SUMMARYExposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson’s disease (PD). Therapies based on PQ’s presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.

We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time. We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds. The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.

By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure was extended from 2.4 to 1.9 A resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13 A away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound to a methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordination of the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrate analogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site of all MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Both substitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme and the comparison of its electronic absorption spectra with other variants suggest that the loss in activity is not due to a conformational change or a defective metal center. Two different reaction mechanisms are proposed and are compared to those of related enzymes. These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.

SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

SummaryHere, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor g coactivator (PGC)-1, PGC-1 and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energydemanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

A variety of studies have implicated the lipid PtdIns(4,5)P 2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P 2 -binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P 2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P 2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P 2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P 2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P 2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P 2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P 2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P 2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization. INTRODUCTIONEndocytosis is an important process in all eukaryotes. It mediates nutrient uptake, regulates responses to extracellular stimuli, and controls the chemical composition and surface area of the plasma membrane. Previous studies in yeast and mammalian cells have led to the identification of many components required for endocytosis (Geli and Riezman, 1998;Munn, 2001;Engqvist-Goldstein and Drubin, 2003). In addition to proteins, lipids, especially PtdIns(4,5)P 2 , are known to be important for the a number of steps in receptormediated endocytosis (D'Hondt et al., 2000). PtdIns(4,5)P 2 is the major polyphosphoinositide found in eukaryotic cell membranes. Early studies identified a role for PtdIns(4,5)P 2 as a precursor to the signaling molecules inositol(1,4,5)-triphosphate and diacylglycerol (Berridge and Irvine, 1984). Subsequent studies showed that PtdIns(4,5)P 2 itself is important for actin assembly, exocytosis, endocytosis, and intracellular membrane trafficking (Cremona and De Camilli, 2001;Martin, 2001;Takenawa and Itoh, 2001;Wenk and De Camilli, 2004). Because PtdIns(4,5)P 2 binding domains are found in many endocytic proteins, the functions of PtdIns(4,5)P 2 in endocytosis are thought to be related to its ability to directly bind to specific proteins (Cullen et al., 2001;Itoh and Takenawa, 2002;Lemmon, 2003).The ENTH (epsin N-terminal homology) domain is a ϳ150 amino acid motif that is highly conserved in a variety of proteins present in yeast, oats, rats, mice, frogs, and...

SUMMARY Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function and highlight the importance of retrotransposon control in somatic SC maintenance.

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