Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.

Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin.

The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-ß (Aß)38 generation by PS2 is accompanied by a reciprocal increase in Aß37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aß38 and Aß37 generation appear to mainly result from altered subsequent stepwise cleavage of Aß peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aß peptides.

Membrane lipid remodeling modulates γ-secretase processivity.

Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.

γ-Secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics.

Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by γ-secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2-TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial γ-secretase cleavage event. These data suggest an unprecedented cleavage mechanism by γ-secretase where flexible TMH regions act as key determinants of substrate cleavage specificity.

Substrate recruitment by γ-secretase.

γ-Secretase is a membrane-embedded protease complex that is crucial for many physiological processes throughout life. Due to its pivotal role in the etiology of Alzheimer's disease (AD), in particular the familial forms of the disease, the enzyme is one of the most studied intramembrane proteases and an important drug target. By cleaving a C-terminal fragment of the ß-amyloid precursor protein (APP), γ-secretase generates several amyloid ß-peptide (Aß) species including longer, neurotoxic forms such as Aß42 that are a widely believed to trigger AD. Besides APP, γ-secretase cleaves numerous other substrates including most prominently Notch1, whose cleavage by γ-secretase is essential for cell differentiation and affected in certain types of cancer. In this review, we will describe the exciting progress made in our understanding of how the γ-secretase complex recognizes and recruits its substrates to its catalytic subunit presenilin for their intramembrane proteolytic cleavage. This complicated process is not well understood and only recently insights from biochemical studies and structural biology are beginning to reveal this secret of γ-secretase.

Aß43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation.

Abnormal generation of neurotoxic amyloid-ß peptide (Aß) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aß43 is still lacking, and it is unclear whether γ-secretase modulators (GSMs) can reduce the levels of this Aß species. By comparing several types of Aß43-generating FAD mutants, we observe that very high levels of Aß43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aß, are found for all mutants and are independent of their particular effect on Aß production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aß43 production of all mutants. Finally, substrate-binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aß43-generating FAD mutations could in principle be treated by GSMs.

The yeast ER-intramembrane protease Ypf1 refines nutrient sensing by regulating transporter abundance.

Proteolysis by aspartyl intramembrane proteases such as presenilin and signal peptide peptidase (SPP) underlies many cellular processes in health and disease. Saccharomyces cerevisiae encodes a homolog that we named yeast presenilin fold 1 (Ypf1), which we verify to be an SPP-type protease that localizes to the endoplasmic reticulum (ER). Our work shows that Ypf1 functionally interacts with the ER-associated degradation (ERAD) factors Dfm1 and Doa10 to regulate the abundance of nutrient transporters by degradation. We demonstrate how this noncanonical branch of the ERAD pathway, which we termed "ERAD regulatory" (ERAD-R), responds to ligand-mediated sensing as a trigger. More generally, we show that Ypf1-mediated posttranslational regulation of plasma membrane transporters is indispensible for early sensing and adaptation to nutrient depletion. The combination of systematic analysis alongside mechanistic details uncovers a broad role of intramembrane proteolysis in regulating secretome dynamics.

Modulation of γ-Secretase Activity by a Carborane-Based Flurbiprofen Analogue.

All over the world, societies are facing rapidly aging populations combined with a growing number of patients suffering from Alzheimer's disease (AD). One focus in pharmaceutical research to address this issue is on the reduction of the longer amyloid-ß (Aß) fragments in the brain by modulation of γ-secretase, a membrane-bound protease. R-Flurbiprofen (tarenflurbil) was studied in this regard but failed to show significant improvement in AD patients in a phase 3 clinical trial. This was mainly attributed to its low ability to cross the blood-brain barrier (BBB). Here, we present the synthesis and in vitro evaluation of a racemic meta-carborane analogue of flurbiprofen. By introducing the carborane moiety, the hydrophobicity could be shifted into a more favourable range for the penetration of the blood-brain barrier, evident by a logD7.4 value of 2.0. Furthermore, our analogue retained γ-secretase modulator activity in comparison to racemic flurbiprofen in a cell-based assay. These findings demonstrate the potential of carboranes as phenyl mimetics also in AD research.

Substrate recruitment of γ-secretase and mechanism of clinical presenilin mutations revealed by photoaffinity mapping.

Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's disease-associated γ-secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to γ-secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N-terminal fragment of the catalytic subunit presenilin was determined as principal substrate-binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN-2 was identified besides nicastrin as additional substrate-binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.

Clinical, pathophysiological and genetic features of motor symptoms in autosomal dominant Alzheimer's disease.

Owing to an early and marked deposition of amyloid-ß in the basal ganglia, autosomal dominant Alzheimer's disease could distinctly involve motor symptoms. Therefore, we aimed to assess the prevalence and characteristics of motor signs in autosomal dominant Alzheimer's disease. Baseline Unified Parkinson Disease Rating Scale part three scores (UPDRS-III) from 433 participants of the Dominantly Inherited Alzheimer's Network observational study were analysed. Motor symptoms were scrutinized with respect to associations with mutation carrier status, mutation site within PSEN1, basal ganglia amyloid-ß as measured by Pittsburgh compound B PET, estimated years to symptom onset and Clinical Dementia Rating Scale-Sum of Boxes. Motor findings in mutation carriers were compared to patients with sporadic Alzheimer's disease using data of the National Alzheimer's Coordination Center. Mutation carriers showed motor findings at a higher frequency (28.4% versus 12.8%; P < 0.001) and severity (mean UPDRS-III scores 2.0 versus 0.4; P < 0.001) compared to non-carriers. Eleven of the 27 UPDRS-III items were statistically more frequently affected in mutation carriers after adjustment for multiple comparisons. Ten of these 11 items were subscale components of bradykinesia. In cognitively asymptomatic mutation carriers, dysdiadochokinesia was more frequent compared to non-carriers (right hand: 3.8% versus 0%; adjusted P = 0.023; left: 4.4% versus 0.6%; adjusted P = 0.031). In this cohort, the positive predictive value for mutation carrier status in cognitively asymptomatic participants (50% a priori risk) of dysdiadochokinesia was 100% for the right and 87.5% for the left side. Mutation carriers with motor findings more frequently were basal ganglia amyloid-ß positive (84% versus 63.3%; P = 0.006) and showed more basal ganglia amyloid-ß deposition (Pittsburgh compound B-standardized uptake value ratio 2.472 versus 1.928; P = 0.002) than those without. Frequency and severity of motor findings were greater in post-codon 200 PSEN1 mutations (36%; mean UPDRS-III score 3.03) compared to mutations pre-codon 200 PSEN1 (19.3%, P = 0.022; 0.91, P = 0.013). In mutation carriers, motor symptom severity was significantly positively correlated with basal ganglia amyloid-ß deposition, Clinical Dementia Rating scores and estimated years to symptom onset. Mutation carriers with a Clinical Dementia Rating global score of 2 exhibited more pronounced motor symptoms than sporadic Alzheimer's disease patients with the same Clinical Dementia Rating global score (mean UPDRS-III scores 20.71 versus 5.96; P < 0.001). With a prevalence of approximately 30% and increasing severity with progression of dementia, motor symptoms are proven as a clinically relevant finding in autosomal dominant Alzheimer's disease, in particular in advanced dementia stages, that correlates with deposition of amyloid-ß in the basal ganglia. In a very small per cent of cognitively asymptomatic members of families with autosomal dominant Alzheimer's disease, dysdiadochokinesia may increase the chance of an individual's status as mutation carrier.

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage.

Intramembrane cleavage of the ß-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. Both mutants impaired γ-secretase cleavage and also altered its cleavage specificity. Circular dichroism, NMR, and backbone amide hydrogen/deuterium exchange measurements as well as molecular dynamics simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the substrate TMD. Although helix destabilization and/or unfolding was not observed at the initial ε-cleavage sites of C99, subtle changes in hinge flexibility were identified that substantially affected helix bending and twisting motions in the entire TMD. These resulted in altered orientation of the distal cleavage domain relative to the N-terminal TMD part. Our data suggest that both enhancing and reducing local helix flexibility of the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can thus be conducive for productive substrate-enzyme interactions.

A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor.

Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.

Noninvasive 3D Field Mapping of Complex Static Electric Fields.

