Identification of immunological patterns characterizing immune-related psoriasis reactions in oncological patients in therapy with anti-PD-1 checkpoint inhibitors.

Introduction: Immunotherapy with biologics targeting programmed cell death protein-1 (PD-1) is highly effective in the treatment of various malignancies. Nevertheless, it is frequently responsible for unexpected cutaneous manifestations, including psoriasis-like dermatitis. The pathogenesis of anti-PD-1-induced psoriasis has yet to be clarified, even though it is plausible that some innate and adaptive immunity processes are in common with canonical psoriasis. The genetic predisposition to psoriasis of patients could also be a contributing factor. Here, we investigated the immunological and genetic profiles of two patients with metastatic melanoma and one patient affected by lung cancer, who developed severe psoriasis after receiving anti-PD-1 nivolumab therapy. Methods: The immune patterns of the three patients were compared with those detectable in classical, chronic plaque-type psoriasis or paradoxical psoriasis induced by anti-TNF-α therapy, mostly sustained by adaptive and innate immunity processes, respectively. Therefore, immunohistochemistry and mRNA analyses of innate and adaptive immunity molecules were conducted on skin biopsy of patients. Genetic analysis of polymorphisms predisposing to psoriasis was carried out by NGS technology. Results: We found that anti-PD-1-induced psoriasis showed immunological features similar to chronic psoriasis, characterized by the presence of cellular players of adaptive immunity, with abundant CD3+, CD8+ T cells and CD11c+ dendritic cells infiltrating skin lesions, and producing IL-23, IL-6, TNF-α, IFN-γ and IL-17. On the contrary, a lower number of innate immunity cells (BDCA2+ plasmacytoid dendritic cells, CD15+ neutrophils, CD117+ mast cells) and reduced IFN-α/ß, lymphotoxin (LT)-α/ß, were observed in anti-PD-1-induced psoriasis lesions, as compared with anti-TNF-α-induced paradoxical psoriasis. Importantly, the disintegrin and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5) psoriasis autoantigen was significantly upregulated in psoriasis lesions of anti-PD-1-treated patients, at levels comparable with chronic plaque-type psoriasis. Finally, NGS analysis revealed that all patients carried several allelic variants in psoriasis susceptibility genes, such as HLA-C, ERAP1 and other genes of the major psoriasis susceptibility PSORS1 locus. Discussion: Our study showed that adaptive immunity predominates over innate immunity in anti-PD-1-induced psoriasis lesions, consistently with the local ADAMTSL5 overexpression. The presence of numerous SNPs in psoriasis susceptibility genes of the three patients also suggested their strong predisposition to the disease.

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

A genetic variant in the immune-related gene ERAP1 affects colorectal cancer prognosis.

BACKGROUND: Findings on the association of genetic factors and colorectal cancer (CRC) survival are limited and inconsistent, and revealing the mechanism underlying their prognostic roles is of great importance. This study aimed to explore the relationship between functional genetic variations and the prognosis of CRC and further reveal the possible mechanism. METHODS: We first systematically performed expression quantitative trait locus (eQTL) analysis using The Cancer Genome Atlas (TCGA) dataset. Then, the Kaplan-Meier analysis was used to filter out the survival-related eQTL target genes of CRC patients in two public datasets (TCGA and GSE39582 dataset from the Gene Expression Omnibus database). The seven most potentially functional eQTL single nucleotide polymorphisms (SNPs) associated with six survival-related eQTL target genes were genotyped in 907 Chinese CRC patients with clinical prognosis data. The regulatory mechanism of the survival-related SNP was further confirmed by functional experiments. RESULTS: The rs71630754 regulating the expression of endoplasmic reticulum aminopeptidase 1 ( ERAP1 ) was significantly associated with the prognosis of CRC (additive model, hazard ratio [HR]: 1.43, 95% confidence interval [CI]: 1.08-1.88, P = 0.012). The results of dual-luciferase reporter assay and electrophoretic mobility shift assay showed that the A allele of the rs71630754 could increase the binding of transcription factor 3 (TCF3) and subsequently reduce the expression of ERAP1 . The results of bioinformatic analysis showed that lower expression of ERAP1 could affect the tumor immune microenvironment and was significantly associated with severe survival outcomes. CONCLUSION: The rs71630754 could influence the prognosis of CRC patients by regulating the expression of the immune-related gene ERAP1 . TRIAL REGISTRATION: No. NCT00454519 ( https://clinicaltrials.gov/ ).

Structure-guided discovery of aminopeptidase ERAP1 variants capable of processing antigens with novel PC anchor specificities.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations: S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.

Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D.

Isozymes are enzymes that catalyze identical biological reactions, yet exhibit slight variations in structures and catalytic efficiency, which enables the precise adjustment of metabolism to fulfill the specific requirements of a particular tissue or stage of development. Methionine aminopeptidase (MetAP) isozymes function a critical role in cleaving N-terminal methionine from nascent proteins to generate functional proteins. In humans, two distinct MetAP types I and II have been identified, with type I further categorized into cytosolic (MetAP1) and mitochondrial (MetAP1D) variants. However, despite extensive structural studies on both bacterial and human cytosolic MetAPs, the structural information remains unavailable for human mitochondrial MetAP. This study was aimed to elucidate the high-resolution structures of human mitochondrial MetAP1D in its apo-, cobalt-, and methionine-bound states. Through a comprehensive analysis of the determined structures and a docking simulation model with mitochondrial substrate peptides, we present mechanistic insights into the cleavage process of the initiator methionine from mitochondrial proteins. Notably, despite the shared features at the active site between the cytosolic and mitochondrial MetAP type I isozymes, we identified distinct structural disparities within the active-site pocket primarily contributed by two specific loops that could play a role in accommodating specific substrates. These structural insights offer a basis for the further exploration of MetAP isozymes as critical players in cellular processes and potential therapeutic applications.

Discovery of Potential Antituberculosis Agents Targeted Methionine Aminopeptidase 1 of Mycobacterium tuberculosis by the Developed Fluorescent Probe.

Tuberculosis (TB) is a chronic systemic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Methionine aminopeptidase 1 (MtMET-AP1) is a hydrolase that mediates the necessary post-translational N-terminal methionine excision (NME) of peptides during protein synthesis, which is necessary for bacterial proliferation and is a potential target for the treatment of tuberculosis. Based on the functional characteristics of MtMET-AP1, we developed an enzymatic activated near-infrared fluorescent probe DDAN-MT for rapid, highly selective, and real-time monitoring of endogenous MtMET-AP1 activity in M. tuberculosis. Using the probe DDAN-MT, a visually high-throughput screening technique was established, which obtained three potential inhibitors (GSK-J4 hydrochchloride, JX06, and lavendustin C) against MtMET-AP1 from a 2560 compounds library. More importantly, these inhibitors could inhibit the growth of M. tuberculosis H37Ra especially (MICs < 5 µM), with low toxicities on intestinal bacteria strains and human cells. Therefore, the visual sensing of MtMET-AP1 was successfully performed by DDAN-MT, and MtMET-AP1 inhibitors were discovered as potential antituberculosis agents.

HLA-C*15:02 and epidermal growth factor receptor inhibitor-induced erosive pustular dermatosis of the scalp.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.

The identification of two M20B family peptidases required for full virulence in Staphylococcus aureus.

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival for both single mutants that is even more pronounced in the double mutant. We show that these enzymes are localized to the cytosol and membrane but are not necessary for peptide-based nutrition, a hallmark of cell-associated aminopeptidases. Furthermore, none of the survival defects appear to be the result of altered virulence factor production. An exploration of their regulation reveals that both are controlled by known regulators of the S. aureus virulence process, including Agr, Rot and/or SarA, and that this cascade may be mediated by FarR. Structural modeling of PepT1 reveals it bears all the hallmarks of a tripeptidase, whilst PepT2 differs significantly in its catalytic pocket, suggesting a broader substrate preference. In sum, we have identified two M20B aminopeptidases that are integral to S. aureus pathogenesis. The future identification of protein and/or peptide targets for these proteases will be critical to understanding their important virulence impacting functions.

Fumagillin regulates stemness and malignancies in cancer stem-like cells derived from liver cancer via targeting to MetAP-2.

