Mapping of interaction domains mediating binding between BACE1 and RTN/Nogo proteins

BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the beta-secretase site. Membrane bound reticulon (RTN) family proteins interact with BACE1 and negatively modulate BACE1 activity through preventing access of BACE1 to its cellular APP substrate. Here, we focused our study on RTN3 and further show that a C-terminal QID triplet conserved among mammalian RTN members is required for the binding of RTN to BACE1. Although RTN3 can form homo- or heterodimers in cells, BACE1 mainly binds to the RTN monomer and disruption of the QID triplet does not interfere with the dimerization. Correspondingly, the C-terminal region of BACE1 is required for the binding of BACE1 to RTNs. Furthermore, we show that the negative modulation of BACE1 by RTN3 relies on the binding of RTN3 to BACE1. The knowledge from this study may potentially guide discovery of small molecules that can mimic the effect of RTN3 on the inhibition of BACE1 activity.     

A metal-binding site in the RTN1-C protein: new perspectives on the physiological role of a neuronal protein

Reticulon 1-C (RTN1-C) is an ER-associated neuronal protein characterized by horse-shoe-like topology with two transmembrane helices and the N- and C-terminal regions which are supposed in the cytosolic side of ER. The physiological role of this protein is not completely clarified, but several studies have suggested its involvement in the neuronal differentiation, membrane vesicle trafficking and induction of apoptosis. The C-terminal region of RTN1-C is characterized by the presence of a H4 histone consensus sequence that makes it able to interact with nucleic acids and HDAC enzymes both in vitro and in vivo. In the present study a potential metal ion binding motif (HxE/D) at the C-terminal of the RTN1-C has been identified and its capability to bind metals investigated by UV-vis, CD, multidimensional NMR spectroscopy and biological assays. The results suggest a possible implication of the metal ions in the mechanisms of formation of the recently observed RTNs multiprotein complexes contributing to understand the structure and function of this neuronal membrane protein, suggesting a possible effect of the metal binding property on its biological function.     

RTN3与细胞凋亡

RTN3是RTN家族的成员之一,因其主要定位于内质网,所以用reticulon来命名.RTN3与RTN4B是RTN家族中目前已知的、唯一一对具有凋亡诱发功能的基因.过表达的RTN3介导了真核细胞的三大凋亡信号转导通路:死亡受体途径、线粒体途径、内质网途径,并使之交联形成凋亡调控网络;RTN3可与RTN4B相互作用,形成同源或异源二聚来调控细胞凋亡.另外,过表达RTN3还参与了细菌的类凋亡作用.RTN3广泛表达于多种组织,其过表达诱发凋亡的机制的总结将让人们更好的了解RTN3及其家族,完善细胞凋亡的信号转导研究.     

RTN4B‐mediated suppression of Sirtuin 2 activity ameliorates β‐amyloid pathology and cognitive impairment in Alzheimer's disease mouse model

Sirtuin 2 (SIRT2) is an NAD+ dependent deacetylase that is the most abundant sirtuin protein in the brain. Accumulating evidence revealed the role of SIRT2 in a wide range of biological processes and age‐related diseases. However, the pivotal mechanism of SIRT2 played in Alzheimer's disease (AD) remains unknown. Here, we report that pharmacological inactivation of SIRT2 has a beneficial effect in AD. The deacetylase inhibitor of SIRT2 rescued the cognitive impairment in amyloid precursor protein/presenilin 1 transgenic mouse (APP/PS1 mouse), and the BACE1 cleavage was weakened to reduce the β‐amyloid (Aβ) production in the hippocampus. Moreover, we firstly identified that Reticulon 4B (RTN4B) played a crucial role between SIRT2/BACE1 regulation in AD. RTN4B, as a deacetylation substrate for SIRT2, the deacetylation by SIRT2 drived the ubiquitination and degradation of RTN4B and then the disturbed RTN4B interacted with and influenced the expression of BACE1. When we overexpressed RTN4B in neurons of the hippocampus in the AD mouse model, the abnormal Aβ accumulation and cognitive impairment were ameliorated, consistent with the results of SIRT2 inhibition in vivo. Moreover, we showed that the regulatory effect of SIRT2 on BACE1 is dependent on RTN4B. When RTN4B was knocked down, the effects of SIRT2 inhibition on the BACE1 level, Aβ pathology, and AD‐liked behaviors were also blocked. Collectively, we provide evidence that SIRT2 may be a potential target for AD; the new found SIRT2/RTN4B/BACE1 pathological pathway is one of the critical mechanisms for the improvement of SIRT2 on AD.     

Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation

Inhibiting the activity of the beta-amyloid converting enzyme 1 (BACE1) or reducing levels of BACE1 in vivo decreases the production of amyloid-beta. The reticulon family of proteins has four members, RTN1, RTN2, RTN3 and RTN4 (also known as Nogo), the last of which is well known for its role in inhibiting neuritic outgrowth after injury. Here we show that reticulon family members are binding partners of BACE1. In brain, BACE1 mainly colocalizes with RTN3 in neurons, whereas RTN4 is more enriched in oligodendrocytes. An increase in the expression of any reticulon protein substantially reduces the production of Abeta. Conversely, lowering the expression of RTN3 by RNA interference increases the secretion of Abeta, suggesting that reticulon proteins are negative modulators of BACE1 in cells. Our data support a mechanism by which reticulon proteins block access of BACE1 to amyloid precursor protein and reduce the cleavage of this protein. Thus, changes in the expression of reticulon proteins in the human brain are likely to affect cellular amyloid-beta and the formation of amyloid plaques.     

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.     

The transmembrane domain of the Alzheimer's beta-secretase (BACE1) determines its late Golgi localization and access to beta -amyloid precursor protein (APP) substrate

Release of Abeta peptides from beta-amyloid precursor protein (APP) requires sequential cleavage by two endopeptidases, beta- and gamma-secretases. beta-Secretase was recently identified as a novel membrane-bound aspartyl protease, named BACE1, Asp2, or memapsin 2. Employing confocal microscopy and subcellular fractionation, we have found that BACE1 is largely situated in the distal Golgi membrane with a minor presence in the endoplasmic reticulum, endosomes, and plasma membrane in human neuroblastoma SHEP cells and in mouse Neuro-2a cell lines expressing either endogenous mouse BACE1 or additional exogenous human BACE1. The major cellular beta-secretase activity is located in the late Golgi apparatus, consistent with its cellular localization. Furthermore, we demonstrate that the single transmembrane domain of BACE1 alone determines the retention of BACE1 to the Golgi compartments, through examination of recombinant proteins of various BACE1 fragments fused to a reporter green fluorescence protein. In addition, we show that the transmembrane domain of BACE1 is required for the access of BACE1 enzymatic activity to the cellular APP substrate and hence for the optimal generation of the C-terminal fragment of APP (CTF99). The results suggest a molecular and cell biological mechanism for the regulation of beta-secretase activity in vivo.     

Adaptor FADD is recruited by RTN3/HAP in ER-bound signaling complexes

It has been well established that FADD plays a critical role in the membrane bound death-inducing signaling complexes. Herein, we report that endogenous FADD could interact with ectopic or endogenous RTN3/HAP. ER-bound RTN3 protein recruited endogenous FADD to the ER membrane and subsequently initiated caspase-8 cascade, including activation of caspase-8, processing of Bid and release of cytochrome c from mitochondria. Furthermore, we demonstrated that endogenous FADD was recruited by ER-bound endogenous RTN3 to the ER membrane under the tunicamycin stimulation. The dominant negative form of FADD containing DD could abolish these RTN3 generated events in the caspase-8 cascade. Moreover, we found that RTN3 induced caspase-9 processing was only partially resulted from caspase-8 activation (data unshown), indicating that multiple caspase cascades participated in the apoptosis from RTN3 over-expression. Furthermore, NogoB/ASY, a homologue of RTN3 and a potential RTN3 interacting protein, also associated with FADD and induced cytochrome c release in a FADD dependent manner. Rong Xiang and Yingle Liu equal contribution to this work.     

BACE2 functions as an alternative alpha-secretase in cells.

