- Email: [email protected]
Nogo Proteins
doi:10.1016/j.jmb.2006.07.094
J. Mol. Biol. (2006) 363, 625–634
Mapping of Interaction Domains Mediating Binding between BACE1 and RTN/Nogo Proteins Wanxia He 1 , Xiangyou Hu 1 , Qi Shi 1 , Xiangdong Zhou 1 , Yifeng Lu 2 Christopher Fisher 2 and Riqiang Yan 1 ⁎ 1
Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA 2
Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, MI 49007, USA
BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the β-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. © 2006 Published by Elsevier Ltd.
*Corresponding author
Keywords: BACE1; RTN; Nogo; reticular; amyloid peptide
Introduction Alzheimer's disease (AD) is the most common neurodegenerative disease. While the exact etiology of AD remains to be identified, the formation of senile plaques in the brains of AD cases has been demonstrated to be an invariant feature of the disease.1 Senile plaques, also called neuritic plaques, contain extracellular aggregates of β-amyloid peptides (Aβ) surrounded by dystrophic neurites, activated microglia and reactive astrocytes. Aβ, typically 39–43 amino acid residues in length, is excised from the larger membrane-bound amyloid precursor protein (APP) through sequential cleavages by two endopeptidases: β and γ-secretase.2–4 Recent studies have demonstrated that the functional γ-secretase activity requires proper assembly of a complex comprising of at least five transmembrane proteins including the presenilins (PSEN1 and
Abbreviations used: APP, amyloid precursor protein; AD, Alzheimer's disease; RHD, reticulon homology domain; TM, transmembrane; co-IP, co-immunoprecipitate(ion); ER, endoplasmic reticulum. E-mail address of the corresponding author: [email protected] 0022-2836/$ - see front matter © 2006 Published by Elsevier Ltd.
PSEN25–7). Various causative mutations in PSEN1 and PSEN2 have been identified in early onset familial AD and all of these mutations lead to increased production of 42 amino acids Aβ (Aβ42).7 The molecular mechanism underlying the cleavage of APP by the γ-secretase complex to result in heterogenous Aβ C-terminal ends remains a mystery, and is an active area of exploration. By contrast, the mechanism of APP processing at the N terminus of Aβ by β-secretase has been clearly established. A membrane-bound aspartyl protease, termed beta-site APP cleaving enzyme 1 (BACE1),8 has been shown to function as the sole β-secretase.8–12 BACE1 cleavage of APP releases a large secreted fragment (sAPPβ) together with a membranebound C-terminal fragment (C99). C99 is the substrate for the γ-secretase complex to release full length Aβ. Several lines of evidence have demonstrated the role of BACE1 in processing APP to Aβ that results in human AD. First, increased expression of BACE1 in cells and in mice significantly facilitates processing of APP at the β-site.13–15 Second, mice lacking BACE1 exhibit almost undetectable levels of Aβ. 16–18 Third, and perhaps most importantly, higher levels of BACE1 have been detected in human AD brains.19–21 Together, these data suggest that inhibition of BACE1 activity will be an
626 effective therapeutic approach for blocking amyloid depositions. Indeed, many potent BACE1 inhibitors have already been shown to reduce Aβ levels both in vitro and in vivo.21–27 In addition to the efforts to identify small molecule BACE1 inhibitors, potential regulation of BACE1 activity by cellular factors also has been explored. For instances, it has been demonstrated that BACE1 activity is regulated by the state of glycosylation,28 composition of lipids in the membrane bilayer,29 interactions with heparin sulfate30 and recently described reticulon proteins.31 The mammalian reticulon family of proteins consists of four members (RTN1, RTN2, RTN3 and RTN4) that have a highly conserved C-terminal region termed the reticulon homology domain (RHD).32,33 The N-terminal domain of each RTN member is unique and may mediate functions specific to each family member. While the specific functions for most of the RTNs remain elusive, RTN4 (also called Nogo) is widely recognized as an inhibitor of axonal extension and neurite outgrowth.34,35 Notably, while the expression pattern of the RTN family members is overlapping in some tissues, there is also regional expression unique to each family member.33 In human and rodent brains, RTN3 is largely expressed in neurons while RTN4-A (Nogo-A) is predominantly expressed in oligodendrocytes.31,33,36 As most Aβ is generated from neurons in the brain, RTN3 is perhaps a main RTN family member that regulates brain BACE1 activity. Therefore, RTN3 is the focus of the current studies. We have previously shown that BACE1 interacts with all four RTN members,31 implying that the conserved RHD may mediate binding of RTNs to BACE1. However, the precise domain responsible for the interaction of RTNs to BACE1 was not determined. Here, by using a mutagenesis approach to delineate binding domains, we demonstrate that residues within the C-terminal regions of both RTN3 and BACE1 mediate this interaction. In addition, we show that, while RTN3 forms both homo- and heterodimers, the dimerization is not a prerequisite for the interaction with BACE1. Moreover, we demonstrate that the RTN3 mediated inhibition of BACE1 activity is dependent upon the binding of RTN3 to BACE1. Together, these studies provide further insights into the regulation of BACE1 activity by RTNs, and that may have direct implications for therapeutic approaches based upon regulating BACE1 activity.
Results Mapping RTN3 binding domain RTN3 has two major domains as illustrated in Figure 1(a). To map the region mediating the binding of RTN proteins (RTNs) to BACE1, we used a Nterminally Xpress epitope tagged full-length RTN3 as a template for generating various truncated RTN3 mutants. In addition, as RHD was anticipated to
Interacting Domains between RTN3 and BACE1
mediate the binding of RTNs to BACE1, we narrowed our mutagenesis to this domain. Our initial studies, based on using RTN3 variants containing mutated transmembrane (TM) domains, suggested that disruption of either TM1 or TM2 significantly reduced expression of RTN3 mutants and thus reduced the binding of RTN3 to BACE1 below the levels of detection (data not shown). To circumvent this, we generated additional truncated RTN3 variants by preserving the TM domains (the deleted regions specified with a bar in Figure 1(a)). A HEK-293 derived cell line, termed HM, which stably expresses HA-tagged BACE1, was used for the binding assay, which utilizes the available specific anti-HA affinity matrix. The mutant RTN3 constructs were subsequently transiently transfected into HM cells and the protein extracts from transfected cells were used for co-immunoprecipitation (co-IP) with the HA antibody. While the expression levels of truncated RTN3 proteins were comparable (Figure 1(b)), RTN3 mutant R3-ΔC36, lacking the last 36 C-terminal amino acid residues, clearly failed to interact with BACE1. All the other RTN3 truncation mutants retained their interactions with BACE1 when compared to full length RTN3 (Ax-RTN3). It should be noted that all RTNs possess a C-terminal KKXX motif, a known endoplasmic reticulum (ER) retention signal (highlighted in Figure 2(a)), but this motif is dispensable for the binding to BACE1 as the R3ΔC5 mutant lacking this domain did co-IP with BACE1. Truncation of the entire loop region of RTN3 did not affect binding of RTN3 to BACE1, implicating that this truncation did not significantly impact the overall folding of RTN3. As RTN3 truncation mutant R3-ΔC30 retained its ability to co-IP with BACE1 while R3-ΔC36 lost its binding ability, we speculated that the loss of the additional six amino acid residues (YKTQID) were likely important for binding to BACE1. Inspection of RTN C termini revealed a highly conserved triplet amino acid QID motif among all RTN proteins (Figure 2(a)). To specifically examine the role of the QID motif, we generated additional RTN3 mutants by either deleting this triplet or converting QID to AAA. Examination of these two RTN3 mutants demonstrated that disruption of the QID motif indeed dramatically reduced the interaction with BACE1 (Figure 2(b)). To further confirm that failure to interact with BACE1 in R3-ΔC36 was not due to aberrant localization, we performed confocal experiments to compare the intracellular localization of wildtype RTN3 and R3-ΔC36 in transiently transfected human neuroblastoma SHEP cells. RTN3 is largely localized in the ER and Golgi compartments (Figure 3(a)–(c)). We found that R3-ΔC36 did not alter its major cellular localization in the ER and Golgi compartments when compared to the wildtype RTN3 (Figure 3(d)). Moreover, subcellular fractionation of HM cells transfected with RTN3 or R3-ΔC36 did not show any changes in subcellular
Interacting Domains between RTN3 and BACE1
627
Figure 1. Mapping the RTN3 binding domain to BACE1. (a) Schematic illustration of RTN3 structures and indicated region truncated. RTN3 has two major domains: a unique N-terminal domain and the C-terminal RHD conserved among RTNs. Two long putative transmembrane domains, highlighted in the corresponding regions are based on predictions by the TM prediction software. The number of amino acid residues (aa) in each domain is shown below the corresponding region. All the constructs in this assay have an Xpress tag (Invitrogen) at the N-terminal end. The bars in the box represent the region deleted. (b) The upper panel shows a Western blot of lysates prepared from the cells transfected with the indicated expression construct. The lower panel shows co-IP from equal amounts of lysates precipitated with the HA antibody matrix. A monoclonal Xpress antibody was used for detecting signals in both panels. An arrowhead denotes non-specific detection of IgG light chain while an arrow indicates a potential homodimer.
