Post-translational Processing Events Research Articles

A single-domain protein catenane of dihydrofolate reductase.

A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is extremely rare in nature. Here, we report a single-domain protein catenane of dihydrofolate reductase (cat-DHFR). This design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement and synthesis was readily accomplished through a series of programmed and streamlined post-translational processing events in cells without any additional in vitro reactions. The target molecule contained few exogenous motifs and was thoroughly characterized using a combination of ultra-performance liquid chromatography-mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease cleavage experiments and ion mobility spectrometry-mass spectrometry. Compared with the linear control, cat-DHFR retained its catalytic capability and exhibited enhanced stability against thermal or chemical denaturation due to conformational restriction. These results suggest that linear proteins may be converted into their concatenated single-domain counterparts with almost identical chemical compositions, well-preserved functions and elevated stabilities, representing an entirely new horizon in protein science.

Exploring the Potential of Chemical Inhibitors for Targeting Post-translational Glycosylation of Coronavirus (SARS-CoV-2).

The Spike (S) protein of SARS-CoV-2 expressed on the viral cell surface is of particular importance as it facilitates viral entry into the host cells. The S protein is heavily glycosylated with 22 N-glycosylation sites and a few N-glycosylation sites. During the viral surface protein synthesis via the host ribosomal machinery, glycosylation is an essential step in post-translational modifications (PTMs) and consequently vital for its life cycle, structure, immune evasion, and cell infection. Interestingly, the S protein of SARS-CoV-2 and the host receptor protein, ACE2, are also extensively glycosylated and these surface glycans are critical for the viral–host cell interaction for viral entry. The glycosylation pathway of both virus (hijacked from the host biosynthetic machinery) and target cells crucially affect SARS-CoV-2 infection at different levels. For example, the glycosaminoglycans (GAGs) of host cells serve as a cofactor as they interact with the receptor-binding domain (RBD) of S-glycoprotein and play a protective role in host immune evasion via masking the viral peptide epitopes. Hence, the post-translational glycan biosynthesis, processing, and transport events could be potential targets for developing therapeutic drugs and vaccines. Especially, inhibition of the N-glycan biosynthesis pathway amplifies S protein proteolysis and, thus, blocks viral entry. The chemical inhibitors of SARS-CoV-2 glycosylation could be evaluated for Covid-19. In this review, we discuss the current status of the chemical inhibitors (both natural and synthetically designed inhibitors) of viral glycosylation for Covid-19 and provide a future perspective. It could be an important strategy in targeting the various emerging SARS-CoV-2 variants of concern (VOCs), as these inhibitors are postulated to aid in reducing the viral load as well as infectivity.

Cellular Synthesis and X-ray Crystal Structure of a Designed Protein Heterocatenane.

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post-translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS-PAGE, LC-MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X-ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein-topology engineering.

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434–440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.

Professor Thue W. Schwartz is the BCPT Nordic Prize Winner 2017 in Basic & Clinical Pharmacology & Toxicology.

Professor Thue W. Schwartz is the BCPT Nordic Prize Winner 2017 in Basic & Clinical Pharmacology & Toxicology.

Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans.

The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.

Experimental annotation of post-translational features and translated coding regions in the pathogen Salmonella Typhimurium

BackgroundComplete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. However, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function.ResultsWe experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function.ConclusionThis work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis.

The secreted form of a melanocyte membrane‐bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding

Ectodomain shedding is a proteolytic mechanism by which a transmembrane protein is converted into a secreted form. Pmel17/gp100 is a melanocyte-specific membrane-bound glycoprotein that has amyloid characteristics and forms fibrillar structures in melanosomes after a complex sequence of post-translational processing and trafficking events, including cleavage by a furin-like proprotein convertase (PC). A secreted form of Pmel17 (termed sPmel17) was also thought to be released due to cleavage by a PC. We used multidisciplinary approaches to demonstrate that sPmel17 is released by ectodomain shedding at the juxtamembrane and/or intramembrane motif and to show that this is independent of cleavage by a PC. We further show that sPmel17 consists of 2 fragments linked by disulfide bonds and that the shedding is inhibited at low temperature but not by metalloproteinase inhibitors. Moreover, treatment with a phorbol ester or a calmodulin inhibitor induces Pmel17 shedding. We also refine the reactivity of HMB50 and NKI/beteb, 2 monoclonal antibodies commonly used as melanoma-specific markers. The fact that those antibodies require physically separated domains of Pmel17 sheds interesting light on its 3-dimensional conformation. We conclude that sPmel17 is released by regulated proteolytic ectodomain shedding.-Hoashi, T., Tamaki, K., Hearing, V. J. The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding.

