Sho Takatori

Cytoplasmic localization and proteasomal degradation of N-terminally cleaved form of PINK1

Mutations in PTEN-induced putative kinase 1 (PINK1) gene have been linked to an autosomal recessive form of familial Parkinsons disease. PINK1 encodes a predicted mitochondrial protein kinase. Although the mitochondrial localization of PINK1 has been suggested, the exact subcellular compartment in which PINK1 exerts its cytoprotective function is elusive. Thus, we studied the subcellular distribution and metabolism of PINK1 in cultured cells. Immunocytochemical analysis showed that PINK1 resides in cytoplasm in addition to mitochondria, and that the mitochondrial localization is dependent on its N-terminal sequence. Cellular expression of PINK1 yielded several N-terminally cleaved fragments as well as the full-length protein, among which the 54 kDa fragment (DeltaN 54 kDa) was highly accumulated in the presence of proteasome inhibitors. Endogenous PINK1 was detected dominantly in the form of DeltaN 54 kDa upon proteasome inhibition. Rapid turnover of DeltaN 54 kDa further supported its higher susceptibility to proteasomal degradation compared with that of full-length protein. These results indicate that DeltaN 54 kDa PINK1 undergoes constitutive degradation by proteasome, and underscore the significance of its localization in cytoplasm, especially in the N-terminally processed form.

Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

Microscopic methods to observe the distribution of lipids in the cellular membrane.

Membrane lipids not only provide the structural framework of cellular membranes but also influence protein functions in several different ways. In comparison to proteins, however, relatively little is known about distribution of membrane lipids because of the insufficiency of microscopic methods. The difficulty in studying lipid distribution results from several factors, including their unresponsiveness to chemical fixation, fast translational movement, small molecular size, and high packing density. In this Current Topic, we consider the major microscopic methods and discuss whether and to what degree of precision these methods can reveal membrane lipid distribution in situ. We highlight two fixation methods, chemical and physical, and compare the theoretical limitations to their spatial resolution. Recognizing the strengths and weaknesses of each method should help researchers interpret their microscopic results and increase our understanding of the physiological functions of lipids.

Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity

γ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.

Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole. DOI: http://dx.doi.org/10.7554/eLife.25960.001

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P2) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze‐fracture electron microscopy. GST‐ATG18‐4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3‐phosphate (PtdIns(3)P) was blocked by an excess of the p40phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)‐deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP‐deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid‐disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP‐deficient, PtdIns(3)P‐rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5‐ and RAB7‐positive endosome/lysosomes of the tubulo‐vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2‐rich and ‐deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.

Partial loss of CALM function reduces Aβ42 production and amyloid deposition in vivo

Aberrant production, clearance and deposition of amyloid-β protein (Aβ) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). γ-Secretase is the enzyme responsible for generating various Aβ species, such as Aβ40 and toxic Aβ42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of Aβ42, through the regulation of the clathrin-mediated endocytosis of γ-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of γ-secretase and to the Aβ42 production ratio. Moreover, reduction of CALM decreases Aβ deposition as well as brain levels of insoluble Aβ42 in vivo These results suggest that CALM expression modifies AD risk by regulating Aβ pathology.

Microscopy of membrane lipids: how precisely can we define their distribution?

Membrane lipids form the basic framework of biological membranes by forming the lipid bilayer, but it is becoming increasingly clear that individual lipid species play different functional roles. However, in comparison with proteins, relatively little is known about how lipids are distributed in the membrane. Several microscopic methods are available to study membrane lipid dynamics in living cells, but defining the distribution of lipids at the submicrometre scale is difficult, because lipids diffuse quickly in the membrane and most lipids do not react with aldehydes that are commonly used as fixatives. Quick-freezing appears to be the only practical method by which to stop the lipid movement instantaneously and capture the molecular localization at the moment of interest. Electron microscopic methods, using cryosections, resin sections, and freeze-fracture replicas are used to visualize lipids in quick-frozen samples. The method that employs the freeze-fracture replica is unique in that it requires no chemical treatment and provides a two-dimensional view of the membrane.

Loss of kallikrein‐related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice

Deposition of amyloid‐β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimers disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein‐related peptidase 7 (KLK7) as an astrocyte‐derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S‐positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatment in the primary astrocyte, suggesting that Klk7 is homeostatically modulated by Aβ‐induced responses. Finally, we found that the Food and Drug Administration‐approved anti‐dementia drug memantine can increase the expression of Klk7 and Aβ degradation activity specifically in the astrocytes. These data suggest that KLK7 is an important enzyme in the degradation and clearance of deposited Aβ species by astrocytes involved in the pathogenesis of AD.

AP180 N-Terminal Homology (ANTH) and Epsin N-Terminal Homology (ENTH) Domains: Physiological Functions and Involvement in Disease

The AP180 N-terminal homology (ANTH) and Epsin N-terminal homology (ENTH) domains are crucially involved in membrane budding processes. All the ANTH/ENTH-containing proteins share the phosphoinositide-binding activity and can interact with clathrin or its related proteins via multiple binding motifs. Their function also include promotion of clathrin assembly, induction of membrane curvature, and recruitment of various effector proteins, such as those involved in membrane fission. Furthermore, they play a role in the sorting of specific cargo proteins, thereby enabling the cargos to be accurately transported and function at their appropriate locations. As the structural bases underlying these functions are clarified, contrary to their apparent similarity, the mechanisms by which these proteins recognize lipids and proteins have unexpectedly been found to differ from each other. In addition, studies using knockout mice have suggested that their physiological roles may be more complicated than merely supporting membrane budding processes. In this chapter, we review the current knowledge on the biochemical features of ANTH/ENTH domains, their functions predicted from the phenotypes of animals deficient in these domain-containing proteins, and recent findings on the structural basis enabling specific recognition of their ligands. We also discuss the association of these domains with human diseases. Here we focus on CALM, a protein containing an ANTH domain, which is implicated in the pathogenesis of blood cancers and Alzheimer disease, and discuss how alteration of CALM function is involved in these diseases.