Figure 2. The conserved QID triplet is required for the binding of RTN3 to BACE1. (a) Sequences of the C terminus of human RTN orthologues were aligned and the identical residues were highlighted with an asterisk(*). The R3-ΔC30 mutant ends after residue D and the terminal residue of R3-ΔC36 mutant is K as indicated in bold. The QID triplet is boxed. The potential ER retention signal (KKXX) at the C terminus is also highlighted. (b) HM cells were transiently transfected with the indicated constructs and lysates were prepared for examination of protein expression and co-IP with the anti-HA matrix. A monoclonal Xpress antibody was used for detecting signals in both panels.
628
Interacting Domains between RTN3 and BACE1
Figure 3. Lacking of C-terminal domain in RTN3 does not alter its cellular distribution. (a)–(c) Human neuroblastomas cells grown on coverslips were transfected with BACE1-HA for 24 h and cells were grown in fresh medium for another 34 hr. Antibody R458 was used to label RTN3 (a) and monoclonal anti-HA recognizes BACE1 (b) in standard confocal experiments. The merged panel showed co-localization of RTN3 with BACE1 (c). (d) The same cell line was transfected with the R3-ΔC36 mutant under the same conditions. An Anti-Xpress tag was used to label mutant RTN3 and showed no gross changes of localization of RTN3. (e) Subcellular distribution of RTN3 and R3-ΔC36 mutant were compared by the step-wise sucrose gradient approach. An equal volume of samples from each fraction was resolved in a 4%–12% NuPage gel followed by Western analysis with antibody R458 for RTN3, Anti-Xpress for R3-ΔC36, and anti-HA for BACE1. Antibodies against calnexin (ER marker) and γ-adaptin (Golgi marker) were used to verify the separation.
distributions (Figure 3(e)). Similarly, experiments using the expression construct (R3-ΔQID) that disrupts the QID motif also showed no changes
in subcellular distribution (Figure 3(e)). Altogether, our results suggest that the QID triplet is necessary for the binding of RTN3 to BACE1.
Interacting Domains between RTN3 and BACE1
629
RTN3 forms homodimers and heterodimers During our co-IP studies, we noticed that BACE1 pulled down a small amount of RTN3 dimers (Figure 1, specified with arrows). In addition, we previously showed that both RTN3 and RTN4-B were present in BACE1 immunoprecipitated material. 31 These observations suggested that BACE1 might interact with RTN dimers as opposed to monomers. To address this question, we first tested whether RTN3 homo- or heterodimerization naturally occurs within cells. HR3M, a stable cell line derived from the HEK293 cell line, was used for co-IP experiments for its expressing ectopically engineered myc-tagged RTN3 in addition to endogenous levels of RTN3 and RTN4B (Figure 4(a), lanes 1 and 3). When the myc antibody was used for co-IP, myc-tagged RTN3 indeed pulled down a significant amount of non-tagged full length RTN3 that migrates clearly faster than the tagged variant (Figure 4(a), lane 2). In addition, RTN4-B was co-IPed with tagged RTN3 as shown on the same blot when antibody R461 was used to specifically react with the N terminus of RTN4-B (Figure 4(a), lane 4). In addition, RTN1 was also co-IPed with RTN3 (Figure 4(a), lane 6). Together, our data suggest that RTNs are capable of forming both a homo- and heterodimers in cells and that the RHD domain appears to mediate this dimerization. To determine whether C-terminally deleted RTN3 variants would form dimers with RTN3, we transiently transfected these constructs in HR3M cells and performed co-IP with the myc antibody. Even though R3-ΔC36 lacked its capability to bind BACE1, it still formed a dimer with myc-tagged RTN3 (Figure 4(b)), suggesting that two different regions within RHD mediate RTN dimerization or the binding of RTNs to BACE1. RTN monomer predominantly binds to BACE1 To further determine whether dimerization is required for the binding of RTN3 to BACE1, we co-
Figure 5. BACE1 mainly interacts with RTN3 monomer. HM cells were transfected with an equal amount of RTN constructs either alone (Ax-RTN3 or R3-ΔC36) or together (Ax-RTN3 + R3-ΔC36) for 48 h and the lysates were prepared for immunoprecipitation with the anti-HA matrix. RTN3 variants on the blot were detected with Xpress antibody.
transfected full length RTN3 either alone or together with R3-ΔC36 in HM cells. We reasoned that BACE1 would pull down R3-ΔC36 only if dimerization was required for this interaction. Interestingly, we found that BACE1 only pulled down full length RTN3 but not R3-ΔC36 or the R3ΔC36/RTN3 dimer (Figure 5). In fact, the expression levels of Ax-RTN3 and R3-ΔC36 were essentially comparable; R3-ΔC36 again was shown
Figure 4. RTN3 forms a homoand heterodimer with RTN orthologues. (a) Cell lysates were prepared from HR3M cells and immunoprecipitated with myc antibody 9E10. Lanes 1, 3 and 5 show expression of proteins and lanes 2, 4 and 6 show proteins co-IPed with myc-tagged RTN3. Antibody R458 was used to detect RTN3 (lanes 1– 2) and R461 was for RTN4-B (lanes 3 and 4). The fragment that migrated faster than RTN-4B is designated RTN-4Bf for a presumably cleaved fragment. In lanes 5 and 6, lysates were prepared from HEK-293 cells transfected with RTN1-myc and immunoprecipitated with antibody R458. Antibody 9E10 was used to detect myc-tagged RTN1. The bands not specified in the panels are non-specific signals from IP. (b) HR3M cells were transfected with R3-ΔC5, R3-ΔC30 and R3-ΔC36 for 48 h and the lysates were immunoprecipated with antibody 9E10 to pull down myc-tagged RTN3. The Western blot was incubated with Xpress antibody to detect Xpress-tagged RTN3 variants.
630 incapable of binding to BACE1. When both AxRTN3 and R3-ΔC36 were expressed in the same cells, BACE1 still did not pull down R3-ΔC36 even though R3-ΔC36 is capable to form a dimer with full length RTN3. Thus, RTN dimerization is unlikely required for the interaction of RTN3 with BACE1. BACE1 may predominantly interact with a RTN monomer. BACE1 C-terminal domain binds to RTN3 Although the membrane topology of RTN3 has not been reported, previous studies have demonstrated that the C-terminal tail of Nogo (RTN4) face cytoplasm. 38 Considering the highly conserved RHD among RTN members, one may reasonably expect the RTN3 C-terminal QID triplet to face the cytoplasmic side. In consistent with the topological orientation, we speculated that the C-terminal region of BACE1, which is predicted to lie on the same side of the plasma membrane, might also be involved in the binding of BACE1 to RTN proteins To test this, we generated a series of BACE1 deletion mutants for co-IP experiments (Figure 6(a)). Similar to the case in RTN3, the amino acid residues near the C-terminal end of BACE1 were found to be dispensable for binding to RTN3 (Figure 6(b)).
Interacting Domains between RTN3 and BACE1
However, further deletions of the BACE1 C-terminal sequences (constructs B1-CLR and B1-QWR; Figure 6(a)) significantly suppressed, perhaps not completely abolished, the binding of BACE1 to RTN3. To further confirm that the C-terminal sequence of BACE1 is important for the binding to RTN3, we generated a construct (B1-CX; Figure 6(a)), which contains a single nucleotide deletion right after residue V477 at the end of the BACE1 TM domain. B1-CX is expected to encode a mutated BACE1 protein differed only at the short C-terminal tail with scrambled sequences (Figure 6(a)). Although this mutant protein was still expressed in the ER and Golgi (data not shown, and similar results were previously presented38), it failed to interact with RTN3 (lane 7 in Figure 6(b)). Together, our results suggest that the sequences immediately adjacent to the TM domain of BACE1 are important for the interaction between BACE1 and RTN3. As discussed above, the TM domains of RTN3 are critical for stably docking the protein in the membrane and for the binding of BACE1 to occur. We found that deletion of the BACE1 TM domain together with the C-terminal region completely abolished the interaction between BACE1 and RTN3 (lane 3 in Figure 6(c)). However, when we expressed the construct STM37 that translates only
Figure 6. The C-terminal region of BACE1 is critical for the binding to RTN3. (a) The C-terminal deleted constructs used for the assay were depicted in partial sequences containing the corresponding ending. The stop codon is denoted with an asterisk (*). The expected scramble sequences due to a frame shift are highlighted in italics. (b) and (c) BACE1 deletion constructs were transfected in HR3M cells for 48 h and protein extracts were used for Western blot analysis and immunoprecipitation with the myc antibody 9E10. Antibody B279 recognizing BACE1 residues 295 to 310 was used for detection. B1-DTMC-KDEL was generated by removal of TM and C-terminal domains followed by addition of a KDEL sequence, an ER retention signal. BACE1 variants are specified with arrows while non-specific bands in all lanes are due to cross-reactions with a secondary antibody.
