Research Update
promoters of herpes simplex virus (HSV), transcription is strongly activated. This differential binding affinity results from a conformational change in Oct-1, induced upon binding to the TAATGARAT sequence, that allows it to bind the HSV VP16 (Vmw65) protein (a strong transactivator), thereby increasing the transactivation potency of Oct-1 (Ref. 11) (Fig. 2b). A recent study12 has extended this to the binding of a cellular transcriptional coactivator, OBF-1 (Oct-binding factor 1), to dimers of Oct-1 that are bound to two distinct sites with different sequences. Thus, when Oct-1 binds as a dimer to a sequence known as the palendromic Oct factor recognition element (PORE), it can then bind OBF-1, resulting in strong activation of transcription. By contrast, when Oct-1 binds as a dimer to the site known as MORE (More PORE), the residues in Oct-1 that interact with OBF-1 on the PORE site are used instead to form the dimer interface between the Oct-1 monomers. Hence, when bound to the MORE site, Oct-1 cannot recruit OBF-1, and strong activation of transcription is precluded (Fig. 2c).
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Conclusion: the key role of the DNA-binding site
A variety of studies into transcriptional regulation indicate that the DNA binding site is not simply a passive partner that is merely recognized by a particular transcription factor. Rather, when transcription factors bind to different sites they assume different protein structures. In turn, these structures determine whether the bound transcription factor can interact with particular coactivator or corepressor proteins. Hence, protein changes, which occur on DNA binding, provide an additional facet to the complexity of transcription factors, allowing them to activate transcription to varying degrees, to have no effect, or to inhibit transcription. This mechanism is one of many that enable transcription factors to control the inducible and cell type-specific gene expression that is central to the complexity of the multicellular eukaryotic organism. References 1 Latchman, D.S. (1998) Gene Regulation – a eukaryotic perspective (3rd edn), Stanley Thorne Publishers 2 Latchman, D.S. (1998) Eukaryotic transcription factors (3rd edn), Academic Press
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3 Latchman, D.S., ed., (1997) Landmarks in Gene Regulation, Portland Press 4 Hanna-Rose, W. and Hansen, U. (1996) Active repression mechanisms of eukaryotic transcription repressors. Trends Genet. 12, 229–234 5 Mangelsdorf, D.J. et al. (1995) The nuclear receptor super family; the second decade. Cell 83, 835–839 6 Lefstin, J.A. and Yamamoto, K.R. (1998) Allosteric effects of DNA on transcriptional regulators. Nature 392, 885–888 7 Beato, M. et al. (1995) Steroid hormone receptors: many actors in search of a plot. Cell 83, 851–857 8 Drouin, J. et al. (1993) Novel glucocorticoid receptor complex with DNA element of the hormone repressed POMC gene. EMBO J. 12, 145–156 9 Scully, K.M. et al. (2000) Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification. Science 290, 1127–1131 10 Ryan, A.K. and Rosenfeld, M.G. (1997) POU domain family values: flexibility, partnerships and developmental codes. Genes Dev. 11, 1207–1225 11 Walker, S. et al. (1994) Site-specific conformational alteration of the Oct-1 POU domain–DNA complex as the basis for differential recognition by Vmw65 (VP16). Cell 741, 841–852 12 Tomilin, A. et al. (2000) Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration. Cell 103, 853–864
David S. Latchman Institute of Child Health, University College London, 30 Guilford Street, London, UK WC1N 1EH. e-mail:
[email protected]
Protein Sequence Motif
Nicastrin, a presenilin-interacting protein, contains an aminopeptidase/transferrin receptor superfamily domain Richard Fagan, Mark Swindells, John Overington and Malcolm Weir Nicastrin, a protein implicated in Alzheimer’s disease, has a domain that is found in the aminopeptidase/transferrin receptor superfamily. In nicastrin, this domain might possess catalytic activity (as observed with aminopeptidases) or it could serve merely as a binding domain (with analogy to the transferrin receptors) for the β-amyloid precursor protein.
