Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.     

The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy

ABSTRACT

Genetic screens have identified two sets of genes that act at distinct steps of basal autophagy in higher eukaryotes: the pan-eukaryotic ATG genes and the metazoan-specific EPG genes. Very little is known about whether these core macroautophagy/autophagy genes are differentially employed during multicellular organism development. Here we analyzed the function of core autophagy genes in autophagic removal of SQST-1/SQSTM1 during C. elegans development. We found that loss of function of genes acting at distinct steps in the autophagy pathway causes different patterns of SQST-1 accumulation in different tissues and developmental stages. We also identified that the calpain protease clp-2 acts in a cell context-specific manner in SQST-1 degradation. clp-2 is required for degradation of SQST-1 in the hypodermis and neurons, but is dispensable in the body wall muscle and intestine. Our results indicate that autophagy genes are differentially employed in a tissue- and stage-specific manner during the development of multicellular organisms.

Abbreviations: ATG: autophagy related; CLP: calpain family; EPG: ectopic PGL granules; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; LGG-1/LC3: LC3, GABARAP and GATE-16 family; MIT: microtubule interacting and transport; PGL: P granule abnormality protein; SQST-1: sequestosome-related; UPS: ubiquitin-proteasome system     

Resveratrol Attenuates Aβ<Subscript>25–35</Subscript> Caused Neurotoxicity by Inducing Autophagy Through the TyrRS-PARP1-SIRT1 Signaling Pathway

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Resveratrol (RSV) is a natural polyphenol that has been found to be beneficial for AD through attenuation of Aβ-induced toxicity in neurons both in vivo and in vitro. However, the specific underlying mechanisms remain unknown. Recently, autophagy was found to protect neurons from toxicity injuries via degradation of impaired proteins and organelles. Therefore, the aim of this study was to determine the role of autophagy in the anti-neurotoxicity effect of RSV in PC12 cells. We found that RSV pretreatment suppressed β-amyloid protein fragment 25–35 (Aβ_25–35)-induced decrease in cell viability. Expression of light chain 3-II, degradation of sequestosome 1, and formation of autophagosomes were also upregulated by RSV. Suppression of autophagy by 3-methyladenine abolished the favorable effects of RSV on Aβ_25–35-induced neurotoxicity. Furthermore, RSV promoted the expression of sirtuin 1 (SIRT1), auto-poly-ADP-ribosylation of poly (ADP-ribose) polymerase 1 (PARP1), as well as tyrosyl transfer-RNA (tRNA) synthetase (TyrRS). Nevertheless, RSV-mediated autophagy was markedly abolished with the addition of inhibitors of SIRT1 (EX527), nicotinamide phosphoribosyltransferase (STF-118804), PARP1 (AG-14361), as well as SIRT1 and TyrRS small interfering RNA transfection, indicating that the action of RSV on autophagy induction was dependent on TyrRS, PARP1 and SIRT1. In conclusion, RSV attenuated neurotoxicity caused by Aβ_25–35 through inducing autophagy in PC12 cells, and the autophagy was partially mediated via activation of the TyrRS-PARP1-SIRT1 signaling pathway.     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

BSN (bassoon) and PRKN/parkin in concert control presynaptic vesicle autophagy

ABSTRACT

Maintaining the integrity and function of the presynaptic neurotransmitter release apparatus is a demanding process for a post-mitotic neuron; the mechanisms behind it are still unclear. BSN (bassoon), an active zone scaffolding protein, has been implicated in the control of presynaptic macroautophagy/autophagy, a process we recently showed depends on poly-ubiquitination of synaptic proteins. Moreover, loss of BSN was found to lead to smaller synaptic vesicle (SV) pools and younger pools of the SV protein SV2. Of note, the E3 ligase PRKN/parkin appears to be involved in BSN deficiency-related changes in autophagy levels, as shRNA-mediated knockdown of PRKN counteracts BSN-deficiency and rescues decreased SV protein levels as well as impaired SV recycling in primary cultured neurons. These data imply that BSN and PRKN act in concert to control presynaptic autophagy and maintain presynaptic proteostasis and SV turnover at the physiologically required levels.     

