accessible quantitative analysis of immunopeptidomes across cellular contexts. In silico analysis of the peptides enriched for each HLA-DM expression conditions suggests a higher affinity of the pool of peptides isolated from the high DM expression samples. Interestingly, our analysis reveals that while for certain autoimmune-relevant epitopes their presentation increases upon DM expression others are clearly edited out from the peptidome.

The Guanine-Nucleotide Exchange Factor Caldag Gefi Fine-Tunes Functional Properties of Regulatory T Cells.

Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3- conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI-/- mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI-/- Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI-/- Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI-/- Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.

Human leukocyte Antigen-DM polymorphisms in autoimmune diseases.

Classical MHC class II (MHCII) proteins present peptides for CD4(+) T-cell surveillance and are by far the most prominent risk factor for a number of autoimmune disorders. To date, many studies have shown that this link between particular MHCII alleles and disease depends on the MHCII's particular ability to bind and present certain peptides in specific physiological contexts. However, less attention has been paid to the non-classical MHCII molecule human leucocyte antigen-DM, which catalyses peptide exchange on classical MHCII proteins acting as a peptide editor. DM function impacts the presentation of both antigenic peptides in the periphery and key self-peptides during T-cell development in the thymus. In this way, DM activity directly influences the response to pathogens, as well as mechanisms of self-tolerance acquisition. While decreased DM editing of particular MHCII proteins has been proposed to be related to autoimmune disorders, no experimental evidence for different DM catalytic properties had been reported until recently. Biochemical and structural investigations, together with new animal models of loss of DM activity, have provided an attractive foundation for identifying different catalytic efficiencies for DM allotypes. Here, we revisit the current knowledge of DM function and discuss how DM function may impart autoimmunity at the organism level.

Quantitative analysis of the human T cell palmitome.

Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize proteins in cellular membranes, affecting the function of receptors and intracellular signaling proteins. The identification of protein "palmitomes" in several cell lines raises the question to what extent this modification is conserved in primary cells. Here we use primary T cells with acyl-biotin exchange and quantitative mass spectrometry to identify a pool of proteins previously unreported as palmitoylated in vivo.