Glycosyltransferases and oligosaccharyltransferases in Archaea: putative components of theN-glycosylation pathway in the third domain of life

The ability of Eukarya, Bacteria and Archaea to perform N-glycosylation underlies the importance and possible antiquity of this post-translational protein modification. However, in contrast to the relatively well-studied eukaryal and bacterial pathways, the archaeal N-glycosylation process is less understood. To remedy this disparity, the following study has examined 56 available archaeal genomes with the aim of identifying glycosyltransferases and oligosaccharyltransferases, including those putatively catalyzing this post-translational processing event. This analysis reveals that while oligosaccharyltransferases, central components of the N-glycosylation pathway, are found across the range of archaeal phenotypes, the N-glycosylation machinery of hyperthermophilic Archaea may well rely on fewer components than do the parallel systems of nonhyperthermophilic Archaea. Moreover, genes encoding predicted glycosyltransferases of hyperthermophilic Archaea tend to be far more scattered within the genome than is the case with nonhyperthermophilic species, where putative glycosyltransferase genes are often clustered around identified oligosaccharyltransferase-encoding sequences.

Quantitative Reverse-Transcription Polymerase Chain Reaction Analysis of Alzheimer's-Associated Genes in Mouse Scrapie

Prion and Alzheimer's diseases are two apparently distinct disorders; however, the two proteinaceous species implicated in disease progression share a number of common features. In prion diseases a ß-rich conformer of the prion protein is the key molecule in the pathogenesis of prion disease, whereas in Alzheimer's disease neurotoxicity is associated with the amyloid-ß peptide. These two molecules share common structural features and post-translational processing events and both undergo structural transition from normal host proteins to a form associated with toxicity, which leads to neurodegeneration. The precise mechanisms leading to neuronal damage and death that are triggered in these diseases are as yet unknown. It is possible, however, that there is a convergence of events in the neurons whereby similar pathways are executed. In this study the expression of a panel of 94 genes associated with the development of Alzheimer's disease was examined using a high-throughput real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Data showed that approximately 31 of these genes are deregulated in the brains of scrapie-infected mice. Among these were genes involved in inflammation, post-translational processing, excitotoxicity, cholesterol metabolism, and neuroprotection. One of the genes showing the greatest degree of upregulation was the cell cycle regulator CDC2. A microarray analysis also revealed deregulation of CDC2 and related genes, including cyclin B and cyclin D, suggesting that in prion disease, as in Alzheimer's disease, misregulation of cell cycle regulators may contribute to neurodegeneration.

Incomplete posttranslational prohormone modifications in hyperactive neuroendocrine cells

BackgroundIn black-background-adapted Xenopus laevis, the intermediate pituitary melanotrope cells are hyperactive, producing large amounts of their major secretory cargo proopiomelanocortin (POMC, representing ~80% of all newly synthesised proteins), whereas in white-adapted frogs these cells are only basally active. Here we explored in the hyperactive and basally active melanotrope cells the capacity for posttranslational POMC processing events in the secretory pathway.ResultsWe found that the hyperactive cells produced mainly non-complex N-glycosylated POMC, whereas in the basally active cells POMC was mostly complex N-glycosylated. Furthermore, the relative level of POMC sulphation was ~5.5-fold lower in the hyperactive than in the basally active cells. When the cargo load in the secretory pathway of the hyperactive cells was pharmacologically reduced, the relative amount of complex glycosylated POMC markedly increased.ConclusionCollectively, our data show that the secretory pathway in hyperactive neuroendocrine secretory cells lacks the capacity to fully comply with the high demands for complex glycosylation and sulphation of the overload of secretory cargo. Thus, a hyperactive secretory cell may run short in providing an output of correctly modified biological signals.

