Combinatorial assembly and design of enzymes.

The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and α,ß-Hydrolase Domain Containing Protein 6.

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

Carfilzomib resistance due to ABCB1/MDR1 overexpression is overcome by nelfinavir and lopinavir in multiple myeloma.

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients' bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

Identification of new quorum sensing autoinducer binding partners in Pseudomonas aeruginosa using photoaffinity probes.

Many bacterial species, including the human pathogen Pseudomonas aeruginosa, employ a mechanism of intercellular communication known as quorum sensing (QS), which is mediated by signalling molecules termed autoinducers. The Pseudomonas Quinolone Signal (PQS) and 2-Heptyl-3H-4-Quinolone (HHQ) are autoinducers in P. aeruginosa, and they are considered important factors in the progress of infections by this clinically relevant organism. Herein, we report the development of HHQ and PQS photoaffinity-based probes for chemical proteomic studies. Application of these probes led to the identification of previously unsuspected putative HHQ and PQS binders, thereby providing new insights into QS at a proteomic level and revealing potential new small molecule targets for virulence attenuation strategies. Notably, we found evidence that PQS binds RhlR, the cognate receptor in the Rhl QS sub-system of P. aeruginosa. This is the first indication of interaction between the Rhl and PQS systems at the protein/ligand level, which suggests that RhlR should be considered a highly attractive target for antivirulence strategies.

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling.

Electron microscopy (EM) is traditionally employed as a follow-up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on-section fluorescence labelling with on-section immunogold labelling. In addition to antibody labelling of LAMP-1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane-associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane-associated antigens.

The influence of acceptor nucleophilicity on the glycosylation reaction mechanism.

A set of model nucleophiles of gradually changing nucleophilicity is used to probe the glycosylation reaction mechanism. Glycosylations of ethanol-based acceptors, bearing varying amounts of fluorine atoms, report on the dependency of the stereochemistry in condensation reactions on the nucleophilicity of the acceptor. Three different glycosylation systems were scrutinized, that differ in the reaction mechanism, that - putatively - prevails during the coupling reaction. It is revealed that the stereoselectivity in glycosylations of benzylidene protected glucose donors are very susceptible to acceptor nucleophilicity whereas condensations of benzylidene mannose and mannuronic acid donors represent more robust glycosylation systems in terms of diastereoselectivity. The change in stereoselectivity with decreasing acceptor nucleophilicity is related to a change in reaction mechanism shifting from the SN2 side to the SN1 side of the reactivity spectrum. Carbohydrate acceptors are examined and the reactivity-selectivity profile of these nucleophiles mirrored those of the model acceptors studied. The set of model ethanol acceptors thus provides a simple and effective "toolbox" to investigate glycosylation reaction mechanisms and report on the robustness of glycosylation protocols.

Simultaneous quantitation of sphingoid bases by UPLC-ESI-MS/MS with identical 13C-encoded internal standards.

Free sphingoid bases (lysosphingolipids) of primary storage sphingolipids are increased in tissues and plasma of several sphingolipidoses. As shown earlier by us, sphingoid bases can be accurately quantified using UPLC-ESI-MS/MS, particularly in combination with identical 13C-encoded internal standards. The feasibility of simultaneous quantitation of sphingoid bases in plasma specimens spiked with a mixture of such standards is here described. The sensitivity and linearity of detection is excellent for all examined sphingoid bases (sphingosine, sphinganine, hexosyl-sphingosine (glucosylsphingosine), hexosyl2-sphingosine (lactosylsphingosine), hexosyl3-sphingosine (globotriaosylsphingosine), phosphorylcholine-sphingosine) in the relevant concentration range and the measurements show very acceptable intra- and inter-assay variation (<10% average). Plasma samples of a series of male and female Gaucher Disease and Fabry Disease patients were analyzed with the multiplex assay. The obtained data compare well to those earlier determined for plasma globotriaosylsphingosine and glucosylsphingosine in GD and FD patients. The same approach can be also applied to measure sphingolipids in the same sample. Following extraction of sphingolipids from the same sample these can be converted to sphingoid bases by microwave exposure and subsequently quantified using 13C-encoded internal standards.

Proteasome inhibitor-adapted myeloma cells are largely independent from proteasome activity and show complex proteomic changes, in particular in redox and energy metabolism.

Adaptive resistance of myeloma to proteasome inhibition represents a clinical challenge, whose biology is poorly understood. Proteasome mutations were implicated as underlying mechanism, while an alternative hypothesis based on low activation status of the unfolded protein response was recently suggested (IRE1/XBP1-low model). We generated bortezomib- and carfilzomib-adapted, highly resistant multiple myeloma cell clones (AMO-BTZ, AMO-CFZ), which we analyzed in a combined quantitative and functional proteomic approach. We demonstrate that proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition, irrespective of a proteasome mutation, and uniformly show an 'IRE1/XBP1-low' signature. Adaptation of myeloma cells to proteasome inhibitors involved quantitative changes in >600 protein species with similar patterns in AMO-BTZ and AMO-CFZ cells: proteins involved in metabolic regulation, redox homeostasis, and protein folding and destruction were upregulated, while apoptosis and transcription/translation were downregulated. The quantitatively most upregulated protein in AMO-CFZ cells was the multidrug resistance protein (MDR1) protein ABCB1, and carfilzomib resistance could be overcome by MDR1 inhibition. We propose a model where proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition owing to metabolic adaptations that favor the generation of reducing equivalents, such as NADPH, which is supported by oxidative glycolysis. Proteasome inhibitor resistance may thus be targeted by manipulating the energy and redox metabolism.

