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.

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.

Cleavage of mitochondrial homeostasis regulator PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state.

The intramembrane protease PARL acts as a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. Depending on the stress level, PGAM5 can either stimulate cell survival or cell death. In contrast to PINK1, which is constantly cleaved in healthy mitochondria and only active when the inner mitochondrial membrane is depolarized, PGAM5 processing is inversely regulated. However, determinants of PGAM5 that indicate it as a conditional substrate for PARL have not been rigorously investigated, and it is unclear how uncoupling the mitochondrial membrane potential affects its processing compared to that of PINK1. Here, we show that several polar transmembrane residues in PGAM5 distant from the cleavage site serve as determinants for its PARL-catalyzed cleavage. Our NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal transmembrane helix harboring the scissile peptide bond are key for a productive interaction with PARL. Furthermore, we also show that PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers, which are then cleaved by PARL. In conclusion, we propose a model in which PGAM5 is slowly processed by PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features, including a membrane potential-dependent oligomeric switch.

Lipase-Catalyzed Production of Sorbitol Laurate in a "2-in-1" Deep Eutectic System: Factors Affecting the Synthesis and Scalability.

Surfactants, such as glycolipids, are specialty compounds that can be encountered daily in cleaning agents, pharmaceuticals or even in food. Due to their wide range of applications and, more notably, their presence in hygiene products, the demand is continuously increasing worldwide. The established chemical synthesis of glycolipids presents several disadvantages, such as lack of specificity and selectivity. Moreover, the solubility of polyols, such as sugars or sugar alcohols, in organic solvents is rather low. The enzymatic synthesis of these compounds is, however, possible in nearly water-free media using inexpensive and renewable building blocks. Using lipases, ester formation can be achieved under mild conditions. We propose, herein, a "2-in-1" system that overcomes solubility problems, as a Deep Eutectic System (DES) made of sorbitol and choline chloride replaces either a purely organic or aqueous medium. For the first time, 16 commercially available lipase formulations were compared, and the factors affecting the conversion were investigated to optimize this process, owing to a newly developed High-Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) method for quantification. Thus, using 50 g/L of lipase formulation Novozym 435® at 50 °C, the optimized synthesis of sorbitol laurate (SL) allowed to achieve 28% molar conversion of 0.5 M of vinyl laurate to its sugar alcohol monoester when the DES contained 5 wt.% water. After 48h, the de novo synthesized glycolipid was separated from the media by liquid-liquid extraction, purified by flash-chromatography and characterized thoroughly by one- and two-dimensional Nuclear Magnetic Resonance (NMR) experiments combined to Mass Spectrometry (MS). In completion, we provide initial proof of scalability for this process. Using a 2.5 L stirred tank reactor (STR) allowed a batch production reaching 25 g/L in a highly viscous two-phase system.

Macrocyclic Tetramers-Structural Investigation of Peptide-Peptoid Hybrids.

Outstanding affinity and specificity are the main characteristics of peptides, rendering them interesting compounds for basic and medicinal research. However, their biological applicability is limited due to fast proteolytic degradation. The use of mimetic peptoids overcomes this disadvantage, though they lack stereochemical information at the α-carbon. Hybrids composed of amino acids and peptoid monomers combine the unique properties of both parent classes. Rigidification of the backbone increases the affinity towards various targets. However, only little is known about the spatial structure of such constrained hybrids. The determination of the three-dimensional structure is a key step for the identification of new targets as well as the rational design of bioactive compounds. Herein, we report the synthesis and the structural elucidation of novel tetrameric macrocycles. Measurements were taken in solid and solution states with the help of X-ray scattering and NMR spectroscopy. The investigations made will help to find diverse applications for this new, promising compound class.

The simulation of NMR data of flexible molecules: sagittamide A as an example for MD simulations with orientational constraints.

The recently developed MDOC (Molecular Dynamics with Orientational Constraints) simulation is applied for the first time to a fully flexible molecule. MDOC simulations aim to single out the naturally existing configuration of molecules and to elucidate conformer populations. The performance of the method was first demonstrated on a well-studied test case, the five-membered ring lactone (α-methylene-γ-butyrolactone). In the case of sagittamide A, one-bond 1H-13C residual dipolar couplings (RDC) are used as orientational constraints that reorient the molecule or parts of it. In addition, NOE distances and 3J scalar couplings are used as constraints. Five possible configurations of sagittamide A (labelled a to e) are considered. One experimental RDC value per flexible unit was available and this was not sufficient to single out one valid configuration. The problem could be solved by including NOE distances as well as 3J couplings as complementary constraints into the MDOC simulations. In accordance with former investigations, we confirmed the configuration a for the natural product. A detailed analysis of conformers of the central chain of 6 chiral carbon atoms could be given by inspecting the MDOC trajectory. The relative abundance of these conformers is crucial in fulfilling all three sets of constraints.

Urinary NMR Profiling in Pediatric Acute Kidney Injury-A Pilot Study.

Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations. In the study reported here, we examined the diagnostic accuracy of NMR urine metabolite patterns for the diagnosis of neonatal and pediatric AKI according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition. A cohort of 65 neonatal and pediatric patients (0-18 years) with established AKI of heterogeneous etiology was compared to both a group of apparently healthy children (n = 53) and a group of critically ill children without AKI (n = 31). Multivariate analysis identified a panel of four metabolites that allowed diagnosis of AKI with an area under the receiver operating characteristics curve (AUC-ROC) of 0.95 (95% confidence interval 0.86-1.00). Especially urinary citrate levels were significantly reduced whereas leucine and valine levels were elevated. Metabolomic differentiation of AKI causes appeared promising but these results need to be validated in larger studies. In conclusion, this study shows that NMR spectroscopy yields high diagnostic accuracy for AKI in pediatric patients.