Distinct Linkage between Post-translational Processing and Differential Secretion of Progastrin Derivatives in Endocrine Cells

Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C-terminal fragments derived from progastrin differ in characteristic manners in levels and patterns of carboxyamidation and tyrosine sulfation in accordance with the secretory pathway taken. Point mutations introduced into a sorting motif disrupt these patterns, suggesting that differences in post-translational modifications are attributable to differential intracellular sorting of precursors. The results suggest a two-step sorting mechanism for progastrin leading to differential secretion of processed fragments via different secretory pathways.

The role of c-Rel in EBV-mediated B cell survival (86.10)

Abstract LMP1 is an Epstein-Barr virus encoded protein that is expressed on the surface of latently infected cells and is known to promote cell survival through activation of the NFκB pathway. Previously our lab developed an immortalized B cell line from a patient with non-X-linked HIGM in which LMP1 is conditionally expressed under the control of a tetracycline-inducible promoter (pt1-LCLtet cells). Analysis of these cells revealed an increased level of apoptosis even in the continuous presence of LMP1 stimulation. In addition, analysis of NFκB/Rel subunits showed a markedly lower expression of c-Rel (c-Rel75) and the presence of a novel 25 kD form (c-Rel25) in pt1but not control-LCLtetcells. Western analysis of immunoprecipitated c-Rel revealed several higher mw forms in pt1- vs. control-LCLtet cells. To investigate the phosphorylation status of c-Rel75 in the pt1-LCLtet cells, in-vivo labeling was carried out and immunoprecipitation of extracts with anti-c-Rel antibodies revealed that c-Rel75 is hyperphosphorylated in these cells. Therefore, we hypothesize that hyperphosphorylation of c-Rel75 targets a post-translational processing event that results in both the reduced level of c-Rel75 and the appearance of the 25 kD form observed in pt1-LCLtet cells. Importantly, the low abundance of c-Rel75 appears to be closely linked to ongoing apoptosis which is not inhibited by LMP1 signaling. (Supported by AI37081)

Analyzing the evolution of β-endorphin post-translational processing events: Studies on reptiles

In many cartilaginous fishes, most ray-finned fishes, lungfishes, and amphibians, the post-translational processing of POMC includes the monobasic cleavage of β-endorphin to yield an opioid that is eight to ten amino acids in length. The amino acid motif within the β-endorphin sequence required for a monobasic cleavage event is -E-R-(S/G)-Q-. Mammals and birds lack this motif and as a result β-endorphin(1–8) is a not an end-product in either group. Since both mammals and birds were derived from ancestors with reptilian origins, an analysis of β-endorphin sequences from extant groups of reptiles should provide insights into the manner in which β-endorphin post-translational processing mechanisms have evolved in amniotes. To this end a POMC cDNA was cloned from the pituitary of the turtle, Chrysemys scripta. The β-endorphin sequence in this species was compared to other reptile β-endorphin sequences (i.e., Chinese soft shell turtle and gecko) and to known bird and mammal sequences. This analysis indicated that either the loss of the arginine residue at the cleavage site (the two turtle species, chick, and human) or a substitution at the glutamine position in the consensus sequence (gecko and ostrich) would account for the loss of the monobasic cleavage reaction in that species. Since amphibians are capable of performing the β-endorphin monobasic reaction, it would appear that the amino acid substitutions that eliminated this post-translational process event in reptilian-related tetrapods must have occurred in the ancestral amniotes.

Photoregulated tyrosinase gene in <i>Polyporus arcularius</i>

tyr1, the gene for tyrosinase, which is related to pigmentation of mycelia in dikaryotic strains, has been cloned and characterized from the basidiomycete Polyporus arcularius. The gene was present in a single copy in the genome. The putative amino acid sequence of Tyr1 was very similar to that of gLeTyr from Lentinula edodes. However, the carboxy-terminal region of the amino acid sequence of Tyr1 was variable among L. edodes, Agaricus bisporus, and this fungus. In the 5’-untranslated region near the initiation codon, a consensus sequence to the Dof1 binding site that is involved in light-regulated gene expression in maize was found. Transcription of tyr1 was photoregulated; transcription of tyr1 in P. arcularius was activated in light mycelia and inactivated in the dark mycelia. These results suggest that tyr1 is a light-regulated gene regulated by a Dof-like transcription factor in P. arcularius. Although the enzyme activity was observed only in a dikaryon, tyr1 was transcripted in both dikaryotic and monokaryotic strains. Thus, activation of the precursor of Tyr1 may require a posttranslational processing event that is developmentally regulated.

