Evolutionary origin of a Kunitz-type trypsin inhibitor domain inserted in the amyloid β precursor protein of Alzheimer's disease

Summary The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid precursor protein (APP). It is known that APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in APP have an evolutionarily close relationship with the inter--trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4⁺ lymphocytes.Offprint requests to: T. Gojobori     

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD.     

New mechanism for post-translational processing during assembly of a cytoplasmic membrane protein?

Insertion of nitrate reductase into the Escherichia coli cytoplasmic membrane was examined by following the fate of pulse-labeled enzyme in both the membrane and cytoplasm during various times after the addition of an unlabeled chase. The polypeptide composition of this labeled enzyme was determined by autoradiography of immunoprecipitated material after separation on sodium dodecyl sulfate-polyacrylamide gels. The data presented here indicate that immediately after appropriate insertion of the enzyme into the membrane, a post-translational event occurs which converts the cytoplasmically synthesized form of subunit B (B') to the form found in the completely assembled enzyme (B). B' is distinguished from B by its more rapid electrophoretic mobility. B' was found in the cytoplasm of all strains tested, in the membrane of strains with defects in enzyme insertion (hemA and chlE), and as a transient component in the membrane of wild-type cells.     

Adaptor protein 2-mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER-Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.     

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.     

Substrate sequence influences γ-secretase modulator activity, role of the transmembrane domain of the amyloid precursor protein

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.     

Direct binding of cholesterol to the amyloid precursor protein: An important interaction in lipid–Alzheimer's disease relationships?

It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the β-C-terminal fragment, or β-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's–cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway—amyloid-β and the intracellular domain of the APP (AICD)—may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.     

Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

Background  

The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated.     

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid β precursor protein (APP) juxtamembrane region

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.     

Does the location of a mutation determine the ability to form amyloid fibrils?

We have previously reported studies of fibril formation by a set of protein G B1 domain (beta1) variants, with mutations located around the central parallel beta-strands. In this study, we designed multiple mutations in the edge strands of beta1 to create proteins with a stability range comparable to that of the set of central mutants. All the edge variants are able to form amyloid fibrils when they are incubated at their melting temperatures. This result suggests that overall protein stability is the key determinant for amyloid formation and not the specific location of destabilizing mutations. The edge strand and variants cross-seed with each other and with members of the central variant family. Interesting fibrillar morphology was observed in some cross-seeding cases and its implications for a better understanding of nucleation and elongation events are discussed.