BCL2L11/BIM

In response to toxic stimuli, BCL2L11 (also known as BIM), a BH3-only protein, is released from its interaction with dynein light chain 1 (DYNLL1 also known as LC8) and can induce apoptosis by inactivating anti-apoptotic BCL2 proteins and by activating BAX-BAK1. Recently, we discovered that BCL2L11 interacts with BECN1 (Beclin 1), and that this interaction is facilitated by DYNLL1. BCL2L11 recruits BECN1 to microtubules by bridging BECN1 and DYNLL1, thereby inhibiting autophagy. In starvation conditions, BCL2L11 is phosphorylated by MAPK8/JNK and this phosphorylation abolishes the BCL2L11-DYNLL1 interaction, allowing dissociation of BCL2L11 and BECN1, thereby ameliorating autophagy inhibition. This finding demonstrates a novel function of BIM beyond its roles in apoptosis, highlighting the crosstalk between autophagy and apoptosis, and suggests that BCL2L11’s dual effects in inhibiting autophagy and promoting apoptosis may have important roles in disease pathogenesis.     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Apoptosis can be modulated by K⁺ and Ca²⁺ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential‐regulated Ca²⁺ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle‐mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K⁺/Ca²⁺ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K⁺]_e and [Ca²⁺]_i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K⁺]_e under conditions of immaturity, is independent of extracellular Ca²⁺ and operates via IP3 channels. The second leads to influx of extracellular Ca²⁺ following activation of ryanodine channels in the presence of physiological [K⁺]_e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum

The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.     

Antagonism of Beclin 1‐dependent autophagy by BCL‐2 at the endoplasmic reticulum requires NAF‐1

In addition to mitochondria, BCL‐2 is located at the endoplasmic reticulum (ER) where it is a constituent of several distinct complexes. Here, we identify the BCL‐2‐interacting protein at the ER, nutrient‐deprivation autophagy factor‐1 (NAF‐1)—a bitopic integral membrane protein whose defective expression underlies the aetiology of the neurodegenerative disorder Wolfram syndrome 2 (WFS2). NAF‐1 contains a two iron–two sulphur coordinating domain within its cytosolic region, which is necessary, but not sufficient for interaction with BCL‐2. NAF‐1 is displaced from BCL‐2 by the ER‐restricted BH3‐only protein BIK and contributes to regulation of BIK‐initiated autophagy, but not BIK‐dependent activation of caspases. Similar to BCL‐2, NAF‐1 is found in association with the inositol 1,4,5‐triphosphate receptor and is required for BCL‐2‐mediated depression of ER Ca²⁺ stores. During nutrient deprivation as a physiological stimulus of autophagy, BCL‐2 is known to function through inhibition of the autophagy effector and tumour suppressor Beclin 1. NAF‐1 is required in this pathway for BCL‐2 at the ER to functionally antagonize Beclin 1‐dependent autophagy. Thus, NAF‐1 is a BCL‐2‐associated co‐factor that targets BCL‐2 for antagonism of the autophagy pathway at the ER.     

骨骼肌中肌浆网/内质网钙ATP酶的功能及其作用机制

骨骼肌是机体生命活动和能量代谢的重要场所,其代谢紊乱会诱发一系列肌肉疾病。Ca²⁺作为肌肉收缩过程的重要调节器,在骨骼肌的功能行使中发挥重要作用。骨骼肌细胞中Ca²⁺浓度主要受肌浆网/内质网钙ATP酶(sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase, SERCA)的调节。SERCA利用ATP水解产生的能量介导胞质Ca²⁺进入肌浆网内腔,维持胞质Ca²⁺平衡。SERCA功能的失调会引发一系列骨骼肌疾病,而SERCA活性受部分肌浆网蛋白质的调控,跨膜蛋白质PLN、SLN、MRLN、DWORF和sAnk1以及胞质蛋白质THADA和SAR,其通过磷酸化,进而调控SERCA的功能。本文对骨骼肌中SERCA的功能、调控SERCA的相关功能蛋白质的结构及其作用机制进行了总结,以期为骨骼肌相关疾病的治疗提供最新的思路和方法。     

Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy

BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis.     

Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos‐2 cells to cannabinoid receptor agonist WIN55,212‐2

WIN55,212‐2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti‐tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212‐2 on osteosarcoma cell line Saos‐2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212‐2. Our results showed that the cell proliferation of Saos‐2 was inhibited by WIN55,212‐2 in a dose‐dependent and time‐dependent manner. WIN55,212‐2‐induced Saos‐2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212‐2 can induce endoplasmic reticulum stress and autophagy in Saos‐2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212‐2 in Saos‐2 cells. These findings indicated that WIN55,212‐2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

白花蛇舌草多糖提取物对喉癌Hep-2细胞内质网自噬的影响*

目的:探讨白花蛇舌草多糖提取物(HDPE)对喉癌Hep-2细胞内质网自噬的影响。方法:实验分为对照组、HDPE 100、200、400 mg/L组和3-MA(自噬抑制剂)组,噻唑盐比色法(MTT)检测各组细胞培养24 h、48 h、72 h后增殖抑制率;原位末端转移酶标记法(TUNEL)法检测各组培养48 h细胞凋亡情况;单丹黄酰尸胺(MDC)染色观察各组培养48 h细胞自噬体及自噬溶酶体的变化;透射电镜观察培养48 h细胞内质网周围自噬囊泡的产生情况;蛋白印迹法(Western blot)检测各组培养48 h细胞Beclin-1蛋白(Beclin-1)、微管相关轻链蛋白3Ⅰ(LC3Ⅰ)、微管相关轻链蛋白3Ⅱ(LC3Ⅱ)、葡萄糖调节蛋白 78(GRP78)、活化转录因子6(ATF6)及CCAAT 增强子结合蛋白同源蛋白(CHOP)表达。结果:与对照组比较,HDPE 100、200、400 mg/L组和3-MA组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率量降低,内质网周围自噬囊泡减少,GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05);与3-MA组比较,HDPE 400 mg/L组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率、GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05)。结论:HDPE可能通过抑制喉癌Hep-2细胞内质网自噬,促进细胞内质网应激凋亡,进而抑制Hep-2细胞增殖能力。     

Darier disease : A disease model of impaired calcium homeostasis in the skin

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca²⁺ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca²⁺ transport by SERCA2 resulting in decreased ER Ca²⁺ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca²⁺ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Disease-causing V(2) vasopressin receptors are retained in different compartments of the early secretory pathway

The G protein-coupled V(2) vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V(2) vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease-causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V(2) vasopressin receptor mutants. We show that mutants L62P, DeltaL62-R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease-causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.     

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors identify a novel calcium pool in the central nervous system

Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.