631
Interacting Domains between RTN3 and BACE1
the BACE1 signal peptide and TM domain, it did not co-IP with RTN3 (data not shown), suggesting that the BACE1 TM alone was insufficient for its interaction with RTN3. Therefore, it is likely that the localization of BACE1 within the membrane, but not the TM itself, is important for binding to RTN3. Thus, both sequences near the TM of BACE within the C terminus and its proper orientation within the membrane are needed for the BACE1/RTN3 interaction to occur. RTN3 mutants in inhibition of BACE1 activity We previously demonstrated that increased expression of RTN3 reduces the production of Aβ peptides by sequestering BACE1 to access its APP substrate.31 To determine whether RTN3 mutants affected BACE1 activity, we transfected the mutant RTN3 constructs into 125.3 cells that stably express APP with the Swedish FAD mutation that results in preferential cleavage by BACE1. Consistent with
prior observations,31 increased expression of wtRTN3 and the N-terminal tagged Ax-RTN3 reduced the steady-state levels of Aβ (Figure 7). However, the RTN3 C-terminally truncated mutant (R3-ΔC36) that was incapable of binding to BACE1 (Figure 1(b)) also did not exhibit significant effects on the levels of Aβ when compared to the control (Figure 7). Similarly, both of the QID mutants also failed to alter the levels of secreted Aβ (Figure 7). Hence, our data strongly suggest that the physical interaction between RTN3 and BACE1 is a prerequisite for the inhibition of BACE1 activity on APP substrates by RTN3.
Discussion Both genetic and biochemical evidence have demonstrated that excessive accumulation of Aβ, especially the longer Aβ42 isoform, leads to amyloid deposition and formation of neuritic plaques in the
Figure 7. ELISA of Aβ peptides. Aliquots of conditioned medium from cells transfected with the indicated constructs for 48 h were used for measuring the levels of Aβ40 (a) and Aβ42 (b) by ELISA. (n = 6, ***<0.001 compared to the vector transfected control).
632 brains of Alzheimer's cases.39 Therefore, inhibition of Aβ is likely an effective therapeutic approach to treat Alzheimer's disease.4,40 BACE1 is widely viewed to be one of the most promising drug targets for this purpose.2 Various approaches for inhibiting BACE1 have been actively pursued. Identification of RTNs as a BACE1 modulator offers an alternative venue to discover molecules that may mimic RTNs' effects. This study demonstrated that small regions located within the C terminus of both BACE1 and RTNs are responsible for their interaction. Our studies suggest that small molecules capable of binding to this small region of BACE1 may have similar inhibitory effects. RTN proteins are characterized to be a group of membrane-bound proteins that share highly conserved C-terminal domains (RHD).32,33 The RHD is comprised of two hydrophobic stretches separated by a 66 amino acid loop followed by a short Cterminal tail. Although the functional role of the RHD is not completely understood, this region may have multiple roles that are likely shared among RTNs. Our data demonstrate that a completely conserved QID triplet within the C-terminal tail of mammalian RTNs is only critical for the interaction of RTNs with BACE1 (Figure 2), but not RTN dimerization. As yet to be identified, a region within RHD may mediate RTN dimerization. Formation of homodimers in RTN1 and RTN4 (Nogo) have been reported,41,42 and our results are consistent with these observations. RTN dimerization appears to occur naturally, but the functional importance has not been defined. Our recent studies suggest that RTN3 dimers may further aggregate to form RTN3 oligomers (X.H. and R.Y., unpublished observations) and oligomerization of RTN1 may also occur.42 Our results suggest that increased RTN3 oligomerization occurs in the brains of AD and is associated with the formation of dystrophic neurites (X.H. and R.Y., unpublished observations). Interestingly, the results from the current study suggest that BACE1 may not be associated with larger RTN3 oligomers as evidenced in Figure 1 that shows no RTN3 oligomers (> 100 kDa) in co-IP with BACE1. This may suggest that modulation of BACE1 activity is largely due to RTN3 monomers while biochemical functions of RTN3 oligomers remain to be determined. The C-terminal domain of BACE1 is short, but increasing evidence suggests that it mediates several different protein–protein interactions. The SLLK sequence within the BACE1 C terminus has already been shown to bind GGA.43,44 Our current studies demonstrate that the SLLK sequences are not required for binding to RTNs, but instead a short C-terminal stretch in immediate proximity to the BACE1 TM are responsible for the binding of BACE1 to RTNs. Despite the fact that a small stretch of BACE1 C-terminal sequence is required for the binding to RTN3, our results cannot exclude the possibility that the proper conformation, due to the effects by these residues, is important for the binding to RTN3. In essence, our results confirm that at least two different domains within the C-
Interacting Domains between RTN3 and BACE1
terminal region of BACE1 are involved in the binding of BACE1 to different proteins. It should be noted that the docking of BACE1 and RTNs within the membrane is essential for their interaction to occur even though the C-terminal sequences in both proteins appear to mediate the binding. We found that disruption of RTN TM domains, particularly the second TM domain, rendered RTN3 unstable (data not shown) and incapable of binding to BACE1. Similarly, the TM domain of BACE1 is critical for localizing BACE1 to the Golgi compartment and efficient processing of APP at the β-secretase site.37 In this study, we found that the BACE1 TM domain alone did not confer the binding of BACE1 to RTNs (data not shown). However, deletion of this TM domain while still artificially retaining the extracellular BACE1 region in the ER compartment completely disrupted the interaction of BACE1 to RTNs (Figure 6(c)). Thus, our study suggests that the interaction between RTNs and BACE1 will likely only occur within the context of the membrane. Although this study has confirmed the interaction between BACE1 and RTNs as we previously reported, the physiological role for RTN3 binding to BACE1 remains unclear. RTN3 was recently implicated to mediate trafficking of proteins between the ER and Golgi.45 The potential function of RTN3 in the cellular trafficking of BACE1 will be examined in future studies. Such studies may further elucidate cellular function of RTN3 and its effect on the on the APP processing by BACE1.
Experimental Procedures Reagents Monoclonal antibody 9E10 recognizing the myc epitope was purchased from Santa Cruz Biotechnology (Sant Cruz, CA); monoclonal anti-HA serum was from Roche Applied Science (Indianapolis, IN); monoclonal antiXpress tag antibody was from Invitrogen (Carlsbad, CA). Polyclonal antibodies R454 and R458 were raised to recognize the N and C terminus of RTN3, respectively, and R461 recognizes the Nterminus of RTN4-B.31 Cell lines Human HEK-293 cells were maintained at 37 °C in a humidified, 5% CO2 controlled atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, 50 μg/ml streptomycin and glutamine. HEK-293 cells were used to generate a stable cell line (HM) expressing BACE1 under the selection of hygromycin B (100 μg/ml) or HR3M expressing myc-tagged RTN3 under the selection of G-418 (80 μg/ml). Enzyme-linked immunosorbent assay (ELISA) The analysis of Aβ1-40 and Aβ1-42 levels from conditioned media from the cell lines was performed as described.9
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Interacting Domains between RTN3 and BACE1
Site-directed mutagenesis
References
Mutagenesis of RTN3 and BACE1 was performed by either using the QuikChange site-directed mutagenesis kit (Strategene, Palo Alto, CA) according to the provided procedure or PCR-based mutagenesis. To make Cterminal deletions a stop codon was introduced by site-directed mutagenesis after the indicated position according to the provided protocol (Strategene, Palo Alto, CA). Deletion of the RTN3 loop region was achieved by standard PCR amplification of two spanning fragments and then ligation of the two fragments via a common restriction site present in the middle. All constructs were validated by double-strand DNA sequencing.