BACKGROUND: Cancer relapse is associated with the presence of cancer stem-like cells (CSCs), which lead to multidirectional differentiation and unrestricted proliferative replication. Fumagillin, a myocotoxin produced by the saprophytic filamentous fungus Aspergillus fumigatus, has been reported to affect malignant characteristics in hepatocellular cancer cells. However, its exact role in CSCs is still unknown. METHODS: CSCs were enriched by culturing cancer cells in serum-free medium. The effects of fumagillin on malignant cell characteristics and mitochondrial function were measured. The regulatory role of fumagillin on methionine aminopeptidase-2 (MetAP-2) was assessed. RESULTS: When it was supplemented in medium, fumagillin treatment inhibited sphere formation and the maintenance of stemness of CSCs without disturbing cell growth. Fumagillin also decreased stemness-related markers and the aldehyde dehydrogenase 1 (ALDH1)-positive proportion, which demonstrated that fumagillin decreases stemness in CSCs. It was also found to inhibit malignant traits in CSCs, including cell proliferation, invasion, and tumor formation, and sensitize CSCs to chemoagents, including sorafenib and doxorubicin, by promoting chemoagent-induced apoptosis. Moreover, fumagillin treatment was found to disturb mitochondrial membrane homeostasis, ATP synthesis and mitochondrial transcriptional activity. In addition, we found that fumagillin decreased MetAP-2 protein levels and exerted anti-CSC effects potentially by regulating MetAP-2. We also found that fumagillin treatment activated p53 and its transcriptional activity and thus caused cell cycle blockade. Moreover, fumagillin treatment significantly decreased tumor formation in nude mice. CONCLUSION: This work offers evidence for fumagillin as a specific inhibitor of liver cancer CSCs and proposes a novel strategy for cancer therapy.

Structure of puromycin-sensitive aminopeptidase and polyglutamine binding.

Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well as peptides released from the proteasome, including polyglutamine. We report the crystal structure of PSA at 2.3 Ǻ. Overall, the enzyme adopts a V-shaped architecture with four domains characteristic of the M1 family aminopeptidases, but it is in a less compact conformation compared to most M1 enzymes of known structure. A microtubule binding sequence is present in a C-terminal HEAT repeat domain of the enzyme in a position where it might serve to mediate interaction with tubulin. In the catalytic metallopeptidase domain, an elongated active site groove lined with aromatic and hydrophobic residues and a large S1 subsite may play a role in broad substrate recognition. The structure with bound polyglutamine shows a possible interacting mode of this peptide, which is supported by mutation.

Aminopeptidase MNP-1 triggers intestine protease production by activating daf-16 nuclear location to degrade pore-forming toxins in Caenorhabditis elegans.

Pore-forming toxins (PFTs) are effective tools for pathogens infection. By disrupting epithelial barriers and killing immune cells, PFTs promotes the colonization and reproduction of pathogenic microorganisms in their host. In turn, the host triggers defense responses, such as endocytosis, exocytosis, or autophagy. Bacillus thuringiensis (Bt) bacteria produce PFT, known as crystal proteins (Cry) which damage the intestinal cells of insects or nematodes, eventually killing them. In insects, aminopeptidase N (APN) has been shown to act as an important receptor for Cry toxins. Here, using the nematode Caenorhabditis elegans as model, an extensive screening of APN gene family was performed to analyze the potential role of these proteins in the mode of action of Cry5Ba against the nematode. We found that one APN, MNP-1, participate in the toxin defense response, since the mnp-1(ok2434) mutant showed a Cry5Ba hypersensitive phenotype. Gene expression analysis in mnp-1(ok2434) mutant revealed the involvement of two protease genes, F19C6.4 and R03G8.6, that participate in Cry5Ba degradation. Finally, analysis of the transduction pathway involved in F19C6.4 and R03G8.6 expression revealed that upon Cry5Ba exposure, the worms up regulated both protease genes through the activation of the FOXO transcription factor DAF-16, which was translocated into the nucleus. The nuclear location of DAF-16 was found to be dependent on mnp-1 under Cry5Ba treatment. Our work provides evidence of new host responses against PFTs produced by an enteric pathogenic bacterium, resulting in activation of host intestinal proteases that degrade the PFT in the intestine.

An anti-biofilm cyclic peptide targets a secreted aminopeptidase from P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.

Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

M1-aminopeptidase family - beyond antigen-trimming activities.

Antigen (Ag)-trimming aminopeptidases belong to the oxytocinase subfamily of M1 metallopeptidases. In humans, this subfamily contains the endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and 2) and the insulin-responsive aminopeptidase (IRAP, synonym oxytocinase), an endosomal enzyme. The ability of these enzymes to trim antigenic precursors and to generate major histocompatibility class-I ligands has been demonstrated extensively for ERAP1, less for ERAP2, which is absent in rodents, and exclusively in the context of cross-presentation for IRAP. During 20 years of research on these aminopeptidases, their enzymatic function has been very well characterized and their genetic association with autoimmune diseases, cancers, and infections is well established. The mechanisms by which these proteins are associated to human diseases are not always clear. This review discusses the Ag-trimming-independent functions of the oxytocinase subfamily of M1 aminopeptidases and the new questions raised by recent publications on IRAP and ERAP2.

Modulation of the 5-Lipoxygenase Pathway by Chalcogen-Containing Inhibitors of Leukotriene A4 Hydrolase.