Nicastrin is a 709 amino acid type I transmembrane glycoprotein that has been recently identified1 as a key component of the Alzheimer-linked multiprotein complex formed with the proteases presenilin 1 and presenilin 2. The formation of this complex is the final step in the production of the neurotoxic β-amyloid peptide (also known as the amyloid), which is observed in brain
plaques of familial Alzheimer’s disease patients. The amyloid protein is produced from the membrane-bound β-amyloid precursor protein, β-APP, in two distinct sequential steps. First, β-APP is cleaved by the protease β-secretase (BACE-2) and second, the amyloid protein is liberated by further γ-secretase processing. Current opinion suggests that presenilin 1 and presenilin 2 possess the protease catalytic activity that is necessary for the production of the neurotoxic β-amyloid peptide (amyloid protein)1,2. It was shown recently that nicastrin binds to β-APP (and its α- and β-cleaved versions) and is able to modulate the production of β-amyloid peptide1. This implicates a direct role for nicastrin in the pathogenesis of Alzheimer’s disease and
suggests that it could be a suitable target for therapeutic intervention. It was speculated that the function of nicastrin might be to bind substrates of presenilin–γ-secretase complexes or, alternatively, to modulate γ-secretase activity. However, no significant amino acid sequence similarity with known proteases, nor indeed with any other functionally annotated proteins, was found. The molecular basis for biological function of this protein therefore remained unclear. We show through the use of Genome Threader (Ref. 3; Fig. 1) that the central region of nicastrin is a new member of the aminopeptidase superfamily, which also includes the non-protease transferrin receptor (TfR). Furthermore, these
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proteins have no relationship with the well-known aspartyl protease family to which γ-secretase belongs. Specifically, the Genome Threader identifies the region of nicastrin between Asn263 and Ala483 as having a fold similar to human TfR (PDB code 1CX8)4, Streptomyces griseus aminopeptidase (PDB code 1XJO; Ref. 5; see www.biochem.ucl.ac.uk/bsm/ pdbsum/1xjo/main.html for images of these structures), and members thereof with 99.9% confidence (Fig. 1). Aminopeptidases and carboxypeptidases from this family bind two co-catalytic zinc ions. Six aminopeptidase residues (His85,247, Asp87,97,160 and Glu132 of 1XJO) are important for coordinating two zinc atoms and are highly conserved in these enzymes; however, only three are present in TfR (specifically TfR that has lost catalytic activity but binds to transferrin)6. By contrast, PSI-BLAST is Q92542 CAB89225.1 1CX8:A
1XJO
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unable to identify any aminopeptidases when the E-value threshold for including sequences in future profiles (-h option) is set at 0.001 and the NCBI non-redundant protein database is searched (-Y set by default at 101588618128). If this E-value is relaxed to 0.002, aminopeptidases are first identified in iteration 6. The alignment of the nicastrin sequence with that of Streptomyces griseus aminopeptidase (Fig. 1) indicates that one of the six zinc-binding residues of the aminopeptidase are conserved in nicastrin (His85Arg, Asp87Asp, Asp97Ser, Glu132Thr, Asp160Gly and His247Gln), as is Glu131, which has been proposed to be a key active-site residue6. Loss of crucial His residues implies that the protease activity will be absent, as in the case of TfR. However, there are conserved residues in the nicastrin sequence that are close to the structurally equivalent
SIQSTF-SINPEIVCDPLSDYNVWSMLKPINTTGTLKP---DDRVVVAATRLDSRSFFWN -----------EGTCLPLGGYSVWSSLPPISVSSSNN----RKPVVLTVASMDTASFFRD SKNVKLT------------VSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDA------
YVKAKLDAAGYTTTLQQFTSGGATGYNLIANWPGG--D---PNKVLMAGAHLDS----VS 40
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Q92542 CAB89225.1 1CX8:A
VAPGAESAVASFVTQLAAAEALQKAPDVTTL--PRNVMFVFFQGETFDYIGSSRMVYDME KSFGADSPISGLVALLGAVDALSRVDGISNL--KKQLVFLVLTGETWGYLGSRRFLHELD WGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYL
1XJO
SGAGINDNGSGSAAVLETALAVSRAGYQ--P--DKHLRFAWWGAEELGLIGSKFYVNNLP
Q92542 CAB89225.1 1CX8:A
KGKFPVQ-LEN--VDSFVELGQVALRTSLELWMHTDPVSQKNESVRNQVEDLLATLEKSG LHSDAVAGLSNTSIETVLEIGSVG--KGLSGGINTFFAHKTRVSSVTNMTLDALKIAQDS S-------SLHLKAFTYINLDKAVLGTS-NFKVSASP-----------------------