The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over‐expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant‐negative Rab7‐interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.     

The Rab-interacting lysosomal protein, a Rab7 and Rab34 effector, is capable of self-interaction

Rab-interacting lysosomal protein (RILP) has been identified as an interacting partner of the small GTPases Rab7 and Rab34. Active Rab7 recruits RILP on the late endosomal/lysosomal membrane and RILP then functions as a Rab7 effector controlling transport to degradative compartments. Indeed, RILP induces recruitment of dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Recently, Rab7 and RILP have been found to be key proteins also for the biogenesis of phagolysosomes. Therefore, RILP represents probably an important factor for all endocytic routes to lysosomes. In this study, we show, using the yeast two-hybrid system, that RILP is able to interact with itself. The data obtained with the two-hybrid system were confirmed using co-immunoprecipitation in HeLa cells. The data together indicate that RILP, as already demonstrated for several other Rab effector proteins, is capable of self-association, thus probably forming a homo-dimer.     

真菌次级代谢产物rasfonin促进舒尼替尼(Sunitinib)诱导的肾癌细胞自噬和凋亡

【目的】明确真菌次级代谢产物rasfonin影响舒尼替尼(Sunitinib,ST)诱导的肾癌细胞自噬和凋亡作用机理。【方法】应用MTS(Methanethiosulfonate assay)和克隆形成实验检测rasfonin和舒尼替尼对肾癌细胞ACHN活性和增殖的影响,通过透射电子显微镜、荧光显微镜、蛋白免疫印迹、免疫荧光方法检测rasfonin和舒尼替尼处理的ACHN细胞自噬、凋亡情况和相关信号通路的变化。【结果】Rasfonin和舒尼替尼能够抑制肾癌细胞ACHN活性和细胞增殖;免疫印迹结果表明,两者均可以引起caspase依赖的凋亡。在rasfonin存在的情况下,不仅舒尼替尼所引起的凋亡和细胞活性丢失明显增加,而且其诱导的自噬流显著提高。无论是rasfonin还是舒尼替尼均明显地抑制哺乳雷帕霉素靶蛋白m TOR(Mammal target of rapamycin)磷酸化,而两者均能促进细胞外调节蛋白激酶(Extracellular regulated protein kinases,ERK)活性增加。【结论】rasfonin促进了舒尼替尼诱导的细胞自噬和凋亡,提高了舒尼替尼抑制肾癌细胞增殖的活性。     

Adenovirus RIDalpha regulates endosome maturation by mimicking GTP-Rab7

The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are guanosine triphosphate (GTP)–Rab7 effectors that instigate minus end–directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation α (RIDα), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135–144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299–8306). RIDα localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDα compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu²⁺ binding to RIDα residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDα–RILP interaction and RIDα activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDα activity during an acute adenovirus infection. We conclude that RIDα coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.     

ZFYVE26/SPASTIZIN and SPG11/SPATACSIN mutations in hereditary spastic paraplegia types AR-SPG15 and AR-SPG11 have different effects on autophagy and endocytosis

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis.

Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A₁; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.     

Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ^~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.     

Differential regulation of autophagy during metabolic stress in astrocytes and neurons

ABSTRACT

Macroautophagy/autophagy is a key homeostatic process that targets cytoplasmic components to the lysosome for breakdown and recycling. Autophagy plays critical roles in glia and neurons that affect development, functionality, and viability of the nervous system. The mechanisms that regulate autophagy in glia and neurons, however, are poorly understood. Here, we define the molecular underpinnings of autophagy in primary cortical astrocytes in response to metabolic stress, and perform a comparative study in primary hippocampal neurons. We find that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes. While both paradigms of metabolic stress dampen MTOR signaling, they affect the autophagy pathway differently. Further, we find that starvation-induced autophagic flux is dependent on the buffering system of the starvation solution. Lastly, starvation conditions that strongly activate autophagy in astrocytes have less pronounced effects on autophagy in neurons. Combined, our study reveals the complexity of regulating autophagy in different paradigms of metabolic stress, as well as in different cell types of the brain. Our findings raise important implications for how neurons and glia may collaborate to maintain homeostasis in the brain.     