1. Selkoe, D. J. (1997). Alzheimer's disease: genotypes, phenotypes, and treatments. Science, 275, 630–631. 2. Citron, M. (2004). Beta-secretase inhibition for the treatment of Alzheimer's disease-promise and challenge. Trends Pharmacol. Sci. 25, 92–97. 3. Sisodia, S. S. & George-Hyslop, P. H. (2002). gammaSecretase, Notch, Abeta and Alzheimer's disease: where do the presenilins fit in? Nature Rev. Neurosci. 3, 281–290. 4. Haass, C. (2004). Take five-BACE and the gammasecretase quartet conduct Alzheimer's amyloid betapeptide generation. EMBO J. 23, 483–488. 5. Kimberly, W. T. & Wolfe, M. S. (2003). Identity and function of gamma-secretase. J. Neurosci. Res. 74, 353–360. 6. Iwatsubo, T. (2004). The gamma-secretase complex: machinery for intramembrane proteolysis. Curr. Opin. Neurobiol. 14, 379–383. 7. Tanzi, R. E. & Bertram, L. (2005). Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell, 120, 545–555. 8. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P. et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286, 735–741. 9. Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M. et al. (1999). Membraneanchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature, 402, 533–537. 10. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C. et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol. Cell Neurosci. 14, 419–427. 11. Sinha, S. (2002). The role of beta-amyloid in Alzheimer's disease. Med. Clin. North Am. 86, 629–639. 12. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A. & Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc. Natl Acad. Sci. USA, 97, 1456–1460. 13. Bodendorf, U., Danner, S., Fischer, F., Stefani, M., Sturchler-Pierrat, C., Wiederhold, K. H. et al. (2002). Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid. J. Neurochem. 80, 799–806. 14. Mohajeri, M. H., Saini, K. D. & Nitsch, R. M. (2004). Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J. Neural Transm. 111, 413–425. 15. Chiocco, M. J., Kulnane, L. S., Younkin, L., Younkin, S., Evin, G. & Lamb, B. T. (2004). Altered amyloidbeta metabolism and deposition in genomic-based beta-secretase transgenic mice. J. Biol. Chem. 279, 52535–52542. 16. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P. et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nature Neurosci. 4, 231–232. 17. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L. & Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nature Neurosci. 4, 233–234. 18. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M. J., Branstetter, D. G., Chen, K. S. et al. (2001). BACE knockout mice are healthy despite lacking the
Subcellular fractionation on a sucrose density gradient Sucrose gradient fractionation was performed according to the procedures described.37 Briefly, two plates of 10 cm dishes of HM cells were transfected with either wtRTN3 or R3-ΔC36 expression constructs for 48 h and cell homogenates were loaded on top of a 10 ml step-wise sucrose gradient. Eleven 1 ml fractions were collected from the top of the gradient after centrifugation. Equal volumes of solution from each fraction were separated on a 4%–12%(w/v) Bis-Tris NuPage gel (Invitrogen, Carlsbad, CA) followed by Western analysis. Antibodies R458 and anti-Xpress tag were used for detection RTN3 and R3ΔC36, respectively. Antibodies against marker proteins for different vesicles such as calnexin, γ-adaptin and EEA1 were used to ensure proper enrichment. An Anti-HA tag was used to locate HA-tagged BACE1. Immunoprecipitation and Western blot assay Cells were first grown in DMEM media for 24 hours in 10 mm plates to about 70% confluence and then transfected with the indicated expression constructs using lipofactamine 2000 (Invitrogen, Carlsbad, CA). After transfection for 48 h, cell lysates were prepared according to the procedures for immunoprecipitation with monoclonal antibody HA, R458 or 9E10 using a standard overnight immunoprecipitation (IP) protocol provided in the Co-IP kit and Application Set (Pierce, Rockford, IL). Immunoprecipitates were resolved on a 4%–12% NuPage Bis-Tris gel from Invitrogen (Carlsbad, CA) and Western blot analysis was performed. Following incubation with the primary antibodies R458 (for RTN3), Xpress (for Xpress tagged RTN3 variants), myc (for tagged RTN proteins) or B278 (for BACE1),37 an appropriate HRPconjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was utilized to detect immunoreactivity via chemiluminescence using SuperSignal West PICO reagent (Pierce, Rockford, IL).
Acknowledgements We thank Dr Bruce Lamb for critical reading of the manuscript and members in Yan Lab for the discussions during the studies. This work is partially supported by NIH grants to R.Y. (AG025493).
634
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum. Mol. Genet. 10, 1317–1324. Fukumoto, H., Cheung, B. S., Hyman, B. T. & Irizarry, M. C. (2002). Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch. Neurol. 59, 1381–1389. Holsinger, R. M., McLean, C. A., Beyreuther, K., Masters, C. L. & Evin, G. (2002). Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann. Neurol. 51, 783–786. Yang, L. B., Lindholm, K., Yan, R., Citron, M., Xia, W., Yang, X. L. et al. (2003). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nature Med. 9, 3–4. Brady, S. F., Singh, S., Crouthamel, M. C., Holloway, M. K., Coburn, C. A., Garsky, V. M. et al. (2004). Rational design and synthesis of selective BACE-1 inhibitors. Bioorg. Med. Chem. Letters, 14, 601–604. Kimura, T., Shuto, D., Kasai, S., Liu, P., Hidaka, K., Hamada, T. et al. (2004). KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine. Bioorg. Med. Chem. Letters, 14, 1527–1531. Lamar, J., Hu, J., Bueno, A. B., Yang, H. C., Guo, D., Copp, J. D. et al. (2004). Phe*-Ala-based pentapeptide mimetics are BACE inhibitors: P2 and P3 SAR. Bioorg. Med. Chem. Letters, 14, 239–243. Chang, W. P., Koelsch, G., Wong, S., Downs, D., Da, H., Weerasena, V. et al. (2004). In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors. J. Neurochem. 89, 1409–1416. Hu, B., Fan, K. Y., Bridges, K., Chopra, R., Lovering, F., Cole, D. et al. (2004). Synthesis and SAR of bis-statine based peptides as BACE 1 inhibitors. Bioorg. Med. Chem. Letters, 14, 3457–3460. John, V., Beck, J. P., Bienkowski, M. J., Sinha, S. & Heinrikson, R. L. (2003). Human beta-secretase (BACE) and BACE inhibitors. J. Med. Chem. 46, 4625–4630. Charlwood, J., Dingwall, C., Matico, R., Hussain, I., Johanson, K., Moore, S. et al. (2001). Characterization of the glycosylation profiles of Alzheimer's betasecretase protein Asp-2 expressed in a variety of cell lines. J. Biol. Chem. 276, 16739–16748. Kalvodova, L., Kahya, N., Schwille, P., Ehehalt, R., Verkade, P., Drechsel, D. & Simons, K. (2005). Lipids as modulators of proteolytic activity Of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro. J. Biol. Chem. 280, 36815–36823. Scholefield, Z., Yates, E. A., Wayne, G., Amour, A., McDowell, W. & Turnbull, J. E. (2003). Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's {beta}-secretase. J. Cell Biol. 163, 97–107. He, W., Lu, Y., Qahwash, I., Hu, X. Y., Chang, A. & Yan, R. (2004). Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nature Med. 10, 959–965.
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32. Oertle, T. & Schwab, M. E. (2003). Nogo and its paRTNers. Trends Cell Biol. 13, 187–194. 33. Yan, R., Shi, Q., Hu, X. & Zhou, X. (2006). Reticulon proteins: emerging players in neurodegenerative diseases. Cell Mol. Life Sci. 33, 877–889. 34. Chen, M. S., Huber, A. B., van der Haar, M. E., Frank, M., Schnell, L., Spillmann, A. A. et al. (2000). Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 403, 434–439. 35. GrandPre, T., Nakamura, F., Vartanian, T. & Strittmatter, S. M. (2000). Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature, 403, 439–444. 36. Wang, X., Chun, S. J., Treloar, H., Vartanian, T., Greer, C. A. & Strittmatter, S. M. (2002). Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J. Neurosci. 22, 5505–5515. 37. Yan, R., Han, P., Miao, H., Greengard, P. & Xu, H. (2001). The transmembrane domain of the Alzheimer's beta-secretase (BACE1) determines its late Golgi localization and access to beta-amyloid precursor protein (APP) substrate. J. Biol. Chem. 276, 36788–36796. 38. Oertle, T., van der Haar, M. E., Bandtlow, C. E., Robeva, A., Burfeind, P., Buss, A. et al. (2003). Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions. J. Neurosci. 23, 5393–5406. 39. Hardy, J. & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297, 353–356. 40. Schenk, D. (2004). Hopes remain for an Alzheimer's vaccine. Nature, 431, 398. 41. Dodd, D. A., Niederoest, B., Bloechlinger, S., Dupuis, L., Loeffler, J. P. & Schwab, M. E. (2005). Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types. J. Biol. Chem. 280, 12494–12502. 42. Senden, N. H., van de Velde, H. J., Broers, J. L., Timmer, E. D., Kuijpers, H. J., Roebroek, A. J. et al. (1994). Subcellular localization and supramolecular organization of neuroendocrine-specific protein B (NSP-B) in small cell lung cancer. Eur. J. Cell Biol. 65, 341–353. 43. He, X., Li, F., Chang, W. & Tang, J. (2004). GGA proteins mediate the recycling pathway of memapsin 2 (BACE). J. Biol. Chem. 280, 11696–11703. 44. von Arnim, C. A., Tangredi, M. M., Peltan, I. D., Lee, B. M., Irizarry, M. C., Kinoshita, A. & Hyman, B. T. (2004). Demonstration of BACE (beta-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy. J. Cell Sci. 117, 5437–5445. 45. Wakana, Y., Koyama, S., Nakajima, K., Hatsuzawa, K., Nagahama, M., Tani, K. et al. (2005). Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. Biochem. Biophys. Res. Commun. 334, 1198–1205.