Expanding the complexity of the human degradome: polyserases and their tandem serine protease domains

The large and growing number of protease genes identified in the human genome, more than 560, reflects the complexity and relevance of these enzymes in multiple biological processes. As part of our studies on the human degradome--which is defined as the complete set of human protease genes--we have recently identified and cloned three complex polyserine proteases called polyserases. Polyserase-1 is a member of the type-II transmembrane serine protease (TTSP) family of proteolytic enzymes that undergoes a series of post-translational processing events to generate three distinct and independent serine protease domains called serase-1, -2, and -3. Polyserase-2 is a secreted enzyme that also possesses three serine protease domains, but they remain as an integral part of the initial protein product. Finally, polyserase-3 is also a secreted enzyme that contains two serine protease domains embedded in the same polypeptide chain. Despite all three human polyserases share this complex molecular design characterized by the presence of several catalytic domains in their structure, they also exhibit distinctive features including unique expression patterns and different enzymatic properties. At present, the putative functional advantages derived from the complex structural organization of polyserases remain unknown, but the widespread occurrence of these enzymes in mammalian degradomes provides additional evidence about the complexity of proteolytic systems in these organisms.

Photoregulated tyrosinase gene in Polyporus arcularius

tyr1, the gene for tyrosinase, which is related to pigmentation of mycelia in dikaryotic strains, has been cloned and characterized from the basidiomycete Polyporus arcularius. The gene was present in a single copy in the genome. The putative amino acid sequence of Tyr1 was very similar to that of gLeTyr from Lentinula edodes. However, the carboxy-terminal region of the amino acid sequence of Tyr1 was variable among L. edodes, Agaricus bisporus, and this fungus. In the 5’-untranslated region near the initiation codon, a consensus sequence to the Dof1 binding site that is involved in light-regulated gene expression in maize was found. Transcription of tyr1 was photoregulated; transcription of tyr1 in P. arcularius was activated in light mycelia and inactivated in the dark mycelia. These results suggest that tyr1 is a light-regulated gene regulated by a Dof-like transcription factor in P. arcularius. Although the enzyme activity was observed only in a dikaryon, tyr1 was transcripted in both dikaryotic and monokaryotic strains. Thus, activation of the precursor of Tyr1 may require a posttranslational processing event that is developmentally regulated.

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

BackgroundWe have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity.ResultsFollowing our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro-urokinase-type plasminogen activator (pro-uPA). Northern blot analysis showed that polyserase-3 exhibits a unique expression pattern among human polyserases, being predominantly detected in testis, liver, heart and ovary, as well as in several tumor cell lines.ConclusionThese findings contribute to define the growing group of human polyserine proteases composed at present by three different proteins. All of them share a complex structural design with several catalytic units in a single polypeptide but also show specific features in terms of enzymatic properties, expression patterns and post-translational maturation mechanisms.

Stimulus-dependent regulation and cellular expression of genes encoding neuropeptides, prohormone convertases, alpha-amidating enzyme and 7B2 in identified Lymnaea neurons.

Synthesis of bioactive peptides is regulated by several post-translational processing events, including cleavage of peptides from a prohormone, and chemical modifications. Using quantitative in situ hybridization and neuron-specific macro-arrays, we first demonstrated cell-type specific expression levels of transcripts encoding prohormone convertases, peptide alpha-amidating enzyme as well as the chaperone 7B2 in Lymnaea neurons. Second, we demonstrated a strict correlation between alpha-amidating enzyme and its neuropeptide substrates. However, this strict relationship of gene expression of the three prohormone convertases and types of cleavage site used is not present. Third, we showed by a physiological stimulus, i.e. clean water, which leads to a stereotyped egg-laying behaviour resulting in successful egg-mass deposition, the co-regulated induction of transcript levels of processing enzymes, 7B2, and egg-laying hormone. These data indicate that (i) these enzymes (and chaperone) are involved in egg-laying hormone neuropeptide biosynthesis, and (ii) neuropeptide release and transcript levels of both prohormones and processing enzymes are regulated in accordance with physiological demands.

A single nucleotide polymorphism in the Emp3 gene defines the H4 minor histocompatibility antigen.

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4b presented on Kb class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to C-->T nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4b while the anti-B6 response directed against H4a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.