Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.

Patient characteristics and eligibility for biologics in severe asthma: Results from the Greek cohort of the RECOGNISE "real world" study.

BACKGROUND: Some patients with severe asthma do not achieve sufficient symptom control despite guideline-based treatment, and therefore receive oral (OCS) and systemic corticosteroids (SCS) on regular basis. The side effects of corticosteroid use negatively impact patients' health-related quality of life (HRQoL) and increase the disease burden. Biologics have shown promise in asthma therapy; however, identifying patients who might benefit from biologic therapy is complex due to the heterogeneous pathophysiology of the disease. METHODS: The European, non-interventional, multicentre RECOGNISE study (NCT03629782) assessed patient characteristics, asthma medication and control, HRQoL as assessed by St. George's Respiratory Questionnaire (SGRQ), and health care resource use in patients with severe asthma, as well as their eligibility for biologic treatment. Here, data from the Greek cohort (N = 97) are reported. RESULTS: In Greece, patients with severe asthma were more often female (71%) and never smokers (68%). 87% of patients were assessed as eligible for biologic treatment by investigator's judgement (per label criteria: 76%). Most patients had been previously treated with SCS (82% eligible vs 85% non-eligible), with OCS use being more common in non-eligible patients (23.1% vs 11.9%). More eligible patients had poorly controlled asthma (76% vs 54%), and more impaired HRQoL (mean total SGRQ score: 46% vs 39%); symptom burden was significantly higher (mean symptom score: 60% vs. 44%, p: 0.0389). CONCLUSIONS: A high proportion of Greek patients with severe asthma are eligible for biologic therapy; however, individual risk factors and differences between asthma types must be considered before the introduction of targeted therapy.

Molecular basis for the regulation of the H3K4 methyltransferase activity of PRDM9.

PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.