Edited by M. Yaniv (Received 8 May 2006; accepted 21 July 2006) Available online 11 August 2006
J. Mol. Biol. (2006) 363, 625–634
Mapping of Interaction Domains Mediating Binding between BACE1 and RTN/Nogo Proteins Wanxia He 1 , Xiangyou Hu 1 , Qi Shi 1 , Xiangdong Zhou 1 , Yifeng Lu 2 Christopher Fisher 2 and Riqiang Yan 1 ⁎ 1
Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA 2
Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, MI 49007, USA
BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the β-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. © 2006 Published by Elsevier Ltd.
*Corresponding author
Keywords: BACE1; RTN; Nogo; reticular; amyloid peptide
Introduction Alzheimer's disease (AD) is the most common neurodegenerative disease. While the exact etiology of AD remains to be identified, the formation of senile plaques in the brains of AD cases has been demonstrated to be an invariant feature of the disease.1 Senile plaques, also called neuritic plaques, contain extracellular aggregates of β-amyloid peptides (Aβ) surrounded by dystrophic neurites, activated microglia and reactive astrocytes. Aβ, typically 39–43 amino acid residues in length, is excised from the larger membrane-bound amyloid precursor protein (APP) through sequential cleavages by two endopeptidases: β and γ-secretase.2–4 Recent studies have demonstrated that the functional γ-secretase activity requires proper assembly of a complex comprising of at least five transmembrane proteins including the presenilins (PSEN1 and
Abbreviations used: APP, amyloid precursor protein; AD, Alzheimer's disease; RHD, reticulon homology domain; TM, transmembrane; co-IP, co-immunoprecipitate(ion); ER, endoplasmic reticulum. E-mail address of the corresponding author: [email protected] 0022-2836/$ - see front matter © 2006 Published by Elsevier Ltd.
PSEN25–7). Various causative mutations in PSEN1 and PSEN2 have been identified in early onset familial AD and all of these mutations lead to increased production of 42 amino acids Aβ (Aβ42).7 The molecular mechanism underlying the cleavage of APP by the γ-secretase complex to result in heterogenous Aβ C-terminal ends remains a mystery, and is an active area of exploration. By contrast, the mechanism of APP processing at the N terminus of Aβ by β-secretase has been clearly established. A membrane-bound aspartyl protease, termed beta-site APP cleaving enzyme 1 (BACE1),8 has been shown to function as the sole β-secretase.8–12 BACE1 cleavage of APP releases a large secreted fragment (sAPPβ) together with a membranebound C-terminal fragment (C99). C99 is the substrate for the γ-secretase complex to release full length Aβ. Several lines of evidence have demonstrated the role of BACE1 in processing APP to Aβ that results in human AD. First, increased expression of BACE1 in cells and in mice significantly facilitates processing of APP at the β-site.13–15 Second, mice lacking BACE1 exhibit almost undetectable levels of Aβ. 16–18 Third, and perhaps most importantly, higher levels of BACE1 have been detected in human AD brains.19–21 Together, these data suggest that inhibition of BACE1 activity will be an
626 effective therapeutic approach for blocking amyloid depositions. Indeed, many potent BACE1 inhibitors have already been shown to reduce Aβ levels both in vitro and in vivo.21–27 In addition to the efforts to identify small molecule BACE1 inhibitors, potential regulation of BACE1 activity by cellular factors also has been explored. For instances, it has been demonstrated that BACE1 activity is regulated by the state of glycosylation,28 composition of lipids in the membrane bilayer,29 interactions with heparin sulfate30 and recently described reticulon proteins.31 The mammalian reticulon family of proteins consists of four members (RTN1, RTN2, RTN3 and RTN4) that have a highly conserved C-terminal region termed the reticulon homology domain (RHD).32,33 The N-terminal domain of each RTN member is unique and may mediate functions specific to each family member. While the specific functions for most of the RTNs remain elusive, RTN4 (also called Nogo) is widely recognized as an inhibitor of axonal extension and neurite outgrowth.34,35 Notably, while the expression pattern of the RTN family members is overlapping in some tissues, there is also regional expression unique to each family member.33 In human and rodent brains, RTN3 is largely expressed in neurons while RTN4-A (Nogo-A) is predominantly expressed in oligodendrocytes.31,33,36 As most Aβ is generated from neurons in the brain, RTN3 is perhaps a main RTN family member that regulates brain BACE1 activity. Therefore, RTN3 is the focus of the current studies. We have previously shown that BACE1 interacts with all four RTN members,31 implying that the conserved RHD may mediate binding of RTNs to BACE1. However, the precise domain responsible for the interaction of RTNs to BACE1 was not determined. Here, by using a mutagenesis approach to delineate binding domains, we demonstrate that residues within the C-terminal regions of both RTN3 and BACE1 mediate this interaction. In addition, we show that, while RTN3 forms both homo- and heterodimers, the dimerization is not a prerequisite for the interaction with BACE1. Moreover, we demonstrate that the RTN3 mediated inhibition of BACE1 activity is dependent upon the binding of RTN3 to BACE1. Together, these studies provide further insights into the regulation of BACE1 activity by RTNs, and that may have direct implications for therapeutic approaches based upon regulating BACE1 activity.
Results Mapping RTN3 binding domain RTN3 has two major domains as illustrated in Figure 1(a). To map the region mediating the binding of RTN proteins (RTNs) to BACE1, we used a Nterminally Xpress epitope tagged full-length RTN3 as a template for generating various truncated RTN3 mutants. In addition, as RHD was anticipated to
Interacting Domains between RTN3 and BACE1
mediate the binding of RTNs to BACE1, we narrowed our mutagenesis to this domain. Our initial studies, based on using RTN3 variants containing mutated transmembrane (TM) domains, suggested that disruption of either TM1 or TM2 significantly reduced expression of RTN3 mutants and thus reduced the binding of RTN3 to BACE1 below the levels of detection (data not shown). To circumvent this, we generated additional truncated RTN3 variants by preserving the TM domains (the deleted regions specified with a bar in Figure 1(a)). A HEK-293 derived cell line, termed HM, which stably expresses HA-tagged BACE1, was used for the binding assay, which utilizes the available specific anti-HA affinity matrix. The mutant RTN3 constructs were subsequently transiently transfected into HM cells and the protein extracts from transfected cells were used for co-immunoprecipitation (co-IP) with the HA antibody. While the expression levels of truncated RTN3 proteins were comparable (Figure 1(b)), RTN3 mutant R3-ΔC36, lacking the last 36 C-terminal amino acid residues, clearly failed to interact with BACE1. All the other RTN3 truncation mutants retained their interactions with BACE1 when compared to full length RTN3 (Ax-RTN3). It should be noted that all RTNs possess a C-terminal KKXX motif, a known endoplasmic reticulum (ER) retention signal (highlighted in Figure 2(a)), but this motif is dispensable for the binding to BACE1 as the R3ΔC5 mutant lacking this domain did co-IP with BACE1. Truncation of the entire loop region of RTN3 did not affect binding of RTN3 to BACE1, implicating that this truncation did not significantly impact the overall folding of RTN3. As RTN3 truncation mutant R3-ΔC30 retained its ability to co-IP with BACE1 while R3-ΔC36 lost its binding ability, we speculated that the loss of the additional six amino acid residues (YKTQID) were likely important for binding to BACE1. Inspection of RTN C termini revealed a highly conserved triplet amino acid QID motif among all RTN proteins (Figure 2(a)). To specifically examine the role of the QID motif, we generated additional RTN3 mutants by either deleting this triplet or converting QID to AAA. Examination of these two RTN3 mutants demonstrated that disruption of the QID motif indeed dramatically reduced the interaction with BACE1 (Figure 2(b)). To further confirm that failure to interact with BACE1 in R3-ΔC36 was not due to aberrant localization, we performed confocal experiments to compare the intracellular localization of wildtype RTN3 and R3-ΔC36 in transiently transfected human neuroblastoma SHEP cells. RTN3 is largely localized in the ER and Golgi compartments (Figure 3(a)–(c)). We found that R3-ΔC36 did not alter its major cellular localization in the ER and Golgi compartments when compared to the wildtype RTN3 (Figure 3(d)). Moreover, subcellular fractionation of HM cells transfected with RTN3 or R3-ΔC36 did not show any changes in subcellular
Interacting Domains between RTN3 and BACE1
627
Figure 1. Mapping the RTN3 binding domain to BACE1. (a) Schematic illustration of RTN3 structures and indicated region truncated. RTN3 has two major domains: a unique N-terminal domain and the C-terminal RHD conserved among RTNs. Two long putative transmembrane domains, highlighted in the corresponding regions are based on predictions by the TM prediction software. The number of amino acid residues (aa) in each domain is shown below the corresponding region. All the constructs in this assay have an Xpress tag (Invitrogen) at the N-terminal end. The bars in the box represent the region deleted. (b) The upper panel shows a Western blot of lysates prepared from the cells transfected with the indicated expression construct. The lower panel shows co-IP from equal amounts of lysates precipitated with the HA antibody matrix. A monoclonal Xpress antibody was used for detecting signals in both panels. An arrowhead denotes non-specific detection of IgG light chain while an arrow indicates a potential homodimer.