BACE1 and BACE2 define a new subfamily of membrane-anchored aspartyl proteases. Both endoproteases share similar structural organization including a prodomain, a catalytic domain formed via DTG and DSG active site motifs, a single transmembrane domain, and a short C-terminal tail. BACE1 has been identified as the Alzheimer's beta-secretase, whereas BACE2 was mapped to the Down's critical region of human chromosome 21. Herein we show that purified BACE2 can be autoactivated in vitro. Purified BACE2 cleaves human amyloid precursor protein (APP) sequences at the beta-secretase site, and near the alpha-secretase site, mainly at A beta-Phe(20)--Ala(21) and also at A beta-Phe(19)--Phe(20). Alternatively, in cells BACE2 has a limited effect on the beta-secretase site but efficiently cleaves the sequences near the alpha-secretase site. The in vitro specificity of APP processing by BACE2 is distinct from that observed in cells. BACE2 localizes in the endoplasmic reticulum, Golgi, trans-Golgi network, endosomes, and plasma membrane, and its cellular localization patterns depend on the presence of its transmembrane domain. BACE2 chimeras that increase localization of BACE2 in the trans-Golgi network do not change its APP processing patterns. Thus, BACE2 can be distinguished from BACE1 on the basis of autoprocessing of the prosegment, APP processing specificity, and subcellular localization patterns.     

Reticulon 3 interacts with NS4B of the hepatitis C virus and negatively regulates viral replication by disrupting NS4B self‐interaction

The non‐structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α‐helical domains (AH1 and AH2). Its self‐oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host‐encoded ER‐associated reticulon 3 (RTN3) protein is involved in the formation of the replication‐associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self‐interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA‐transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.     

Increased Expression of Reticulon 3 in Neurons Leads to Reduced Axonal Transport of β Site Amyloid Precursor Protein-cleaving Enzyme 1

BACE1 is the sole enzyme responsible for cleaving amyloid precursor protein at the β-secretase site, and this cleavage initiates the generation of β-amyloid peptide (Aβ). Because amyloid precursor protein is predominantly expressed by neurons and deposition of Aβ aggregates in the human brain is highly correlated with the Aβ released at axonal terminals, we focused our investigation of BACE1 localization on the neuritic region. We show that BACE1 was not only enriched in the late Golgi, trans-Golgi network, and early endosomes but also in both axons and dendrites. BACE1 was colocalized with the presynaptic vesicle marker synaptophysin, indicating the presence of BACE1 in synapses. Because the excessive release of Aβ from synapses is attributable to an increase in amyloid deposition, we further explored whether the presence of BACE1 in synapses was regulated by reticulon 3 (RTN3), a protein identified previously as a negative regulator of BACE1. We found that RTN3 is not only localized in the endoplasmic reticulum but also in neuritic regions where no endoplasmic reticulum-shaping proteins are detected, implicating additional functions of RTN3 in neurons. Coexpression of RTN3 with BACE1 in cultured neurons was sufficient to reduce colocalization of BACE1 with synaptophysin. This reduction correlated with decreased anterograde transport of BACE1 in axons in response to overexpressed RTN3. Our results in this study suggest that altered RTN3 levels can impact the axonal transport of BACE1 and demonstrate that reducing axonal transport of BACE1 in axons is a viable strategy for decreasing BACE1 in axonal terminals and, perhaps, reducing amyloid deposition.     

双转基因小鼠模型中microRNA-153对RTN4的表达调控

目的探讨mir-153在阿尔茨海默病发病机制中的作用。方法通过microRNA芯片及Real-timePCR检测APPswe/PSΔE9双转基因小鼠脑内mir-153的表达;构建高表达mir-153的稳转细胞系,通过western-blot检测稳转细胞系内RTN4的蛋白表达;构建野生型及突变型RTN4的3’UTR荧光素酶报告载体,分别将其与mir-153表达载体或microRNA阴性对照载体共转染入293T细胞内,检测海肾荧光素酶的相对活性以验证mir-153对RTN4 mRNA的作用靶点。结果 microRNA芯片及Real-time PCR检测均证实3月龄APPswe/PSΔE9双转基因小鼠脑内mir-153的表达较同龄野生对照显著降低;在高表达mir-153的稳转细胞系内RTN4的蛋白水平明显降低;共转染野生型RTN4的3’UTR/mir-153表达载体可使海肾荧光素酶相对活性较共转染野生型RTN4的3’UTR/miRNA阴性对照质粒组显著降低（P〈0.05）,而共转染突变型RTN4的3’UTR/mir-153表达载体组则较之无明显差异。结论在3月龄APPswe/PSΔE9双转基因小鼠脑内存在mir-153的异常表达;mir-153可调控RTN4的蛋白表达;mir-153对RTN4的表达调控是通过结合RTN4 3’UTR 839-845碱基处的作用位点而实现的。     

Two hydrophobic segments of the RTN1 family determine the ER localization and retention

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.     

