Recipient HLA-B Leader Genotype, and Its Relationship to Total KIR Missing Ligand, Informs Relapse and Survival Following Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

In addition to donor T cells, natural killer (NK) cells are proposed to play a significant role in the graft-versus-leukemia (GVL) effect following haploidentical donor transplantation (HIDT). Following HIDT, donor NK cells express activating (NKG2C) receptors (Ciurea, Leukemia 2021), whose ligand is the non-classical human leukocyte antigen, HLA-E. For surface expression, HLA-E requires binding of leader peptides derived from class I HLA molecules. The rs1050458C/T dimorphism at position -21 of exon 1 of HLA-B gives rise to leader peptides with either methionine (M) or threonine (T) at the second residue of the processed leader peptide. M-containing HLA-B leader peptides promote higher HLA-E expression than T-leaders, potentially favoring more robust natural NK cell recognition of HLA-E-expressing tumor cells. Alternatively, donor NK cells can be activated through inhibitory killer cell immunoglobulin-like receptors (iKIR: 2DL1, 2DL23, 3DL1, 3DL2), if they fail to engage a corresponding class I HLA ligand on recipient leukemia cells (missing ligand (ML) paradigm). We hypothesized that M-containing B-leaders (either MM or MT genotype), and potentially its association with KIR ML, may inform relapse and survival after HIDT using post-transplant cyclophosphamide (PTCy). A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. M-containing B-leader genotype (either MM or MT) was seen in 42% and 44% of recipients and donors, respectively. The B-leader on the unshared donor-recipient haplotypes was matched (either T or M) in 61% of transplants. ML for iKIR 2DL1, 2DL23, 3DL1, and 3DL2 was noted in 29%, 20%, 24% and 72% respectively. Total ML was 0, 1, 2 and 3 in 10%, 43%, 40% and 7% respectively. In univariate analysis, an M-containing recipient B-leader genotype [R(M+)] improved OS and DFS compared to a recipient TT genotype [R(M-)] (75 vs. 53%, p<0.001; 66 vs. 47%, p<0.001), which was primarily due to a lower risk of relapse/progression (24% vs. 38%, p=0.012). In regard to total iKIR ML, the presence of 2-3 ML was associated with better overall (OS) and disease-free (DFS) survival compared with 0-1 (67% vs. 57%, p=0.08; 62% vs. 48%, p=0.019), which was due to lower relapse/progression (25% vs. 38%, p=0.015). There was no association of either recipient B-leader or total iKIR ML with the incidence of NRM, acute or chronic GVHD. When recipient B-leader and ML were combined, the detrimental effect of a R(M-) genotype was seen exclusively in patients with ML 0-1 (see figure). DFS for R(M-)/ML(0-1), R(M+)/ML(0-1), R(M-)/ML(2-3), R(M+)/ML(2-3) was 36%, 68%, 65% and 60%, respectively (p<0.001). Corresponding relapse/progression rates were 49%, 22%, 26% and 25%, respectively (P<0.001). In multivariate analysis, controlling for patient age/sex/race, disease risk index, donor age, HLA-DR mm, HLA-DP npmm and year of transplantation, both recipient B leader and total iKIR ML were independently associated with DFS (HR 0.57, p=0.002 and HR 0.67, p=0.026) and relapse/progression (HR 0.55, p=0.006 and HR 0.55, p=0.007). In summary, a recipient M-containing B-leader genotype (MM or MT) reduces relapse and improves survival following HIDT-PTCy, presumably through HLA-E-mediated effector cell engagement. Furthermore, the negative consequences of low total ML for iKIR, in terms of increased relapse risk and lower DFS, can be mitigated when a R(M+) B-leader is present. In addition to the clinical importance of this novel finding for optimizing HIDT-PTCy, it further supports the role of alloreactive donor NK cells for optimal GVL in this context. Figure 1 Figure 1. Disclosures Solh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; ADCT Therapeutics: Consultancy, Research Funding; BMS: Consultancy.

