TGFbeta2 mediates rapid inhibition of calcium influx in identified cholinergic basal forebrain neurons.

Transforming growth factors betas (TGFbetas) are known to have important roles in neuronal survival and can be upregulated in disease. However, unlike many other trophic factors, nothing is known about the rapid neurotransmitter-like actions of TGFbeta in the CNS. We explored this by examining the effects of TGFbeta on calcium influx of large enzymatically dissociated basal forebrain neurons. We show that brief application of TGFbeta2, but not TGFbeta1, to fura-2AM-loaded neurons reversibly and acutely (within seconds) inhibited K(+)-evoked calcium influx. Moreover, using single-cell RT-PCR, we confirmed that the large TGFbeta2-responsive neurons presented a cholinergic phenotype. Investigation of the signaling mechanism underlying TGFbeta2 actions using whole-cell recordings of calcium currents revealed that TGFbeta2-mediated responses were insensitive to the nonhydrolyzable GTP analogue GTPgammaS. However, TGFbeta2-mediated calcium current reductions were prevented by intracellular perfusion of a Smad2/3 peptide antagonist. Together, these results suggest that TGFbeta2 can acutely regulate the excitability of basal forebrain cholinergic neurons through an atypical signaling mechanism.     

FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport

Autophagy is the main eukaryotic degradation pathway for long-lived proteins, protein aggregates, and cytosolic organelles. Although the protein machinery involved in the biogenesis of autophagic vesicles is well described, very little is known about the mechanism of cytosolic transport of autophagosomes. In this study, we have identified an adaptor protein complex, formed by the two autophagic membrane-associated proteins LC3 and Rab7 and the novel FYVE and coiled-coil (CC) domain–containing protein FYCO1, that promotes microtubule (MT) plus end–directed transport of autophagic vesicles. We have characterized the LC3-, Rab7-, and phosphatidylinositol-3-phosphate–binding domains in FYCO1 and mapped part of the CC region essential for MT plus end–directed transport. We also propose a mechanism for selective autophagosomal membrane recruitment of FYCO1.     

Glycoproteome profiling of transforming growth factor-beta (TGFbeta) signaling: nonglycosylated cell death-inducing DFF-like effector A inhibits TGFbeta1-dependent apoptosis

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.