Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

This is the first study to show the direct effects of α-synuclein on synaptic vesicle trafficking and to elucidate the underlying structural mechanisms. Acutely increasing α-synuclein severely inhibits synaptic vesicle recycling from the plasma membrane. The endocytic defects require a properly folded N-terminal α-helical domain of α-synuclein.

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models. [Mol Cancer Ther 2009;8(4):921-9] IntroductionHeat shock protein 90 (Hsp90) is a widely expressed molecular chaperone that functions in the maturation and stabilization of cellular proteins (1-3). Hsp90, in complex with other cochaperone proteins, catalyzes the conformational changes of client proteins via its ATPase activity (4). The activity of Hsp90 maintains a variety of client proteins in their active conformation (5). Hsp90 also plays an important role in the regulation of several key oncogenic signaling proteins (6-8) and steroid receptors (9). Mutated proteins are particularly dependent on Hsp90 for the maintenance of the active conformation (2, 3).Ansamycin drugs such as geldanamycin bind in the ATPbinding site in the NH 2 terminus of Hsp90 (6, 10). This binding inhibits the chaperone activity of Hsp90 and results in proteasomal degradation of the client proteins (5, 11-13). Because tumor cells rely on the activity of client proteins for cell proliferation and survival, drug-induced client protein degradation leads to cytostasis and/or selective cell killing of tumor cell in vitro and in vivo (14-16).The semisynthetic Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials for cancer (17)(18)(19). However, 17-AAG is expensive to prepare and difficult to formulate. The problematic nature of the formulations may well contribute to the dose-limiting toxicity observed with this compound. 17-AAG is also susceptible to metabolism by NQO1/DT-diaphorase enzymes (20) and to efflux by P-glycoprotein (21). The identification of a synthetic Hsp90 inhibitor would be of great therapeutic interest as it would circumvent t...

Spinal cord injury axotomizes neurons and induces many of them to die, whereas others survive. Therefore, it is important to identify factors that lead to neuronal death after injury as a first step toward developing better strategies for increasing neuronal survival and functional recovery. However, the intrinsic molecular pathways that govern whether an injured neuron lives or dies remain surprisingly unclear. To address this question, we took advantage of the large size of giant reticulospinal (RS) neurons in the brain of the lamprey, Petromyzon marinus. We report that axotomy of giant RS neurons induces a select subset of them to accumulate high levels of synuclein, a synaptic vesicle-associated protein whose abnormal accumulation is linked to Parkinson's disease. Injury-induced synuclein accumulation occurred only in neurons that were classified as "poor survivors" by both histological and Fluoro-Jade C staining. In contrast, post-injury synuclein immunofluorescence remained at control levels in neurons that were identified as "good survivors." Synuclein accumulation appeared in the form of aggregated intracellular inclusions. Cells that accumulated synuclein also exhibited more ubiquitin-containing inclusions, similar to what occurs during disease states. When synuclein levels and cell vitality were measured in the same neurons, it became clear that synuclein accumulation preceded and strongly correlated with subsequent neuronal death. Thus, synuclein accumulation is identified as a marker and potential risk factor for forthcoming neuronal death after axotomy, expanding its implications beyond the neurodegenerative diseases.

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.