Figure 2. The conserved QID triplet is required for the binding of RTN3 to BACE1. (a) Sequences of the C terminus of human RTN orthologues were aligned and the identical residues were highlighted with an asterisk(*). The R3-ΔC30 mutant ends after residue D and the terminal residue of R3-ΔC36 mutant is K as indicated in bold. The QID triplet is boxed. The potential ER retention signal (KKXX) at the C terminus is also highlighted. (b) HM cells were transiently transfected with the indicated constructs and lysates were prepared for examination of protein expression and co-IP with the anti-HA matrix. A monoclonal Xpress antibody was used for detecting signals in both panels.
628
Interacting Domains between RTN3 and BACE1
Figure 3. Lacking of C-terminal domain in RTN3 does not alter its cellular distribution. (a)–(c) Human neuroblastomas cells grown on coverslips were transfected with BACE1-HA for 24 h and cells were grown in fresh medium for another 34 hr. Antibody R458 was used to label RTN3 (a) and monoclonal anti-HA recognizes BACE1 (b) in standard confocal experiments. The merged panel showed co-localization of RTN3 with BACE1 (c). (d) The same cell line was transfected with the R3-ΔC36 mutant under the same conditions. An Anti-Xpress tag was used to label mutant RTN3 and showed no gross changes of localization of RTN3. (e) Subcellular distribution of RTN3 and R3-ΔC36 mutant were compared by the step-wise sucrose gradient approach. An equal volume of samples from each fraction was resolved in a 4%–12% NuPage gel followed by Western analysis with antibody R458 for RTN3, Anti-Xpress for R3-ΔC36, and anti-HA for BACE1. Antibodies against calnexin (ER marker) and γ-adaptin (Golgi marker) were used to verify the separation.
distributions (Figure 3(e)). Similarly, experiments using the expression construct (R3-ΔQID) that disrupts the QID motif also showed no changes
in subcellular distribution (Figure 3(e)). Altogether, our results suggest that the QID triplet is necessary for the binding of RTN3 to BACE1.
Interacting Domains between RTN3 and BACE1
629
RTN3 forms homodimers and heterodimers During our co-IP studies, we noticed that BACE1 pulled down a small amount of RTN3 dimers (Figure 1, specified with arrows). In addition, we previously showed that both RTN3 and RTN4-B were present in BACE1 immunoprecipitated material. 31 These observations suggested that BACE1 might interact with RTN dimers as opposed to monomers. To address this question, we first tested whether RTN3 homo- or heterodimerization naturally occurs within cells. HR3M, a stable cell line derived from the HEK293 cell line, was used for co-IP experiments for its expressing ectopically engineered myc-tagged RTN3 in addition to endogenous levels of RTN3 and RTN4B (Figure 4(a), lanes 1 and 3). When the myc antibody was used for co-IP, myc-tagged RTN3 indeed pulled down a significant amount of non-tagged full length RTN3 that migrates clearly faster than the tagged variant (Figure 4(a), lane 2). In addition, RTN4-B was co-IPed with tagged RTN3 as shown on the same blot when antibody R461 was used to specifically react with the N terminus of RTN4-B (Figure 4(a), lane 4). In addition, RTN1 was also co-IPed with RTN3 (Figure 4(a), lane 6). Together, our data suggest that RTNs are capable of forming both a homo- and heterodimers in cells and that the RHD domain appears to mediate this dimerization. To determine whether C-terminally deleted RTN3 variants would form dimers with RTN3, we transiently transfected these constructs in HR3M cells and performed co-IP with the myc antibody. Even though R3-ΔC36 lacked its capability to bind BACE1, it still formed a dimer with myc-tagged RTN3 (Figure 4(b)), suggesting that two different regions within RHD mediate RTN dimerization or the binding of RTNs to BACE1. RTN monomer predominantly binds to BACE1 To further determine whether dimerization is required for the binding of RTN3 to BACE1, we co-
Figure 5. BACE1 mainly interacts with RTN3 monomer. HM cells were transfected with an equal amount of RTN constructs either alone (Ax-RTN3 or R3-ΔC36) or together (Ax-RTN3 + R3-ΔC36) for 48 h and the lysates were prepared for immunoprecipitation with the anti-HA matrix. RTN3 variants on the blot were detected with Xpress antibody.
transfected full length RTN3 either alone or together with R3-ΔC36 in HM cells. We reasoned that BACE1 would pull down R3-ΔC36 only if dimerization was required for this interaction. Interestingly, we found that BACE1 only pulled down full length RTN3 but not R3-ΔC36 or the R3ΔC36/RTN3 dimer (Figure 5). In fact, the expression levels of Ax-RTN3 and R3-ΔC36 were essentially comparable; R3-ΔC36 again was shown
Figure 4. RTN3 forms a homoand heterodimer with RTN orthologues. (a) Cell lysates were prepared from HR3M cells and immunoprecipitated with myc antibody 9E10. Lanes 1, 3 and 5 show expression of proteins and lanes 2, 4 and 6 show proteins co-IPed with myc-tagged RTN3. Antibody R458 was used to detect RTN3 (lanes 1– 2) and R461 was for RTN4-B (lanes 3 and 4). The fragment that migrated faster than RTN-4B is designated RTN-4Bf for a presumably cleaved fragment. In lanes 5 and 6, lysates were prepared from HEK-293 cells transfected with RTN1-myc and immunoprecipitated with antibody R458. Antibody 9E10 was used to detect myc-tagged RTN1. The bands not specified in the panels are non-specific signals from IP. (b) HR3M cells were transfected with R3-ΔC5, R3-ΔC30 and R3-ΔC36 for 48 h and the lysates were immunoprecipated with antibody 9E10 to pull down myc-tagged RTN3. The Western blot was incubated with Xpress antibody to detect Xpress-tagged RTN3 variants.
630 incapable of binding to BACE1. When both AxRTN3 and R3-ΔC36 were expressed in the same cells, BACE1 still did not pull down R3-ΔC36 even though R3-ΔC36 is capable to form a dimer with full length RTN3. Thus, RTN dimerization is unlikely required for the interaction of RTN3 with BACE1. BACE1 may predominantly interact with a RTN monomer. BACE1 C-terminal domain binds to RTN3 Although the membrane topology of RTN3 has not been reported, previous studies have demonstrated that the C-terminal tail of Nogo (RTN4) face cytoplasm. 38 Considering the highly conserved RHD among RTN members, one may reasonably expect the RTN3 C-terminal QID triplet to face the cytoplasmic side. In consistent with the topological orientation, we speculated that the C-terminal region of BACE1, which is predicted to lie on the same side of the plasma membrane, might also be involved in the binding of BACE1 to RTN proteins To test this, we generated a series of BACE1 deletion mutants for co-IP experiments (Figure 6(a)). Similar to the case in RTN3, the amino acid residues near the C-terminal end of BACE1 were found to be dispensable for binding to RTN3 (Figure 6(b)).
Interacting Domains between RTN3 and BACE1
However, further deletions of the BACE1 C-terminal sequences (constructs B1-CLR and B1-QWR; Figure 6(a)) significantly suppressed, perhaps not completely abolished, the binding of BACE1 to RTN3. To further confirm that the C-terminal sequence of BACE1 is important for the binding to RTN3, we generated a construct (B1-CX; Figure 6(a)), which contains a single nucleotide deletion right after residue V477 at the end of the BACE1 TM domain. B1-CX is expected to encode a mutated BACE1 protein differed only at the short C-terminal tail with scrambled sequences (Figure 6(a)). Although this mutant protein was still expressed in the ER and Golgi (data not shown, and similar results were previously presented38), it failed to interact with RTN3 (lane 7 in Figure 6(b)). Together, our results suggest that the sequences immediately adjacent to the TM domain of BACE1 are important for the interaction between BACE1 and RTN3. As discussed above, the TM domains of RTN3 are critical for stably docking the protein in the membrane and for the binding of BACE1 to occur. We found that deletion of the BACE1 TM domain together with the C-terminal region completely abolished the interaction between BACE1 and RTN3 (lane 3 in Figure 6(c)). However, when we expressed the construct STM37 that translates only
Figure 6. The C-terminal region of BACE1 is critical for the binding to RTN3. (a) The C-terminal deleted constructs used for the assay were depicted in partial sequences containing the corresponding ending. The stop codon is denoted with an asterisk (*). The expected scramble sequences due to a frame shift are highlighted in italics. (b) and (c) BACE1 deletion constructs were transfected in HR3M cells for 48 h and protein extracts were used for Western blot analysis and immunoprecipitation with the myc antibody 9E10. Antibody B279 recognizing BACE1 residues 295 to 310 was used for detection. B1-DTMC-KDEL was generated by removal of TM and C-terminal domains followed by addition of a KDEL sequence, an ER retention signal. BACE1 variants are specified with arrows while non-specific bands in all lanes are due to cross-reactions with a secondary antibody.
