The emerging multifunctional roles of ERAP1, ERAP2 and IRAP between antigen processing and renin-angiotensin system modulation.

The Endoplasmic Reticulum Aminopeptidase 1 and 2 (ERAP1 and ERAP2) and Insulin Regulated Aminopeptidase (IRAP) are three M1 zinc metalloproteases whose role in antigen processing is the refining of peptidome either in the Endoplasmic reticulum (ERAP1 and ERAP2), or in the endosomes (IRAP). However, other novel and distinct functions are emerging. Here, we focus specifically on ERAP2. This gene has a peculiar evolutionary history, being absent in rodents and undergoing in humans to a balanced selection of two haplotypes, one of which not expressing the full length ERAP2. These observations suggest that its role in antigen presentation is not essential. An additional, less investigated role is in the regulation of the Renin Angiotensin System (RAS). ERAP1 and ERAP2 cleave Angiotensin II (Ang II) into Ang III and IV, which counteract the action of Ang II whereas IRAP is itself the receptor for Ang IV. We have recently reported that macrophages, independently from the haplotype, express and release a N-terminus ERAP2 "short" form which directly binds IRAP and the two molecules are co-expressed in the endosomes and on the cell membrane. This new evidence suggests that the maintenance of the ERAP2 gene in humans could be due to its activity in the regulation of the RAS system, possibly as an Ang IV agonist. Its role in the immune-mediated diseases as well as in disorders more specifically related to an imbalance of the RAS system, including hypertension, pre-eclampsia but also viral infections such as COVID-19, is discussed here.

Genome-wide association study of SARS-CoV-2 infection in Chinese population.

Coronavirus disease 2019 (COVID-19) is a global public health concern. The purpose of this study was to investigate the association between genetic variants and SARS-CoV-2 infection and the COVID-19 severity in Chinese population. A total of 256 individuals including 87 symptomatic patients (tested positive for SARS-CoV-2), 84 asymptomatic cases, and 85 close contacts of confirmed patients (tested negative for SARS-CoV-2) were recruited from February 2020 to May 2020. We carried out the whole exome genome sequencing between the individuals and conducted a genetic association study for SARS-CoV-2 infection and the COVID-19 severity. In total, we analyzed more than 100,000 single-nucleotide polymorphisms. The genome-wide association study suggested potential correlation between genetic variability in POLR2A, ANKRD27, MAN1A2, and ERAP1 genes and SARS-CoV-2 infection susceptibility. The most significant gene locus associated with SARS-CoV-2 infection was located in POLR2A (p = 5.71 × 10-6). Furthermore, genetic variants in PCNX2, CD200R1L, ZMAT3, PLCL2, NEIL3, and LINC00700 genes (p < 1 × 10-5) were closely associated with the COVID-19 severity in Chinese population. Our study confirmed that new genetic variant loci had significant association with SARS-CoV-2 infection and the COVID-19 severity in Chinese population, which provided new clues for the studies on the susceptibility of SARS-CoV-2 infection and the COVID-19 severity. These findings may give a better understanding on the molecular pathogenesis of COVID-19 and genetic basis of heterogeneous susceptibility, with potential impact on new therapeutic options.

Impact of the COVID-19 pandemic on access to the cerliponase alfa managed access agreement in England for CLN2 treatment.

BACKGROUND: Cerliponase alfa, an enzyme replacement therapy for neuronal ceroid lipofuscinosis type 2 (CLN2), is currently available in England through a managed access agreement (MAA). It is administered every 2 weeks via an intracerebroventricular device. Here we report qualitative research with families of children with CLN2 disease and healthcare professionals (HCPs) who run the MAA, to understand how access to cerliponase alfa via the MAA at Great Ormond Street Hospital (GOSH) in London, and the overall management of CLN2 disease, was affected during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Telephone interviews were conducted with nine families, representing 11 children with CLN2 disease, and two HCPs in November and December 2020. RESULTS: Children had received cerliponase alfa treatment for a mean (SD) of 23.1 ± 24.7 months (7.1 ± 4.6 months in the MAA). Families travelled 7-398 km for treatment (mean 210 ± 111 km). Treatment with cerliponase alfa was designated "essential" by GOSH and continued as normal during the pandemic but with extra safety precautions, and no children missed any treatments. Families were highly motivated to continue treatment, despite considerable anxiety about the risk of coronavirus infection from travelling and staying overnight but were reassured by communications from GOSH and the safety precautions put in place. Support therapy services were widely compromised, causing families concern about deterioration in their children's condition. Families were confused about COVID-19 testing and shielding, and were unclear whether children with CLN2 disease were vulnerable to COVID-19. CONCLUSIONS: Looking forward, advice for children with CLN2 disease should be specific and tailored, taking into account the family unit. Support therapies should be considered essential alongside cerliponase alfa treatment.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

Generation of SARS-CoV-2 S1 Spike Glycoprotein Putative Antigenic Epitopes in Vitro by Intracellular Aminopeptidases.

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

Antigen presentation in SARS-CoV-2 infection: the role of class I HLA and ERAP polymorphisms.

