Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation-induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy-related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin-associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitinrelated proteins in this process.

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or β-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N-acetyl-d-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.

Phagocytosis, which is essential for the immune response to pathogens, is initiated by specific interactions between pathogens and cell surface receptors expressed by phagocytes. This study identifies triggering receptor expressed on myeloid cells 2 (TREM-2) and its signaling counterpart DAP12 as a molecular complex that promotes phagocytosis of bacteria. Expression of TREM-2–DAP12 enables nonphagocytic Chinese hamster ovary cells to internalize bacteria. This function depends on actin cytoskeleton dynamics and the activity of the small guanosine triphosphatases Rac and Cdc42. Internalization also requires src kinase activity and tyrosine phosphorylation. In bone marrow–derived macrophages, phagocytosis is decreased in the absence of DAP12 and can be restored by expression of TREM-2–DAP12. Depletion of TREM-2 inhibits both binding and uptake of bacteria. Finally, TREM-2–dependent phagocytosis is impaired in Syk-deficient macrophages. This study highlights a novel role for TREM-2–DAP12 in the immune response to bacterial pathogens.

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O2 −. Zymosan, which is composed of α-mannan and β-glucan, was internalized by the MR and a β-glucan receptor, but the production of O2 − was triggered only by phagocytosis through the β-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.

Abbreviations used in this paper: BMDM, bone marrow -derived macrophage; cyto D, cytochalasin D; dn, dominant negative; ITAM, immunoreceptor tyrosinebased activation motif; KD, knockdown; TREM, triggering receptor expressed on myeloid cells; wt, wild type.

Transforming growth factor–β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2–anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and ␤-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1α and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1α–induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1α receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gβγ-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gβγ function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gβγ in pull downs. Interaction with Gβγ did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1α activation of phospholipase C, indicating that PLIC-1's inhibition of Gβγ did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gβγ; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.