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H₂O₂) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H₂O₂-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.     

Insights from modeling the tertiary structure of human BACE2

BACE1, or beta-secretase, is a putative prime therapeutic target for the treatment of Alzheimer's disease. Mapping to the Down syndrome critical region (chromosome 21) and identified as a homologue of BACE1, BACE2 also cleaves amyloid precursor protein at the beta-site. Thus, BACE2, named also as Asp1 or Memapsin1, represents a second beta-secretase candidate. In this paper, the tertiary structure of the protease domain of BACE2 was developed. Although the overall structural topology between BACE1 and BACE2 protease domains is quite similar, the former contains 3 disulfide bonds but the latter only two. Particularly, a subtle structural difference around the DTG/DSG active site between the two structures has been observed that is useful for the in-depth selectivity study of BACE1 and BACE2 inhibitors, stimulating new therapeutic strategies for the treatment of Alzheimer's disease and Down syndrome as well.     

Genetic and biochemical analysis of the transmembrane domain of Arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase

We have examined the amino terminal membrane anchoring domain of Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmg1p), a key enzyme of the isoprenoid biosynthetic pathway. Using both in vitro and in vivo approaches, we have analyzed a series of recombinant derivatives to identify key structural elements which play a role in defining Hmg1p transmembrane topology. Based on our results, we have proposed a topological model for Hmg1p in which the enzyme spans the lipid bilayer twice. We have shown the two transmembrane segments, designated TMS1 and TMS2, to be structurally and functionally inequivalent in their ability to direct the targeting and orientation of reporter proteins. Furthermore, we provide evidence indicating both the extreme amino terminal end and carboxyl terminal domain of the protein reside in the cytosol. This model therefore provides a key basis for the future examination of the role of the transmembrane domain in the targeting and regulation of Hmg1p in plant cells. J. Cell. Biochem. 65:443–459. © 1997 Wiley-Liss Inc.     

CutA divalent cation tolerance homolog (Escherichia coli) (CUTA) regulates β-cleavage of β-amyloid precursor protein (APP) through interacting with β-site APP cleaving protein 1 (BACE1)

Accumulation of the neurotoxic β-amyloid (Aβ) peptide in the brain is central to the pathogenesis of Alzheimer disease. Aβ is derived from the β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, and the production of Aβ is greatly affected by the subcellular localization of these factors. CUTA, the mammalian CutA divalent cation tolerance homolog (E. coli), has been proposed to mediate acetylcholinesterase activity and copper homeostasis, which are important in Alzheimer disease pathology. However, the exact function of CUTA remains largely unclear. Here we show that human CUTA has several variants that differ in their N-terminal length and are separated as heavy (H) and light (L) components. The H component has the longest N terminus and is membrane-associated, whereas the L component is N-terminally truncated at various sites and localized in the cytosol. Importantly, we demonstrate that the H component of CUTA interacts through its N terminus with the transmembrane domain of β-site APP cleaving enzyme 1 (BACE1), the putative β-secretase, mainly in the Golgi/trans-Golgi network. Overexpression and RNA interference knockdown of CUTA can reduce and increase BACE1-mediated APP processing/Aβ secretion, respectively. RNA interference of CUTA decelerates intracellular trafficking of BACE1 from the Golgi/trans-Golgi network to the cell surface and reduces the steady-state level of cell surface BACE1. Our results identify the H component of CUTA as a novel BACE1-interacting protein that mediates the intracellular trafficking of BACE1 and the processing of APP to Aβ.     

Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics

β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer''s disease as the β-secretase responsible for generating the amyloid-β protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few β-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative β-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.     

The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.