Remodeling of Conformational Dynamics Enhances Catalytic Activities of M1 Zinc-metallopeptidases from Lanthipeptide Biosynthesis

Lanthipeptides are an important group of natural products with diverse biological functions, and their biosynthesis requires the removal of N-terminal leader peptides (LPs) by designated proteases. LanPM1 enzymes, a subgroup of M1 zinc-metallopeptidases, are recently identified as bifunctional proteases with both endo- and aminopeptidase activities to remove LPs of class III and class IV lanthipeptides. Herein, we report the biochemical and structural characterization of EryP as the LanPM1 enzyme from the biosynthesis of class III lanthipeptide erythreapeptin. We determined X-ray crystal structures of EryP in three conformational states, the open, intermediate and closed states and identified a unique inter-domain Ca binding site as a regulatory element to modulate its domain dynamics and proteolytic activity. Inspired by the regulatory Ca binding, we develop a strategy to engineer LanPM1 enzymes for enhanced catalytic activities by strengthening inter-domain associations and driving the conformational equilibrium toward their closed forms.

Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien

Bacterial transcription attenuators are a source of small RNAs (sRNAs) and leader peptides, for which no own functions were known. However, the attenuator sRNA of the tryptophan (Trp) biosynthesis operon regulates gene expression in trans according to the Trp-availability. Moreover, the cognate leader peptide adopted Trp-independent functions. It builds antibiotic-dependent ribonucleoprotein complexes (ARNPs) for sRNA reprogramming and regulation of ribosomal and multiresistance genes.

Structural basis of substrate recognition by a polypeptide processing and secretion transporter

The peptidase-containing ATP-binding cassette transporters (PCATs) are unique members of the ABC transporter family that proteolytically process and export peptides and proteins. Each PCAT contains two peptidase domains that cleave off the secretion signal, two transmembrane domains forming a translocation pathway, and two nucleotide-binding domains that hydrolyze ATP. Previously the crystal structures of a PCAT from Clostridium thermocellum (PCAT1) were determined in the absence and presence of ATP, revealing how ATP binding regulates the protease activity and access to the translocation pathway. However, how the substrate CtA, a 90-residue polypeptide, is recognized by PCAT1 remained elusive. To address this question, we determined the structure of the PCAT1-CtA complex by electron cryo-microscopy (cryo-EM) to 3.4 Å resolution. The structure shows that two CtAs are bound via their N-terminal leader peptides, but only one is positioned for cleavage and translocation. Based on these results, we propose a model of how substrate cleavage, ATP hydrolysis, and substrate translocation are coordinated in a transport cycle.

Zn-dependent bifunctional proteases are responsible for leader peptide processing of class III lanthipeptides

Lanthipeptides are an important subfamily of ribosomally synthesized and posttranslationally modified peptides, and the removal of their N-terminal leader peptides by a designated protease(s) is a key step during maturation. Whereas proteases for class I and II lanthipeptides are well-characterized, the identity of the protease(s) responsible for class III leader processing remains unclear. Herein, we report that the class III lanthipeptide NAI-112 employs a bifunctional Zn-dependent protease, AplP, with both endo- and aminopeptidase activities to complete leader peptide removal, which is unprecedented in the biosynthesis of lanthipeptides. AplP displays a broad substrate scope in vitro by processing a number of class III leader peptides. Furthermore, our studies reveal that AplP-like proteases exist in the genomes of all class III lanthipeptide-producing strains but are usually located outside the biosynthetic gene clusters. Biochemical studies show that AplP-like proteases are universally responsible for the leader removal of the corresponding lanthipeptides. In addition, AplP-like proteases are phylogenetically correlated with aminopeptidase N from Escherichia coli, and might employ a single active site to catalyze both endo- and aminopeptidyl hydrolysis. These findings solve the long-standing question as to the mechanism of leader peptide processing during class III lanthipeptide biosynthesis, and pave the way for the production and bioengineering of this class of natural products.