Cell-free protein production of a gamma secretase homolog.

Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

High throughput and targeted screens for prepilin peptidase inhibitors do not identify common inhibitors of eukaryotic gamma-secretase.

INTRODUCTION: Prepilin peptidases (PPP) are essential enzymes for the biogenesis of important virulence factors, such as type IV pili (T4P), type II secretion systems, and other T4P-related systems of bacteria and archaea. PPP inhibitors could be valuable pharmaceuticals, but only a few have been reported. Interestingly, PPP share similarities with presenilin enzymes from the gamma-secretase protease complex, which are linked to Alzheimer's disease. Numerous gamma-secretase inhibitors have been reported, and some have entered clinical trials, but none has been tested against PPP. OBJECTIVE: The objective of this study is to develop a high-throughput screening (HTS) method to search for inhibitors of PPP from various chemical libraries and reported gamma-secretase inhibitors. METHOD: More than 15,000 diverse compounds, including 13 reported gamma-secretase inhibitors and other reported peptidase inhibitors, were screened to identify potential PPP inhibitors. RESULTS: The authors developed a novel screening method and screened 15,869 compounds. However, the screening did not identify a PPP inhibitor. Nevertheless, the study suggests that gamma-secretase is sufficiently different from PPP that specific inhibitors may exist in a larger chemical space. CONCLUSION: The authors believe that the HTS method that they describe has numerous advantages and encourage others to consider its application in the search for PPP inhibitors.

Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro.

Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-ß plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.

Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis.

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here, we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

Cutting proteins into pieces is a crucial process in the cell, allowing several important processes to take place, including cell differentiation (which allows cells to develop into specific types), cell death, protein quality control, or even where in the cell a protein will end up. However, the specialized proteins that carry out this task, known as proteases, can also be involved in the development of disease. For example, in the brain, a protease called γ-secretase cuts up the amyloid-ß protein precursor, producing toxic forms of amyloid-ß peptides that are widely believed to cause Alzheimer's disease. Proteases like γ-secretase carry out their role in the membrane, the layer of fats (also known as lipids) that forms the outer boundary of the cell. The environment in this area of the cell can influence the activity of proteases, but it is poorly understood how this happens. One way to address this question would be to compare the activity of γ-secretase in the lipid environment of the membrane to its activity when it is entirely surrounded by different molecules, such as detergent molecules. Unfortunately, γ-secretase is not active when it is removed from its lipid environment by a detergent, making it difficult to perform this comparison. To overcome this issue, Feilen et al. chose to study PSH, a protease similar to γ-secretase that produces the same amyloid-ß peptides but remains active in detergent. When Feilen et al. mixed PSH with lipid molecules like those found in the membrane and amyloid-ß precursor protein, PSH produced amyloid-ß peptides including those that are thought to cause Alzheimer's. However, when a detergent was substituted for the lipid molecules this led to longer amyloid-ß peptides than usual, indicating that PSH was not able to cut proteins as effectively. The change in environment appeared to reduce PSH's ability to progressively trim small segments from the peptides. Computer modelling of the protease's structure in lipids versus detergent supported the experimental findings: the model predicted that the areas of PSH important for recognizing and cutting other proteins would be more stable in the membrane compared to the detergent. These results indicate that the cell membrane plays a vital role in the stability of the active regions of proteases that are cleaving in this environment. In the future, this could help to better understand how changes to the lipid molecules in the membrane may contribute to the activity of γ-secretase and its role in Alzheimer's disease.

Preparation of a Deuterated Membrane Protein for Small-Angle Neutron Scattering.

This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370-377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.

Archaeal roots of intramembrane aspartyl protease siblings signal peptide peptidase and presenilin.

Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD…GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.

A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins.

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased koff rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.

Complex Stability is Encoded in Binding Patch Softness: a Monomer-Based Approach to Predict Inter-Subunit Affinity of Protein Dimers.

Knowledge about the structure and stability of protein-protein interactions is vital to decipher the behavior of protein systems. Prediction of protein complexes' stability is an interesting topic in the field of structural biology. There are some promising published computational approaches that predict the affinity between subunits of protein dimers using 3D structures of both subunits. In the current study, we classify protein complexes with experimentally measured affinities into distinct classes with different mean affinities. By predicting the mechanical stiffness of the protein binding patch (PBP) region on a single subunit, we successfully predict the assigned affinity class of the PBP in the classification step. Now to predict the experimentally measured affinity between protein monomers in solution, we just need the 3D structure of the suggested PBP on one subunit of the proposed dimer. We designed the SEPAS software and have made the software freely available for academic non-commercial research purposes at " http://biophysics.ir/affinity ". SEPAS predicts the stability of the intended dimer in a classwise manner by utilizing the computed mechanical stiffness of the introduced binding site on one subunit with the minimum accuracy of 0.72.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aß42 peptide (Ala-Thr) and the other to the non-pathogenic Aß48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Influence of solubilization and AD-mutations on stability and structure of human presenilins.