The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.

The role of endoplasmic reticulum aminopeptidases in type 1 diabetes mellitus.

Type-I diabetes mellitus (T1DM) is generally considered as a chronic, T-cell mediated autoimmune disease. This notwithstanding, both the endogenous characteristics of ß-cells, and their response to environmental factors and exogenous inflammatory stimuli are key events in disease progression and exacerbation. As such, T1DM is now recognized as a multifactorial condition, with its onset being influenced by both genetic predisposition and environmental factors, among which, viral infections represent major triggers. In this frame, endoplasmic reticulum aminopeptidase 1 (ERAP1) and 2 (ERAP2) hold center stage. ERAPs represent the main hydrolytic enzymes specialized in trimming of N-terminal antigen peptides to be bound by MHC class I molecules and presented to CD8+ T cells. Thus, abnormalities in ERAPs expression alter the peptide-MHC-I repertoire both quantitatively and qualitatively, fostering both autoimmune and infectious diseases. Although only a few studies succeeded in determining direct associations between ERAPs variants and T1DM susceptibility/outbreak, alterations of ERAPs do impinge on a plethora of biological events which might indeed contribute to the disease development/exacerbation. Beyond abnormal self-antigen peptide trimming, these include preproinsulin processing, nitric oxide (NO) production, ER stress, cytokine responsiveness, and immune cell recruitment/activity. The present review brings together direct and indirect evidence focused on the immunobiological role of ERAPs in T1DM onset and progression, covering both genetic and environmental aspects.

Variation in ERAP2 has opposing effects on severe respiratory infection and autoimmune disease.

ERAP2 is an aminopeptidase involved in immunological antigen presentation. Genotype data in human samples from before and after the Black Death, an epidemic due to Yersinia pestis, have marked changes in allele frequency of the single-nucleotide polymorphism (SNP) rs2549794, with the T allele suggested to be deleterious during this period, while ERAP2 is also implicated in autoimmune diseases. This study explored the association between variation at ERAP2 and (1) infection, (2) autoimmune disease, and (3) parental longevity. Genome-wide association studies (GWASs) of these outcomes were identified in contemporary cohorts (UK Biobank, FinnGen, and GenOMICC). Effect estimates were extracted for rs2549794 and rs2248374, a haplotype tagging SNP. Additionally, cis expression and protein quantitative trait loci (QTLs) for ERAP2 were used in Mendelian randomization (MR) analyses. Consistent with decreased survival in the Black Death, the T allele of rs2549794 showed evidence of association with respiratory infection (odds ratio; OR for pneumonia 1.03; 95% CI 1.01-1.05). Effect estimates were larger for more severe phenotypes (OR for critical care admission with pneumonia 1.08; 95% CI 1.02-1.14). In contrast, opposing effects were identified for Crohn disease (OR 0.86; 95% CI 0.82-0.90). This allele was shown to associate with decreased ERAP2 expression and protein levels, independent of haplotype. MR analyses suggest that ERAP2 expression may be mediating disease associations. Decreased ERAP2 expression is associated with severe respiratory infection with an opposing association with autoimmune diseases. These data support the hypothesis of balancing selection at this locus driven by autoimmune and infectious disease.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

Optimized extraction and large-scale proteomics of pig jejunum brush border membranes for use in in vitro digestion models.

Despite the physiological importance of the hydrolases from the intestinal brush border membrane (BBM), a step simulating the intestinal digestion has not been included yet in the harmonized protocols of in vitro digestion, due to commercial unavailability of these enzymes and lack of consensus for the conditions of use. The proper utilize of BBM requires a detailed investigation of their enzymatic composition. BBM vesicles were purified from specimens of pig jejunum optimizing previously described methods and assayed for aminopeptidase N and dipeptidyl peptidase IV activity. Large-scale proteomics was carried out with a bottom-up shotgun approach, also performing a rough quantification with the iBAQ (intensity Based Absolute Quantification). Overall, 1428 proteins were identified and functionally classified by gene ontology enrichment analysis. The predominant enzyme fraction (220 gene products) was represented by hydrolases, including peptidases, glycosidases, and lipases. Aminopeptidase N and sucrase-isomaltase represented 52.9 % and 50.2 % of the peptidase and glycosidase abundance, respectively. In addition to expected transporters and cytoskeletal actin-binding proteins, purified BBM vesicles also contains a complex array of protease inhibitors, here described for the first time, that may modulate the activity of hydrolases. Considering the similarity with the human counterpart, intestinal porcine BBM are suited for simulating the human small intestinal digestion.