Given the highly polymorphic nature of Human Leukocyte Antigen (HLA) molecules, it is not surprising that they function as key regulators of the host immune response to almost all invading pathogens, including SARS-CoV-2, the etiological agent responsible for the recent COVID-19 pandemic. Several correlations have already been established between the expression of a specific HLA allele/haplotype and susceptibility/progression of SARS-CoV-2 infection and new ones are continuously emerging. Protective and harmful HLA variants have been described in both mild and severe forms of the disease, but considering the huge amount of existing variants, the data gathered in such a brief span of time are to some extent confusing and contradictory. The aim of this mini-review is to provide a snap-shot of the main findings so far collected on the HLA-SARS-CoV-2 interaction, so as to partially untangle this intricate yarn. As key factors in the generation of antigenic peptides to be presented by HLA molecules, ERAP1 and ERAP2 role in SARS-CoV-2 infection will be revised as well.

ERAP1 and ERAP2 Enzymes: A Protective Shield for RAS against COVID-19?

Patients with coronavirus disease 2019 (COVID-19) have a wide variety of clinical outcomes ranging from asymptomatic to severe respiratory syndrome that can progress to life-threatening lung lesions. The identification of prognostic factors can help to improve the risk stratification of patients by promptly defining for each the most effective therapy to resolve the disease. The etiological agent causing COVID-19 is a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that enters cells via the ACE2 receptor. SARS-CoV-2 infection causes a reduction in ACE2 levels, leading to an imbalance in the renin-angiotensin system (RAS), and consequently, in blood pressure and systemic vascular resistance. ERAP1 and ERAP2 are two RAS regulators and key components of MHC class I antigen processing. Their polymorphisms have been associated with autoimmune and inflammatory conditions, hypertension, and cancer. Based on their involvement in the RAS, we believe that the dysfunctional status of ERAP1 and ERAP2 enzymes may exacerbate the effect of SARS-CoV-2 infection, aggravating the symptomatology and clinical outcome of the disease. In this review, we discuss this hypothesis.

Metabolism of angiotensin peptides by angiotensin converting enzyme 2 (ACE2) and analysis of the effect of excess zinc on ACE2 enzymatic activity.

After decades of notoriety for its adverse cardiovascular, proinflammatory and profibrotic actions, the renin-angiotensin system (RAS) began to be cast in a more favorable light with the discovery of angiotensin-converting enzyme-2 (ACE2) in 2000. This monocarboxypeptidase, best known for its ability to metabolize angiotensin (Ang) II to Ang 1-7, counteracts the adverse effects of Ang II mediated by the AT1 Ang II receptor. Ang peptides are classically considered to be metabolized by aminopeptidases, by which the nomenclature Ang III (des-Asp1Ang II, 2-8 heptapeptide) and Ang IV (des-Asp1des-Arg2Ang II, 3-8 hexapeptide) are derived. This report compares the ability of recombinant human ACE2 (rhACE2) to metabolize Ang III, Ang IV and Ang V, (4-8 pentapeptide) relative to Ang II to form corresponding des-omega-Phe metabolites. rhACE2 has highest affinity (lowest Km) for Ang III, followed by Ang II ∼ Ang V, followed by Ang IV. However, rhACE2 has the highest Kcat for metabolising Ang IV followed by Ang V, Ang III and Ang II. The enzymatic efficiency (Kcat/Km) is highest for Ang V and Ang III followed by Ang IV and is lowest for Ang II. As a gluzincin metallopeptidase, ACE2 requires a zinc molecule at its active site for catalysis. This report also documents inhibition of ACE2 activity by concentrations of zinc exceeding 10 µM. These observations extend the functional significance of ACE2 to include the metabolic inactivation of Ang III, Ang IV and Ang V, reemphasizing the importance of monitoring zinc intake to maintain metabolic homeostasis.

A New ERAP2/Iso3 Isoform Expression Is Triggered by Different Microbial Stimuli in Human Cells. Could It Play a Role in the Modulation of SARS-CoV-2 Infection?

Following influenza infection, rs2248374-G ERAP2 expressing cells may transcribe an alternative spliced isoform: ERAP2/Iso3. This variant, unlike ERAP2-wt, is unable to trim peptides to be loaded on MHC class I molecules, but it can still dimerize with both ERAP2-wt and ERAP1-wt, thus contributing to profiling an alternative cellular immune-peptidome. In order to verify if the expression of ERAP2/Iso3 may be induced by other pathogens, PBMCs and MDMs isolated from 20 healthy subjects were stimulated with flu, LPS, CMV, HIV-AT-2, SARS-CoV-2 antigens to analyze its mRNA and protein expression. In parallel, Calu3 cell lines and PBMCs were in vitro infected with growing doses of SARS-CoV-2 (0.5, 5, 1000 MOI) and HIV-1BAL (0.1, 1, and 10 ng p24 HIV-1Bal/1 × 106 PBMCs) viruses, respectively. Results showed that: (1) ERAP2/Iso3 mRNA expression can be prompted by many pathogens and it is coupled with the modulation of several determinants (cytokines, interferon-stimulated genes, activation/inhibition markers, antigen-presentation elements) orchestrating the anti-microbial immune response (Quantigene); (2) ERAP2/Iso3 mRNA is translated into a protein (western blot); (3) ERAP2/Iso3 mRNA expression is sensitive to SARS-CoV-2 and HIV-1 concentration. Considering the key role played by ERAPs in antigen processing and presentation, it is conceivable that these enzymes may be potential targets and modulators of the pathogenicity of infectious diseases and further analyses are needed to define the role played by the different isoforms.