Presenilin (PS1 or PS2) functions as the catalytic subunit of γ-secretase, which produces the toxic amyloid beta peptides in Alzheimer's disease (AD). The dependence of folding and structural stability of PSs on the lipophilic environment and mutation were investigated by far UV CD spectroscopy. The secondary structure content and stability of PS2 depended on the lipophilic environment. PS2 undergoes a temperature-dependent structural transition from α-helical to ß-structure at 331 K. The restructured protein formed structures which tested positive in spectroscopic amyloid fibrils assays. The AD mutant PS1L266F, PS1L424V and PS1ΔE9 displayed reduced stability which supports a proposed 'loss of function' mechanism of AD based on protein instability. The exon 9 coded sequence in the inhibitory loop of the zymogen was found to be required for the modulation of the thermal stability of PS1 by the lipophilic environment.

Probing the Structure and Function Relationships of Presenilin by Substituted-Cysteine Accessibility Method.

Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position. In combination with small molecule inhibitors/modulators and cross-linking experiments, we were able to identify certain residues and regions of presenilin that contribute to its intramembrane-cleaving activity. In addition, we revealed the structural dynamics of the transmembrane domains of presenilin during the formation of the complex and its proteolytic process. The SCAM provides new insights into the relationship between the structure and activity of presenilin, and is useful for probing the protein dynamics of the membrane-embedded enzymes.

Screening and Characterization Strategies for Nanobodies Targeting Membrane Proteins.

The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.

Presenilin adopts the ClC channel fold.

Presenilin is an integral membrane aspartate protease that regulates cellular processes by cleaving proteins within the cell membrane. The recent crystal structure of presenilin reveals a conspicuous pore in a bundle of nine α-helices, which was originally thought to adopt a novel protein fold. However, here I show that the presenilin fold is a variant of the ClC chloride channel/transporter fold. This observation may have important implications for presenilin's postulated biological role as a calcium leak channel.

Sampling the conformational space of the catalytic subunit of human γ-secretase.

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins.

Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca(2+) release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aß) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca(2+) signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca(2+) signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca(2+) and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca(2+) concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca(2+) and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca(2+) signals, which can account for the more frequent global Ca(2+) signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca(2+) signals in cells expressing FAD-causing mutant PS.

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH.

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer's disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aß42, Aß40, and Aß38. The molar ratio of the cleavage products Aß42 over Aß40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aß42/Aß40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase.

Presenilin-like GxGD membrane proteases have dual roles as proteolytic enzymes and ion channels.

The GxGD proteases function to cleave protein substrates within the membrane. As these proteases contain multiple transmembrane domains typical of ion channels, we examined if GxGD proteases also function as ion channels. We tested the putative dual function by examining two archeobacterial GxGD proteases (PSH and FlaK), with known three-dimensional structures. Both are in the same GxGD family as presenilin, a protein mutated in Alzheimer Disease. Here, we demonstrate that PSH and FlaK form cation channels in lipid bilayers. A mutation that affected the enzymatic activity of FlaK rendered the channel catalytically inactive and altered the ion selectivity, indicating that the ion channel and the catalytic activities are linked. We report that the GxGD proteases, PSH and FlaK, are true "chanzymes" with interdependent ion channel and protease activity conferred by a single structural domain embedded in the membrane, supporting the proposal that higher-order proteases, including presenilin, have channel function.

Presenilins regulate the cellular activity of ryanodine receptors differentially through isotype-specific N-terminal cysteines.

Presenilins (PS), endoplasmic reticulum (ER) transmembrane proteins, form the catalytic core of γ-secretase, an amyloid precursor protein processing enzyme. Mutations in PS lead to Alzheimer's disease (AD) by altering γ-secretase activity to generate pathologic amyloid beta and amyloid plaques in the brain. Here, we identified a novel mechanism where binding of a soluble, cytosolic N-terminal domain fragment (NTF) of PS to intracellular Ca(2+) release channels, ryanodine receptors (RyR), controls Ca(2+) release from the ER. While PS1NTF decreased total RyR-mediated Ca(2+) release, PS2NTF had no effect at physiological Ca(2+) concentrations. This differential function and isotype-specificity is due to four cysteines absent in PS1NTF, present, however, in PS2NTF. Site-directed mutagenesis targeting these cysteines converted PS1NTF to PS2NTF function and vice versa, indicating differential RyR binding. This novel mechanism of intracellular Ca(2+) regulation through the PS-RyR interaction represents a novel target for AD drug development and the treatment of other neurodegenerative disorders that critically depend on RyR and PS signaling.

Presenilins and calcium signaling-systems biology to the rescue.

Mutations in presenilins result in familial Alzheimer's disease (FAD). Presenilins encode a catalytic subunit of the γ-secretase complex, and FAD mutations in presenilins alter γ-secretase activity. Many FAD mutations in presenilins also affect intracellular calcium signaling. To explain these results, it was proposed that presenilins also function as endoplasmic reticulum (ER) calcium leak channels and that this function is disrupted by FAD mutations. Although this hypothesis has been controversial, new research supports the calcium leak channel hypothesis. One group reported the presence of putative ion-conduction pore in the high-resolution crystal structure of bacterial presenilin homolog PSH1. Another group identified an essential role for presenilins in mediating ER calcium leak in a cell-based screen for calcium homeostasis modulators. These results should enable the field to move forward and to focus on exploring connections between FAD mutations in presenilins, changes in γ-secretase and ER Ca(2+) leak functions, and development of the disease.

Structural biology of presenilins and signal peptide peptidases.

Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of γ-secretase, a protease responsible for the generation of amyloid-ß peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.