Many upcoming experiments in antimatter research require low-energy antiproton beams. With a kinetic energy in the order of 100 keV, the standard magnetic components to control and focus the beams become less effective. Therefore, electrostatic components are being developed and installed in transfer lines and storage rings. However, there is no equipment available to precisely map and check the electric field generated by these elements. Instead, one has to trust in simulations and, therefore, depend on tight fabrication tolerances. Here we present, for the first time, a noninvasive way to experimentally probe the electrostatic field in a 3D volume with a microsensor. Using the example of an electrostatic quadrupole focusing component, we find excellent agreement between a simulated and real field. Furthermore, it is shown that the spatial resolution of the probe is limited by the electric field curvature which is almost zero for the quadrupole. With a sensor resolution of 61 V/m/sqrt[Hz], the field deviation due to a noncompliance with the tolerances can be resolved. We anticipate that this compact and practical field strength probe will be relevant also for other scientific and technological disciplines such as atmospheric electricity or safeguarding near power infrastructure.

A Thermal Flow Sensor Based on Printed Circuit Technology in Constant Temperature Mode for Various Fluids.

We present a thermal flow sensor designed for measuring air as well as water flow velocities in heating, ventilation, and air conditioning (HVAC) systems. The sensor is designed to integrate the flow along the entire diameter of the pipe also quantifying the volume flow rate of the streaming fluid where the calorimetric principle in constant temperature operation is utilized as a readout method. In the constant temperature mode, a controller keeps a specific excess temperature between sensing elements at a constant level resulting in a flow dependent heater voltage. To achieve cost-effective sensors, the fabrication of the transducer is fully based on printed circuit board technology allowing low-cost mass production with different form factors. In addition, 2D-FEM simulations were carried out in order to predict the sensor characteristic of envisaged setups. The simulation enables a fast and easy way to evaluate the sensor's behaviour in different fluids. The results of the FEM simulations are compared to measurements in real environments, proving the credibility of the model.

Dual Resonator MEMS Magnetic Field Gradiometer.

Accurate knowledge of the spatial magnetic field distribution is necessary when measuring field gradients. Therefore, a MEMS magnetic field gradiometer is reported, consisting of two identical, but independent laterally oscillating masses on a single chip. The sensor is actuated by Lorentz force and read out by modulation of the light flux passing through stationary and moving arrays of the chip. This optical readout decouples the transducer from the electronic components. Both phase and intensity are recorded which reveals information about the uniformity of the magnetic field. The magnetic flux density is measured simultaneously at two points in space and the field gradient is evaluated locally. The sensor was characterised at ambient pressure by performing frequency and magnitude response measurements with coil and various different permanent magnet arrangements, resulting in a responsivity of 35.67 V/T and detection limit of 3.07 µT/ Hz (@ 83 Hz ENBW). The sensor is compact, offers a large dynamic measurement range and can be of low-cost by using conventional MEMS batch fabrication technology.

Comparison of Strategies for the Determination of Sterol Sulfates via GC-MS Leading to a Novel Deconjugation-Derivatization Protocol.

Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages and disadvantages of trimethylsilyl (TMS), methyloxime-trimethylsilyl (MO-TMS), and trifluoroacetyl (TFA) derivatives were examined. Different published procedures of sterol sulfate deconjugation, including enzymatic and chemical cleavage, were reinvestigated and examined for diverse sterol sulfates. Finally, we present a new protocol for the chemical cleavage of sterol sulfates, allowing for simultaneous deconjugation and derivatization, simplifying GC-MS based sterol sulfate analysis.

Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Proteolytic Processing of Neuregulin 1 Type III by Three Intramembrane-cleaving Proteases.

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of ß-secretase (ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III ß-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.

Substrate processing in intramembrane proteolysis by γ-secretase - the role of protein dynamics.

Intramembrane proteases comprise a number of different membrane proteins with different types of catalytic sites. Their common denominator is cleavage within the plane of the membrane, which usually results in peptide bond scission within the transmembrane helices of their substrates. Despite recent progress in the determination of high-resolution structures, as illustrated here for the γ-secretase complex and its substrate C99, it is still unknown how these enzymes function and how they distinguish between substrates and non-substrates. In principle, substrate/non-substrate discrimination could occur at the level of substrate binding and/or cleavage. Focusing on the γ-secretase/C99 pair, we will discuss recent observations suggesting that global motions within a substrate transmembrane helix may be much more important for defining a substrate than local unraveling at cleavage sites.