Andrew C. Nyborg

Androgens modulate β-amyloid levels in male rat brain

As a normal consequence of aging, men experience a significant decline in androgen levels. Although the neural consequences of age‐related androgen depletion remain unclear, recent evidence suggests a link between low androgen levels and the development of Alzheimers disease (AD). Here, we test the hypothesis that androgens act as endogenous modulators of β‐amyloid protein (Aβ) levels. To investigate this possibility, brain and plasma levels of Aβ were measured in male rats with varying hormonal conditions. Depletion of endogenous sex steroid hormones via gonadectomy (GDX) resulted in increased brain levels of Aβ in comparison to gonadally intact male rats. This GDX‐induced increase in Aβ levels was reversed by DHT supplementation, demonstrating a functional role for androgens in modulating brain levels of Aβ. These findings suggest that age‐related androgen depletion may result in accumulation of Aβ in the male brain and thereby act as a risk factor for the development of AD.

Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel γ-secretase substrates

BackgroundThe mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimers disease brains, interact with ApoE, and modulate Aβ production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75NTR and proNGF. We have investigated and provide evidence for γ-secretase cleavage of this family of proteins.ResultsWe provide evidence that these receptors are substrates for presenilin dependent γ-secretase cleavage. γ-Secretase cleavage of these sorting receptors is inhibited by γ-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most γ-secretase substrates, we find that ectodomain shedding precedes γ-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an α-secretase like cleavage.ConclusionThese data indicate that the α- and γ-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known γ-secretase substrates, and could possibly regulate the biological functions of these proteins.

C‐terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimers disease‐associated β‐amyloid peptide is produced through cleavage of amyloid precursor protein by β‐secretase and γ‐secretase. γ‐Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH‐1) and presenilin enhancer‐2 (PEN‐2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane‐embedded aspartic acid residues for catalysis. Apart from the two aspartate‐containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C‐terminus. Although it has been well documented that this motif is essential for γ‐secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of γ‐secretase resulting in a complete loss of PS binding to a γ‐secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and γ‐secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of γ‐secretase and of SPP.

A signal peptide peptidase (SPP) reporter activity assay based on the cleavage of type II membrane protein substrates provides further evidence for an inverted orientation of the SPP active site relative to presenilin.

Signal peptide peptidase (SPP) is an intramembrane-cleaving protease identified by its cleavage of several type II membrane signal peptides after signal peptidase cleavage. Here we describe a novel, quantitative, cell-based SPP reporter assay. This assay utilizes a substrate consisting of the NH2 terminus of the ATF6 transcription factor fused to a transmembrane domain susceptible to SPP cleavage in vitro. In cells, cleavage of the substrate releases ATF6 from the membrane. This cleavage can be monitored by detection of an epitope that is unmasked in the cleaved substrate or by luciferase activity induced by the cleaved ATF6 substrate binding to and activating an ATF6 luciferase reporter construct. Using this assay we show that (i) SPP is the first aspartyl intramembrane-cleaving protease whose activity increases proportionally to its overexpression and (ii) selectivity of various SPP and γ-secretase inhibitors can be rapidly evaluated. Because this assay was designed based on data suggesting that SPP has an orientation distinct from presenilin and cleaves type II membrane proteins, we determined whether the segment of SPP located between the two presumptive catalytic aspartates was in the lumen or cytoplasm. Using site-directed mutagenesis to insert an N-linked glycosylation site we show that a portion of this region is present in the lumen. These data provide strong evidence that although the SPP and presenilin active sites have some similarities, their presumptive catalytic domains are inverted. This assay should prove useful for additional functional studies of SPP as well as evaluation of SPP and γ-secretase inhibitors.

Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase

Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I‐CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.—Nyborg, A. C., Ladd, T. B., Jansen, K., Kukar, T., Golde, T. E. Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase. FASEB J. 20, 1671–1679 (2006)

Overexpression of Nicastrin increases Aβ production

γ‐Secretase cleavage is the final proteolytic step that releases the amyloid β‐peptide (Aβ) from the amyloid β‐protein precursor (APP). Significant evidence indicates that the presenilins (PS) are catalytic components of a high molecular weight γ‐secretase complex. The glycoprotein nicastrin was recently identified as a functional unit of this complex based on 1) binding to PS and 2) the ability to modulate Aβ production following mutation of a conserved DYIGS region. In contrast to the initial report, we find that overexpression of wild‐type (WT) nicastrin increases Aβ production, whereas DYIGS mutations (MT) have little or no effect. The increase in Aβ production is associated with an increase in γ‐secretase activity but not with a detectable increase in PS1 levels. Subcellular fractionation studies show that WT but not MT nicastrin matures into buoyant membrane fractions enriched in γ‐secretase activity. These data support the hypothesis that nicastrin is an essential component of the γ‐secretase complex. The finding that WT nicastrin overexpression can increase γ‐secretase activity without altering levels of the presumed catalytic component (PS) of the enzyme may point to a role for nicastrin in facilitating cleavage by regulating substrate interactions with the γ‐secretase complex.

Signal peptide peptidase (SPP) dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM) in intact cells

BackgroundSignal peptide peptidase (SPP) is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs) make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo.ResultsFluorescence lifetime imaging microscopy (FLIM) can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET) pairs; cyan fluorescent protein (CFP) with yellow fluorescent protein (YFP) or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs.ConclusionOur FLIM data strongly suggest dimer formation between two separate SPP proteins. Although the tagged SPP constructs are expressed throughout the cell, SPP dimer detection by FLIM is seen predominantly at or near the plasma membrane.

In vivo and ex vivo imaging of amyloid-β cascade aggregates with a Pronucleon™ peptide.

Accumulation of amyloid-β (Aβ) cascade aggregates is considered a hallmark of Alzheimers disease (AD). Current dogma holds that the appearance of Aβ oligomers and larger aggregates occur many years prior to plaque formation associated with the advanced and irreparable neurocognitive decline characteristic of AD. This premise is the impetus to identify these Aβ precursor structures prior to advanced plaque development. The Pronucleon™ technology platform is comprised of a novel series of engineered peptides that provide a unique readout when associated with beta-rich fiber and oligomeric Aβ. This technology has been applied to Ex Vivo tissue sections and In Vivo mouse models of AD to determine the potential utility of these synthetic peptides as potential imaging agents. In Ex Vivo studies, the Pronucleon™ peptide binds plaque like structures in brain sections obtained from transgenic mice overexpressing hAPP with both the human Swedish and London Aβ mutations. In Vivo, Pronucleon™ peptide administered peripherally can localize to the brain and label plaques throughout the brain in transgenic mice. Taken together, the data suggest that Pronucleon™ could provide a new imaging tool for Aβ cascade elements that precede advanced plaque and fibril formation, thereby advancing early diagnosis and treatment opportunities.

SIGNAL PEPTIDE PEPTIDASES

Signal peptide peptidases (SPPs) are the most recently identified members of a protease family of integral membrane proteins that includes the intensively studied presenilin 1 (PS1) and presenilin (PS2) proteins. There are 5 human genes encoding SPPs which can be divided into two branches based on homology and initial functional studies. One branch, which is the focus of this chapter, consists of the SPP and SPPL3 proteins. The second branch will be the focus of a subsequent chapter, and consists of the three SPPL2 proteins (SPPL2a, b, and c). The SPP proteins are conserved through evolution with family members found in fungi, archaea and plants. Presenilins (PSs) and SPPs cleave substrate polypeptides within a transmembrane region, but differ in that PSs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. SPPs and PSs have low overall sequence homology, yet exhibit considerable structural similarity as well as strict conservation of several small motifs. They are both multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their C-termini. They differ in that the active site topology of SPPs is inverted relative to PSs. Moreover, SPP and SPPL3 appear to function as proteases without the need for additional cofactors. In contrast, PSs function as the UPgamma-secretase protease only when complexed with three accessory proteins. Although the biological roles of PSs are reasonably well understood, the biological roles of SPP are largely unknown, and only a few endogenous substrates for SPP have been identified. SPP and possibly SPPL3 appear to cleave a number of endogenous type 2 signal peptides and these genes are essential genes in the development of several model organisms. In addition, in many human parasites, there is only a single SPP gene that is most closely related to the human SPP. Thus, SPPs may be novel antiviral drug targets in humans and represent a novel drug target for major human pathogens such as malaria