Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.

Epithelial IL-33 appropriates exosome trafficking for secretion in chronic airway disease.

IL-33 is a key mediator of chronic airway disease driven by type 2 immune pathways, yet the nonclassical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is cosecreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared with non-COPD specimens. Using an Alternaria-induced airway disease model, we found that the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a potentially novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type 2 inflammation.

Functional insights from biophysical study of TREM2 interactions with apoE and Aß1-42.

INTRODUCTION: Triggering receptor expressed on myeloid cells-2 (TREM2) is an immune receptor expressed on microglia that also can become soluble (sTREM2). How TREM2 engages different ligands remains poorly understood. METHODS: We used comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 and sTREM2 interactions with apolipoprotein E (apoE) and monomeric amyloid beta (Aß) (mAß42). RESULTS: TREM2 engagement of apoE was protein mediated with little effect of lipidation, showing slight affinity differences between isoforms (E4 > E3 > E2). Another family member, TREML2, did not bind apoE. Disease-linked TREM2 variants within a "basic patch" minimally impact apoE binding. Instead, TREM2 uses a unique hydrophobic surface to bind apoE, which requires the apoE hinge region. TREM2 and sTREM2 directly bind mAß42 and potently inhibit Aß42 polymerization, suggesting a potential role for soluble sTREM2 in preventing AD pathogenesis. DISCUSSION: These findings demonstrate that TREM2 has at least two ligand-binding surfaces that might be therapeutic targets and uncovers a potential function for sTREM2 in directly inhibiting Aß polymerization.