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Interacting Domains between RTN3 and BACE1
the BACE1 signal peptide and TM domain, it did not co-IP with RTN3 (data not shown), suggesting that the BACE1 TM alone was insufficient for its interaction with RTN3. Therefore, it is likely that the localization of BACE1 within the membrane, but not the TM itself, is important for binding to RTN3. Thus, both sequences near the TM of BACE within the C terminus and its proper orientation within the membrane are needed for the BACE1/RTN3 interaction to occur. RTN3 mutants in inhibition of BACE1 activity We previously demonstrated that increased expression of RTN3 reduces the production of Aβ peptides by sequestering BACE1 to access its APP substrate.31 To determine whether RTN3 mutants affected BACE1 activity, we transfected the mutant RTN3 constructs into 125.3 cells that stably express APP with the Swedish FAD mutation that results in preferential cleavage by BACE1. Consistent with
prior observations,31 increased expression of wtRTN3 and the N-terminal tagged Ax-RTN3 reduced the steady-state levels of Aβ (Figure 7). However, the RTN3 C-terminally truncated mutant (R3-ΔC36) that was incapable of binding to BACE1 (Figure 1(b)) also did not exhibit significant effects on the levels of Aβ when compared to the control (Figure 7). Similarly, both of the QID mutants also failed to alter the levels of secreted Aβ (Figure 7). Hence, our data strongly suggest that the physical interaction between RTN3 and BACE1 is a prerequisite for the inhibition of BACE1 activity on APP substrates by RTN3.
Discussion Both genetic and biochemical evidence have demonstrated that excessive accumulation of Aβ, especially the longer Aβ42 isoform, leads to amyloid deposition and formation of neuritic plaques in the
Figure 7. ELISA of Aβ peptides. Aliquots of conditioned medium from cells transfected with the indicated constructs for 48 h were used for measuring the levels of Aβ40 (a) and Aβ42 (b) by ELISA. (n = 6, ***<0.001 compared to the vector transfected control).
632 brains of Alzheimer's cases.39 Therefore, inhibition of Aβ is likely an effective therapeutic approach to treat Alzheimer's disease.4,40 BACE1 is widely viewed to be one of the most promising drug targets for this purpose.2 Various approaches for inhibiting BACE1 have been actively pursued. Identification of RTNs as a BACE1 modulator offers an alternative venue to discover molecules that may mimic RTNs' effects. This study demonstrated that small regions located within the C terminus of both BACE1 and RTNs are responsible for their interaction. Our studies suggest that small molecules capable of binding to this small region of BACE1 may have similar inhibitory effects. RTN proteins are characterized to be a group of membrane-bound proteins that share highly conserved C-terminal domains (RHD).32,33 The RHD is comprised of two hydrophobic stretches separated by a 66 amino acid loop followed by a short Cterminal tail. Although the functional role of the RHD is not completely understood, this region may have multiple roles that are likely shared among RTNs. Our data demonstrate that a completely conserved QID triplet within the C-terminal tail of mammalian RTNs is only critical for the interaction of RTNs with BACE1 (Figure 2), but not RTN dimerization. As yet to be identified, a region within RHD may mediate RTN dimerization. Formation of homodimers in RTN1 and RTN4 (Nogo) have been reported,41,42 and our results are consistent with these observations. RTN dimerization appears to occur naturally, but the functional importance has not been defined. Our recent studies suggest that RTN3 dimers may further aggregate to form RTN3 oligomers (X.H. and R.Y., unpublished observations) and oligomerization of RTN1 may also occur.42 Our results suggest that increased RTN3 oligomerization occurs in the brains of AD and is associated with the formation of dystrophic neurites (X.H. and R.Y., unpublished observations). Interestingly, the results from the current study suggest that BACE1 may not be associated with larger RTN3 oligomers as evidenced in Figure 1 that shows no RTN3 oligomers (> 100 kDa) in co-IP with BACE1. This may suggest that modulation of BACE1 activity is largely due to RTN3 monomers while biochemical functions of RTN3 oligomers remain to be determined. The C-terminal domain of BACE1 is short, but increasing evidence suggests that it mediates several different protein–protein interactions. The SLLK sequence within the BACE1 C terminus has already been shown to bind GGA.43,44 Our current studies demonstrate that the SLLK sequences are not required for binding to RTNs, but instead a short C-terminal stretch in immediate proximity to the BACE1 TM are responsible for the binding of BACE1 to RTNs. Despite the fact that a small stretch of BACE1 C-terminal sequence is required for the binding to RTN3, our results cannot exclude the possibility that the proper conformation, due to the effects by these residues, is important for the binding to RTN3. In essence, our results confirm that at least two different domains within the C-
Interacting Domains between RTN3 and BACE1
terminal region of BACE1 are involved in the binding of BACE1 to different proteins. It should be noted that the docking of BACE1 and RTNs within the membrane is essential for their interaction to occur even though the C-terminal sequences in both proteins appear to mediate the binding. We found that disruption of RTN TM domains, particularly the second TM domain, rendered RTN3 unstable (data not shown) and incapable of binding to BACE1. Similarly, the TM domain of BACE1 is critical for localizing BACE1 to the Golgi compartment and efficient processing of APP at the β-secretase site.37 In this study, we found that the BACE1 TM domain alone did not confer the binding of BACE1 to RTNs (data not shown). However, deletion of this TM domain while still artificially retaining the extracellular BACE1 region in the ER compartment completely disrupted the interaction of BACE1 to RTNs (Figure 6(c)). Thus, our study suggests that the interaction between RTNs and BACE1 will likely only occur within the context of the membrane. Although this study has confirmed the interaction between BACE1 and RTNs as we previously reported, the physiological role for RTN3 binding to BACE1 remains unclear. RTN3 was recently implicated to mediate trafficking of proteins between the ER and Golgi.45 The potential function of RTN3 in the cellular trafficking of BACE1 will be examined in future studies. Such studies may further elucidate cellular function of RTN3 and its effect on the on the APP processing by BACE1.
Experimental Procedures Reagents Monoclonal antibody 9E10 recognizing the myc epitope was purchased from Santa Cruz Biotechnology (Sant Cruz, CA); monoclonal anti-HA serum was from Roche Applied Science (Indianapolis, IN); monoclonal antiXpress tag antibody was from Invitrogen (Carlsbad, CA). Polyclonal antibodies R454 and R458 were raised to recognize the N and C terminus of RTN3, respectively, and R461 recognizes the Nterminus of RTN4-B.31 Cell lines Human HEK-293 cells were maintained at 37 °C in a humidified, 5% CO2 controlled atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, 50 μg/ml streptomycin and glutamine. HEK-293 cells were used to generate a stable cell line (HM) expressing BACE1 under the selection of hygromycin B (100 μg/ml) or HR3M expressing myc-tagged RTN3 under the selection of G-418 (80 μg/ml). Enzyme-linked immunosorbent assay (ELISA) The analysis of Aβ1-40 and Aβ1-42 levels from conditioned media from the cell lines was performed as described.9
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Interacting Domains between RTN3 and BACE1
Site-directed mutagenesis
References
Mutagenesis of RTN3 and BACE1 was performed by either using the QuikChange site-directed mutagenesis kit (Strategene, Palo Alto, CA) according to the provided procedure or PCR-based mutagenesis. To make Cterminal deletions a stop codon was introduced by site-directed mutagenesis after the indicated position according to the provided protocol (Strategene, Palo Alto, CA). Deletion of the RTN3 loop region was achieved by standard PCR amplification of two spanning fragments and then ligation of the two fragments via a common restriction site present in the middle. All constructs were validated by double-strand DNA sequencing.
