Unconventional Secretion Mediates the Trans-cellular Spreading of Tau.

The progressive deposition of misfolded hyperphosphorylated tau is a pathological hallmark of tauopathies, including Alzheimer's disease. However, the underlying molecular mechanisms governing the intercellular spreading of tau species remain elusive. Here, we show that full-length soluble tau is unconventionally secreted by direct translocation across the plasma membrane. Increased secretion is favored by tau hyperphosphorylation, which provokes microtubule detachment and increases the availability of free protein inside cells. Using a series of binding assays, we show that free tau interacts with components enriched at the inner leaflet of the plasma membrane, finally leading to its translocation across the plasma membrane mediated by sulfated proteoglycans. We provide further evidence that secreted soluble tau species spread trans-cellularly and are sufficient for the induction of intracellular tau aggregation in adjacent cells. Our study demonstrates the mechanistic details of tau secretion and provides insights into the initiation and progression of tau pathology.

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand.

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P2-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.

Hydrophobic Mismatch Drives the Interaction of E5 with the Transmembrane Segment of PDGF Receptor.

The oncogenic E5 protein from bovine papillomavirus is a short (44 amino acids long) integral membrane protein that forms homodimers. It activates platelet-derived growth factor receptor (PDGFR) ß in a ligand-independent manner by transmembrane helix-helix interactions. The nature of this recognition event remains elusive, as numerous mutations are tolerated in the E5 transmembrane segment, with the exception of one hydrogen-bonding residue. Here, we examined the conformation, stability, and alignment of the E5 protein in fluid lipid membranes of substantially varying bilayer thickness, in both the absence and presence of the PDGFR transmembrane segment. Quantitative synchrotron radiation circular dichroism analysis revealed a very long transmembrane helix for E5 of ∼26 amino acids. Oriented circular dichroism and solid-state (15)N-NMR showed that the alignment and stability of this unusually long segment depend critically on the membrane thickness. When reconstituted alone in exceptionally thick DNPC lipid bilayers, the E5 helix was found to be inserted almost upright. In moderately thick bilayers (DErPC and DEiPC), it started to tilt and became slightly deformed, and finally it became aggregated in conventional DOPC, POPC, and DMPC membranes due to hydrophobic mismatch. On the other hand, when E5 was co-reconstituted with the transmembrane segment of PDGFR, it was able to tolerate even the most pronounced mismatch and was stabilized by binding to the receptor, which has the same hydrophobic length. As E5 is known to activate PDGFR within the thin membranes of the Golgi compartment, we suggest that the intrinsic hydrophobic mismatch of these two interaction partners drives them together. They seem to recognize each other by forming a closely packed bundle of mutually aligned transmembrane helices, which is further stabilized by a specific pair of hydrogen-bonding residues.

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion.

HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel ß sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.

Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) ß transmembrane segment.

The platelet-derived growth factor receptor ß is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor ß transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.