1XJO
SADRS-------KLAGYLNFDXIGSPNPGYFVYDDDP----VIEKTFKNYFAGLNVPTEI
100
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130
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Q92542 CAB89225.1 1CX8:A
AGVPAVILRRPNQSQP-LPPSSLQRFLRAR-N-ISGVVLADHSGAFHNKYYQ-------LASKNIKILSADTANPGIPPSSLMAFMRKNPQ-TSAVVLEDFDTNFVNKFYH--------LLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAV------
1XJO
RSDHAPFKNV------GVPVGGLFTGAGYTKSAAQAQKWGGTAGQAFDRCYHSSCDSLSN 210
220
230
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Ti BS
Fig. 1. Sequence alignment of human nicastrin (SWISS-PROT: Q92542, Asn263–Gln534), a putative Arabidopsis thaliana protein (GenBank: CAB89225.1, Glu33–His244), human transferrin receptor ectodomain (PDB: 1CX8:A, Ser370–Pro551) and Streptomyces griseus aminopeptidase (PDB: 1XJO), highlighting residues by 1XJO residue numbering. Results are taken from an all-by-all set of comparisons in which every unique sequence of unknown structure from GenBank and SWISS-PROT has been compared with every unique sequence of known structure from the PDB. Comparisons are performed by Genome Threader, an enhanced version of the GenTHREADER algorithm originally published by Jones3. The two approaches differ in that Genome Threader profiles are generated by a PSI-BLAST-based algorithm whereas a local Smith–Waterman alignment is subsequently performed between each sequence and the profile. Confidence values for the Genome Threader were derived by training the Genome Threader neural network to distinguish between a set of 3000 true distant relationships and 3000 known false relationships extracted from the CATH structure classification. Real levels of confidence could be lower because of limitations in assuming that PDB provides a representative benchmark for all sequences in a genome. Secondary structure is represented as follows: α helices are shown as barrels and β strands are depicted as arrows. 1XJO zinc-coordinating residues are highlighted in yellow, the catalytic Glu131 is highlighted in red and the DYIGS domain is over- or underlined in nicastrin and aminopeptidase, respectively. A chain break is indicated by black circles. The DYIGS motif is the site of missense mutation1 that has been associated with increased amyloid secretion.
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positions of the aminopeptidase and that could play a role in zinc coordination. In either case, these results imply that this domain of nicastrin will bind peptides or proteins. Loss of catalytic activity while retaining specific binding of peptides is evolutionally well precedented, for example in the case of haptoglobin and serine proteases7. Interestingly, a motif identified as being important for the presenilin–nicastrin interaction, Asp336–Ser340 (Ref. 1), lies in the same loop between strands four and five as does the active site Glu131 of the aminopeptidase, 1XJ0. We observe that in nicastrin this residue is conserved (Fig. 1). It follows that future strategies for screening and inhibition that focus on this domain, for example peptidomimetics targeted at the presumed vestigial active site of nicastrin, could prove fruitful in the search for smallmolecule drugs against Alzheimer’s disease. Nicastrin also provides an interesting link between the aminopeptidase and TfR families showing how active-site modification and subsequent loss of crucial ‘fingerprint’residues might occur through evolution. References 1 Yu, G. et al. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and βAPP processing. Nature 407, 48–54 2 Li, Y.M. et al. (2000) Photoactivated γ-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405, 689–694 3 Jones, D.T. (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J. Mol. Biol. 287, 797–815 4 Lawrence, C.M. et al. (1999) Crystal structure of the ectodomain of human transferrin receptor. Science 286, 779–782 5 Greenblatt, H.M. et al. (1997) Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 Å resolution. J. Mol. Biol. 265, 620–636 6 Mahadevan, D. and Saldanha, J. (1999) The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design. Protein Sci. 8, 2546–2549 7 Tosi, M. et al. (1989) Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin. J. Mol. Biol. 208, 709–714
Richard Fagan* Mark Swindells John Overington Malcolm Weir Inpharmatica, 60 Charlotte St, London, UK W1T 2NU. *e-mail:
[email protected]