1. Selkoe, D. J. (1997). Alzheimer's disease: genotypes, phenotypes, and treatments. Science, 275, 630–631. 2. Citron, M. (2004). Beta-secretase inhibition for the treatment of Alzheimer's disease-promise and challenge. Trends Pharmacol. Sci. 25, 92–97. 3. Sisodia, S. S. & George-Hyslop, P. H. (2002). gammaSecretase, Notch, Abeta and Alzheimer's disease: where do the presenilins fit in? Nature Rev. Neurosci. 3, 281–290. 4. Haass, C. (2004). Take five-BACE and the gammasecretase quartet conduct Alzheimer's amyloid betapeptide generation. EMBO J. 23, 483–488. 5. Kimberly, W. T. & Wolfe, M. S. (2003). Identity and function of gamma-secretase. J. Neurosci. Res. 74, 353–360. 6. Iwatsubo, T. (2004). The gamma-secretase complex: machinery for intramembrane proteolysis. Curr. Opin. Neurobiol. 14, 379–383. 7. Tanzi, R. E. & Bertram, L. (2005). Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell, 120, 545–555. 8. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P. et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science, 286, 735–741. 9. Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M. et al. (1999). Membraneanchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature, 402, 533–537. 10. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C. et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol. Cell Neurosci. 14, 419–427. 11. Sinha, S. (2002). The role of beta-amyloid in Alzheimer's disease. Med. Clin. North Am. 86, 629–639. 12. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A. & Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc. Natl Acad. Sci. USA, 97, 1456–1460. 13. Bodendorf, U., Danner, S., Fischer, F., Stefani, M., Sturchler-Pierrat, C., Wiederhold, K. H. et al. (2002). Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid. J. Neurochem. 80, 799–806. 14. Mohajeri, M. H., Saini, K. D. & Nitsch, R. M. (2004). Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J. Neural Transm. 111, 413–425. 15. Chiocco, M. J., Kulnane, L. S., Younkin, L., Younkin, S., Evin, G. & Lamb, B. T. (2004). Altered amyloidbeta metabolism and deposition in genomic-based beta-secretase transgenic mice. J. Biol. Chem. 279, 52535–52542. 16. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P. et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nature Neurosci. 4, 231–232. 17. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L. & Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nature Neurosci. 4, 233–234. 18. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M. J., Branstetter, D. G., Chen, K. S. et al. (2001). BACE knockout mice are healthy despite lacking the
Subcellular fractionation on a sucrose density gradient Sucrose gradient fractionation was performed according to the procedures described.37 Briefly, two plates of 10 cm dishes of HM cells were transfected with either wtRTN3 or R3-ΔC36 expression constructs for 48 h and cell homogenates were loaded on top of a 10 ml step-wise sucrose gradient. Eleven 1 ml fractions were collected from the top of the gradient after centrifugation. Equal volumes of solution from each fraction were separated on a 4%–12%(w/v) Bis-Tris NuPage gel (Invitrogen, Carlsbad, CA) followed by Western analysis. Antibodies R458 and anti-Xpress tag were used for detection RTN3 and R3ΔC36, respectively. Antibodies against marker proteins for different vesicles such as calnexin, γ-adaptin and EEA1 were used to ensure proper enrichment. An Anti-HA tag was used to locate HA-tagged BACE1. Immunoprecipitation and Western blot assay Cells were first grown in DMEM media for 24 hours in 10 mm plates to about 70% confluence and then transfected with the indicated expression constructs using lipofactamine 2000 (Invitrogen, Carlsbad, CA). After transfection for 48 h, cell lysates were prepared according to the procedures for immunoprecipitation with monoclonal antibody HA, R458 or 9E10 using a standard overnight immunoprecipitation (IP) protocol provided in the Co-IP kit and Application Set (Pierce, Rockford, IL). Immunoprecipitates were resolved on a 4%–12% NuPage Bis-Tris gel from Invitrogen (Carlsbad, CA) and Western blot analysis was performed. Following incubation with the primary antibodies R458 (for RTN3), Xpress (for Xpress tagged RTN3 variants), myc (for tagged RTN proteins) or B278 (for BACE1),37 an appropriate HRPconjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was utilized to detect immunoreactivity via chemiluminescence using SuperSignal West PICO reagent (Pierce, Rockford, IL).
Acknowledgements We thank Dr Bruce Lamb for critical reading of the manuscript and members in Yan Lab for the discussions during the studies. This work is partially supported by NIH grants to R.Y. (AG025493).
634
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum. Mol. Genet. 10, 1317–1324. Fukumoto, H., Cheung, B. S., Hyman, B. T. & Irizarry, M. C. (2002). Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch. Neurol. 59, 1381–1389. Holsinger, R. M., McLean, C. A., Beyreuther, K., Masters, C. L. & Evin, G. (2002). Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann. Neurol. 51, 783–786. Yang, L. B., Lindholm, K., Yan, R., Citron, M., Xia, W., Yang, X. L. et al. (2003). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nature Med. 9, 3–4. Brady, S. F., Singh, S., Crouthamel, M. C., Holloway, M. K., Coburn, C. A., Garsky, V. M. et al. (2004). Rational design and synthesis of selective BACE-1 inhibitors. Bioorg. Med. Chem. Letters, 14, 601–604. Kimura, T., Shuto, D., Kasai, S., Liu, P., Hidaka, K., Hamada, T. et al. (2004). KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine. Bioorg. Med. Chem. Letters, 14, 1527–1531. Lamar, J., Hu, J., Bueno, A. B., Yang, H. C., Guo, D., Copp, J. D. et al. (2004). Phe*-Ala-based pentapeptide mimetics are BACE inhibitors: P2 and P3 SAR. Bioorg. Med. Chem. Letters, 14, 239–243. Chang, W. P., Koelsch, G., Wong, S., Downs, D., Da, H., Weerasena, V. et al. (2004). In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors. J. Neurochem. 89, 1409–1416. Hu, B., Fan, K. Y., Bridges, K., Chopra, R., Lovering, F., Cole, D. et al. (2004). Synthesis and SAR of bis-statine based peptides as BACE 1 inhibitors. Bioorg. Med. Chem. Letters, 14, 3457–3460. John, V., Beck, J. P., Bienkowski, M. J., Sinha, S. & Heinrikson, R. L. (2003). Human beta-secretase (BACE) and BACE inhibitors. J. Med. Chem. 46, 4625–4630. Charlwood, J., Dingwall, C., Matico, R., Hussain, I., Johanson, K., Moore, S. et al. (2001). Characterization of the glycosylation profiles of Alzheimer's betasecretase protein Asp-2 expressed in a variety of cell lines. J. Biol. Chem. 276, 16739–16748. Kalvodova, L., Kahya, N., Schwille, P., Ehehalt, R., Verkade, P., Drechsel, D. & Simons, K. (2005). Lipids as modulators of proteolytic activity Of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro. J. Biol. Chem. 280, 36815–36823. Scholefield, Z., Yates, E. A., Wayne, G., Amour, A., McDowell, W. & Turnbull, J. E. (2003). Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's {beta}-secretase. J. Cell Biol. 163, 97–107. He, W., Lu, Y., Qahwash, I., Hu, X. Y., Chang, A. & Yan, R. (2004). Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nature Med. 10, 959–965.
Interacting Domains between RTN3 and BACE1
32. Oertle, T. & Schwab, M. E. (2003). Nogo and its paRTNers. Trends Cell Biol. 13, 187–194. 33. Yan, R., Shi, Q., Hu, X. & Zhou, X. (2006). Reticulon proteins: emerging players in neurodegenerative diseases. Cell Mol. Life Sci. 33, 877–889. 34. Chen, M. S., Huber, A. B., van der Haar, M. E., Frank, M., Schnell, L., Spillmann, A. A. et al. (2000). Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 403, 434–439. 35. GrandPre, T., Nakamura, F., Vartanian, T. & Strittmatter, S. M. (2000). Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature, 403, 439–444. 36. Wang, X., Chun, S. J., Treloar, H., Vartanian, T., Greer, C. A. & Strittmatter, S. M. (2002). Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J. Neurosci. 22, 5505–5515. 37. Yan, R., Han, P., Miao, H., Greengard, P. & Xu, H. (2001). The transmembrane domain of the Alzheimer's beta-secretase (BACE1) determines its late Golgi localization and access to beta-amyloid precursor protein (APP) substrate. J. Biol. Chem. 276, 36788–36796. 38. Oertle, T., van der Haar, M. E., Bandtlow, C. E., Robeva, A., Burfeind, P., Buss, A. et al. (2003). Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions. J. Neurosci. 23, 5393–5406. 39. Hardy, J. & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297, 353–356. 40. Schenk, D. (2004). Hopes remain for an Alzheimer's vaccine. Nature, 431, 398. 41. Dodd, D. A., Niederoest, B., Bloechlinger, S., Dupuis, L., Loeffler, J. P. & Schwab, M. E. (2005). Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types. J. Biol. Chem. 280, 12494–12502. 42. Senden, N. H., van de Velde, H. J., Broers, J. L., Timmer, E. D., Kuijpers, H. J., Roebroek, A. J. et al. (1994). Subcellular localization and supramolecular organization of neuroendocrine-specific protein B (NSP-B) in small cell lung cancer. Eur. J. Cell Biol. 65, 341–353. 43. He, X., Li, F., Chang, W. & Tang, J. (2004). GGA proteins mediate the recycling pathway of memapsin 2 (BACE). J. Biol. Chem. 280, 11696–11703. 44. von Arnim, C. A., Tangredi, M. M., Peltan, I. D., Lee, B. M., Irizarry, M. C., Kinoshita, A. & Hyman, B. T. (2004). Demonstration of BACE (beta-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy. J. Cell Sci. 117, 5437–5445. 45. Wakana, Y., Koyama, S., Nakajima, K., Hatsuzawa, K., Nagahama, M., Tani, K. et al. (2005). Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. Biochem. Biophys. Res. Commun. 334, 1198–1205.
Edited by M. Yaniv (Received 8 May 2006; accepted 21 July 2006) Available online 11 August 2006