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.

Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion.

Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

Autoinduced catalysis and inverse equilibrium isotope effect in the frustrated Lewis pair catalyzed hydrogenation of imines.

The frustrated Lewis pair (FLP)-catalyzed hydrogenation and deuteration of N-benzylidene-tert-butylamine (2) was kinetically investigated by using the three boranes B(C6F5)3 (1), B(2,4,6-F3-C6H2)3 (4), and B(2,6-F2-C6H3)3 (5) and the free activation energies for the H2 activation by FLP were determined. Reactions catalyzed by the weaker Lewis acids 4 and 5 displayed autoinductive catalysis arising from a higher free activation energy (2 kcal mol(-1)) for the H2 activation by the imine compared to the amine. Surprisingly, the imine reduction using D2 proceeded with higher rates. This phenomenon is unprecedented for FLP and resulted from a primary inverse equilibrium isotope effect.

Dendrimer-type peptoid-decorated hexaphenylxylenes and tetraphenylmethanes: synthesis and structure in solution and in the gas phase.

Branched organic nanostructures are useful scaffolds that find multiple applications in a variety of fields. Here, we present a novel approach to dendrimer-like structures. Our design contains a rigid hydrocarbon-based core (hexaphenylxylylene/tetraethynylphenylmethane) combined with a library of N-substituted oligoglycines (so-called peptoids) providing a flexible shell. The use of click chemistry allows rapid assembly of the nanostructures. The possibility of tuning the size and the solubility of this new type of nanostructure will be advantageous for future applications such as heterogeneous catalysis.

Trehalose lipid biosurfactants produced by the actinomycetes Tsukamurella spumae and T. pseudospumae.

Actinomycetales are known to produce various secondary metabolites including products with surface-active and emulsifying properties known as biosurfactants. In this study, the nonpathogenic actinomycetes Tsukamurella spumae and Tsukamurella pseudospumae are described as producers of extracellular trehalose lipid biosurfactants when grown on sunflower oil or its main component glyceryltrioleate. Crude extracts of the trehalose lipids were purified using silica gel chromatography. The structure of the two trehalose lipid components (TL A and TL B) was elucidated using a combination of matrix-assisted laser desorption/ionization time-of-flight/time-of-flight/tandem mass spectroscopy (MALDI-ToF-ToF/MS/MS) and multidimensional NMR experiments. The biosurfactants were identified as 1-α-glucopyranosyl-1-α-glucopyranosid carrying two acyl chains varying of C4 to C6 and C16 to C18 at the 2' and 3' carbon atom of one sugar unit. The trehalose lipids produced demonstrate surface-active behavior and emulsifying capacity. Classified as risk group 1 organisms, T. spumae and T. pseudospumae hold potential for the production of environmentally friendly surfactants.

Substrate Selection Criteria in Regulated Intramembrane Proteolysis.

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aß peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) ß transmembrane segment.

The platelet-derived growth factor receptor ß is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor ß transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.

Structure analysis of the membrane-bound PhoD signal peptide of the Tat translocase shows an N-terminal amphiphilic helix.

Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution ¹³C/¹5N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.

Structural characterization of a peptoid with lysine-like side chains and biological activity using NMR and computational methods.

N-substituted glycine oligomers or peptoids with charged side chains are a novel class of cell penetrating peptide mimetics and have been shown to serve as drug delivery agents. Here, we investigated by NMR spectroscopy and quantum chemical calculations whether a Rhodamine B labelled peptoid [RhoB(Spiro)-Ahx]-[But](6A)NH(2) with lysine-like side chains adopts structural motifs similar to regular peptides. Due to a low chemical shift dispersion, high resolution structure determination with conventional NMR-derived distance restraints and J-couplings was not possible. Instead, a combined assignment and structure refinement strategy using the QM/MM force field COSMOS-NMR was developed to interpret the highly ambiguous chemical shift and distance constraints and obtain a medium resolution three-dimensional structural model. This allowed us to select for the all cis-amide conformation of the peptide with a pseudo-helical arrangement of extended side chains as a faithful representative structure of [RhoB(Spiro)-Ahx]-[But](6A)NH(2). We tested the biological activity of the peptoid by live-cell imaging, which showed that the cellular uptake of the peptoid was comparable to conventional cell-penetrating peptides.

Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase.

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite ß-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.

Rapid calculation of protein chemical shifts using bond polarization theory and its application to protein structure refinement.

Although difficult to analyze, NMR chemical shifts provide detailed information on protein structure. We have adapted the semi-empirical bond polarization theory (BPT) to protein chemical shift calculation and chemical shift driven protein structure refinement. A new parameterization for BPT amide nitrogen chemical shift calculation has been derived from MP2 ab initio calculations and successfully evaluated using crystalline tripeptides. We computed the chemical shifts of the small globular protein ubiquitin, demonstrating that BPT calculations can match the results obtained at the DFT level of theory at very low computational cost. In addition to the calculation of chemical shift tensors, BPT allows the calculation of chemical shift gradients and consequently chemical shift driven geometry optimizations. We applied chemical shift driven protein structure refinement to the conformational analysis of a set of Trypanosoma brucei (the causative agent of African sleeping sickness) tryparedoxin peroxidase Px III structures. We found that the interaction of Px III with its reaction partner Tpx seems to be governed by conformational selection rather than by induced fit.