CCR6 activation links innate immune responses to mucosa-associated lymphoid tissue lymphoma development.

The genesis of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is driven by oncogenic co-operation among immunological stimulations and acquired genetic changes. We previously identified recurrent CCR6 mutations in MALT lymphoma, with majority predicted to result in truncated proteins lacking the phosphorylation motif important for receptor desensitization. Functional consequences of these mutational changes, the molecular mechanisms of CCR6 activation and how this receptor signaling contributes to MALT lymphoma development remain to be investigated. In the present study, we demonstrated that these mutations impaired CCR6 receptor internalization and were activating changes, being more potent in apoptosis resistance, malignant transformation, migration and intracellular signaling, particularly in the presence of the ligands CCL20, HBD2 (human b defensin 2) and HD5 (human a defensin 5). CCR6 was highly expressed in malignant B cells irrespective of the lymphoma sites. HBD2 and CCL20 were constitutively expressed by the duct epithelial cells of salivary glands, and also those involved in lymphoepithelial lesions (LEL) in salivary gland MALT lymphoma. While in the gastric setting, HBD2, and HD5, to a less extent CCL20, were highly expressed in epithelial cells of pyloric and intestinal metaplasia respectively including those involved in LEL, which are adaptive responses to chronic Helicobacter pylori infection. These findings suggest that CCR6 signaling is most likely active in MALT lymphoma, independent of its mutation status. The observations explain why the emergence of malignant B cells and their clonal expansion in MALT lymphoma are typically around LEL, linking the innate immune responses to lymphoma genesis.

GPR34 activation potentially bridges lymphoepithelial lesions to genesis of salivary gland MALT lymphoma.

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-lymphoma). The majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-lymphoma and how these mutations contribute to the development of lymphoma. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants and performed a range of in vitro assays. We found that the GPR34 Q340X truncation, but not the R84H and D151A mutants, conferred a significantly increased resistance to apoptosis and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization compared with the wild type after ligand (lysophosphatidylserine) stimulation. Among the 9 signaling pathways examined, the GPR34 Q340X truncation, and to a lesser extent the D151A mutant, significantly activated CRE, NF-κB, and AP1 reporter activities, particularly in the presence of ligand stimulation. We further described the enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, as well as their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B cells via GPR34, in which phospholipase A is released by LELs and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-lymphoma.

The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.

Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called ß1-2 loop that is involved in phospholipid binding. Mutations in the ß1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the ß1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.

Correction: A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool.

[This corrects the article DOI: 10.1371/journal.pbio.3000329.].

A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool.

Premature release of nascent ribosomes into the translating pool must be prevented because these do not support viability and may be prone to mistakes. Here, we show that the kinase Rio1, the nuclease Nob1, and its binding partner Pno1 cooperate to establish a checkpoint that prevents the escape of immature ribosomes into polysomes. Nob1 blocks mRNA recruitment, and rRNA cleavage is required for its dissociation from nascent 40S subunits, thereby setting up a checkpoint for maturation. Rio1 releases Nob1 and Pno1 from pre-40S ribosomes to discharge nascent 40S into the translating pool. Weak-binding Nob1 and Pno1 mutants can bypass the requirement for Rio1, and Pno1 mutants rescue cell viability. In these strains, immature ribosomes escape into the translating pool, where they cause fidelity defects and perturb protein homeostasis. Thus, the Rio1-Nob1-Pno1 network establishes a checkpoint that safeguards against the release of immature ribosomes into the translating pool.

Structural Insights into Notch Receptor-Ligand Interactions.

Pioneering cell aggregation experiments from the Artavanis-Tsakonas group in the late 1980's localized the core ligand recognition sequence in the Drosophila Notch receptor to epidermal growth factor-like (EGF) domains 11 and 12. Since then, advances in protein expression, structure determination methods and functional assays have enabled us to define the molecular basis of the core receptor/ligand interaction and given new insights into the architecture of the Notch complex at the cell surface. We now know that Notch EGF11 and 12 interact with the Delta/Serrate/LAG-2 (DSL) and C2 domains of ligand and that membrane-binding, together with additional protein-protein interactions outside the core recognition domains, are likely to fine-tune generation of the Notch signal. Furthermore, structure determination of O-glycosylated variants of Notch alone or in complex with receptor fragments, has shown that these sugars contribute directly to the binding interface, as well as to stabilizing intra-molecular domain structure, providing some mechanistic insights into the observed modulatory effects of O-glycosylation on Notch activity.Future challenges lie in determining the complete extracellular architecture of ligand and receptor in order to understand (i) how Notch/ligand complexes may form at the cell surface in response to physiological cues, (ii) the role of lipid binding in stabilizing the Notch/ligand complex, (iii) the impact of O-glycosylation on binding and signalling and (iv) to dissect the different pathologies that arise as a consequence of mutations that affect proteins involved in the Notch pathway.

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands.

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N-terminus of Notch ligands, which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and Delta-like4 and human Notch2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signalling in different physiological contexts.

Natural genetic variation in yeast longevity.

The genetics of aging in the yeast Saccharomyces cerevisiae has involved the manipulation of individual genes in laboratory strains. We have instituted a quantitative genetic analysis of the yeast replicative lifespan by sampling the natural genetic variation in a wild yeast isolate. Haploid segregants from a cross between a common laboratory strain (S288c) and a clinically derived strain (YJM145) were subjected to quantitative trait locus (QTL) analysis, using 3048 molecular markers across the genome. Five significant, replicative lifespan QTL were identified. Among them, QTL 1 on chromosome IV has the largest effect and contains SIR2, whose product differs by five amino acids in the parental strains. Reciprocal gene swap experiments showed that this gene is responsible for the majority of the effect of this QTL on lifespan. The QTL with the second-largest effect on longevity was QTL 5 on chromosome XII, and the bulk of the underlying genomic sequence contains multiple copies (100-150) of the rDNA. Substitution of the rDNA clusters of the parental strains indicated that they play a predominant role in the effect of this QTL on longevity. This effect does not appear to simply be a function of extrachromosomal ribosomal DNA circle production. The results support an interaction between SIR2 and the rDNA locus, which does not completely explain the effect of these loci on longevity. This study provides a glimpse of the complex genetic architecture of replicative lifespan in yeast and of the potential role of genetic variation hitherto unsampled in the laboratory.