Degradation of heparan sulfate proteoglycans enhances oxidized-LDL-mediated autophagy and apoptosis in human endothelial cells

BackgroundCell surface heparan sulfate proteoglycans (HSPG) play an important role in atherogenesis. We hypothesized that degradation of HSPG may increase the binding of atherogenic oxidized low density lipoprotein (ox-LDL) to endothelial cells, and result in extensive HSPG degradation as well as autophagy and apoptosis.MethodsPrimary human umbilical vein endothelial cells (HUVECs) were used to study the expression of lectin-like ox-LDL receptor-1 (LOX-1), HSPG, autophagy and apoptosis in response to ox-LDL and heparinase III (Hep III).ResultsAs expected, ox-LDL treatment resulted in LOX-1 expression, ox-LDL uptake and reactive oxygen species (ROS) generation. Ox-LDL treatment also resulted in a modest degradation of HSPG and increase in autophagy (expression of LC3, beclin-1 and Atg5) and apoptosis (enhanced expression of caspases and Bax, and reduced expression of Bcl-2 and Bcl-xL). The effects of ox-LDL were blocked by pretreatment of cells with LOX-1 antibody or apocynin, an NADPH oxidase inhibitor. Hep III alone caused HSPG degradation and slightly, but significantly, increased ROS generation, and induced autophagy and caspase expression. However, autophagy and apoptosis induced by Hep III were not affected by apocynin or LOX-1 antibody. Importantly, Hep III pretreatment of cells significantly enhanced ox-LDL-induced HSPG degradation, LOX-1 expression, ox-LDL uptake and ROS generation as well as autophagy and apoptosis.ConclusionThese data demonstrate that Hep III enhances the pro-atherosclerotic characteristics in HUVECs induced by ox-LDL.     

Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin

The small GTPase Rab7 controls late endocytic transport by the minus end-directed motor protein complex dynein-dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP-Rab7-ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150(Glued), which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150(Glued) recruitment by Rab7-RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as betaIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150(Glued) dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7-RILP is transferred by ORP1L to betaIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with betaIII spectrin, which requires the activities of Rab7-RILP and ORP1L.     

A novel dual luciferase based high throughput assay to monitor autophagy in real time in yeast S. cerevisiae

BackgroundMacroautophagy is a cellular response to starvation wherein superfluous and damaged cytoplasmic constituents are degraded to provide energy for survival and to maintain cellular homeostasis. Dysfunctional autophagy is attributed to disease progression in several pathological conditions and therefore, autophagy has appeared as a potential pharmacological target for such conditions.ObjectiveIn search of potential drugs that modulate autophagy, identifying small molecule effectors of autophagy is the primary step. The conventional autophagy assays have a limitation that they cannot be scaled down to a high throughput format, therefore, novel sensitive assays are needed to discover new candidate molecules. Keeping this rationale in mind, a dual luciferase based assay was developed in the yeast S. cerevisiae that could measure both selective and general autophagy in real time.MethodsFirefly and Renilla luciferase reporter genes were cloned under POT-1 promoter. Using fatty acid medium the promoter was induced and the luciferase cargo was allowed to build up. The cells were then transferred to starvation conditions to stimulate autophagy and the degradation of luciferase markers was followed with time.Results and conclusionThe assay was more sensitive than conventional assays and could be scaled down to a 384 well format using an automated system. A good Z-factor score indicated that the assay is highly suitable for High Throughput Screening (HTS) of small molecule libraries. Screening of a small molecule library with our assay identified several known and novel modulators of autophagy.