Immunoproteasome dysfunction augments alternative polarization of alveolar macrophages.

The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes involved in shaping adaptive immune responses, but their role in innate immune signaling is still elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages, highly specialized tissue macrophages of the alveolar lung surface. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits, low molecular mass protein 2 (LMP2), LMP7 and multicatalytic endopeptidase complex-like 1, which was accompanied by increased immunoproteasome activity in M1 cells. Deficiency of LMP7 had no effect on the LPS/IFNγ-triggered M1 profile indicating that immunoproteasome function is dispensable for classical alveolar macrophage activation. In contrast, IL-4 triggered alternative (M2) activation of primary alveolar macrophages was accompanied by a transcriptionally independent amplified expression of LMP2 and LMP7 and an increase in immunoproteasome activity. Alveolar macrophages from LMP7 knockout mice disclosed a distorted M2 profile upon IL-4 stimulation as characterized by increased M2 marker gene expression and CCL17 cytokine release. Comparative transcriptome analysis revealed enrichment of IL-4-responsive genes and of genes involved in cellular response to defense, wounding and inflammation in LMP7-deficient alveolar macrophages indicating a distinct M2 inflammation resolving phenotype. Moreover, augmented M2 polarization was accompanied by amplified AKT/STAT6 activation and increased RNA and protein expression of the M2 master transcription factor interferon regulatory factor 4 in LMP7(-/-) alveolar macrophages. IL-13 stimulation of LMP7-deficient macrophages induced a similar M2-skewed profile indicative for augmented signaling via the IL-4 receptor α (IL4Rα). IL4Rα expression was generally elevated only on protein but not RNA level in LMP7(-/-) alveolar macrophages. Importantly, specific catalytic inhibition with an LMP7-specific proteasome inhibitor confirmed augmented IL-4-mediated M2 polarization of alveolar macrophages. Our results thus suggest a novel role of immunoproteasome function for regulating alternative activation of macrophages by limiting IL4Rα expression and signaling.

Gaucher disease and Fabry disease: new markers and insights in pathophysiology for two distinct glycosphingolipidoses.

Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Multigram-scale synthesis of an orthogonally protected 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) building block.

Reported is the gram-scale synthesis of tert-butyldiphenylsilyl 4-(N-benzyloxycarbonyl)-amino-2-azido-2,4,6-trideoxy-ß-D-galactopyranoside, which represents an orthogonally protected 2,4-diamino-D-fucose building block, a common constituent of various zwitterionic polysaccharides. The building block has been synthesized from D-glucosamine in 19% overall yield over 14 steps, requiring 5 chromatographic purifications. The key step in the synthesis is the introduction of the C-4 amino substituent, which has been accomplished by a one-pot three step procedure, involving regioselective C-3-O-trichloroacetimidate formation, C-4-O-triflation, and intramolecular substitution. The building block can be used as an acceptor and is readily transformed into a donor glycoside.

Light fluorous synthesis of glucosylated glycerol teichoic acids.

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.

Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells.

Resistance towards the proteasome inhibitor bortezomib is poorly understood. We adapted the HL-60, ARH-77 and AMO-1 cell lines (myeloid leukemia, plasmocytoid lymphoma, myeloma) to bortezomib exceeding therapeutic plasma levels, and compared characteristics of the ubiquitin-proteasome system, alternative proteases and the unfolded protein response (UPR) between adapted cells and parental lines. Adapted cells showed increased transcription rates, activities and polypeptide levels of the bortezomib-sensitive beta5, but also of the beta2 proteasome subunit and consistently retained elevated levels of active beta1/beta5-type proteasome subunits in the presence of therapeutic levels of bortezomib. Bortezomib-adapted HL-60 cells showed increased expression and proteasome association of the 11S proteasome activator, and did not accumulate poly-ubiquitinated protein, activate the UPR or UPR-mediated apoptosis in response to bortezomib. The rate of protein biosynthesis was reduced, and the transcription of chaperone genes downmodulated. We did not observe major changes in the activities of TPPII, cathepsins or deubiquitinating proteases. We conclude that different types of bortezomib-adapted cell lines, including myeloma, show similar patterns of changes in the proteasomal machinery which result in residual proteasome activity in the presence of bortezomib and a quantitative balance between protein biosynthesis and destruction.

Leukocyte cathepsin S is a potent regulator of both cell and matrix turnover in advanced atherosclerosis.

OBJECTIVE: A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr(-/-) mice deficient in leukocyte CatS by transplantation with CatS(-/-)xLDLr(-/-) or with LDLr(-/-) bone marrow and administered a high-fat diet. METHODS AND RESULTS: No difference in aortic root lesion size could be detected between CatS(+/+) and CatS(-/-) chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS(-/-) chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix. CONCLUSIONS: Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits.

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14.

A C-terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site-directed probe that irreversibly modifies a subset of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [(125)I]UbVS labeling with N-ethylmaleimide and Ub aldehyde, but also by genetic analysis. [(125)I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [(125)I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S-associated USP14 with [(125)I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

The p41 isoform of invariant chain is a chaperone for cathepsin L.

The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41-CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack p41. This defect mainly concerns the mature two-chain forms of CatL, which depend on p41 to be expressed at wild-type levels. Indeed, pulse-chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.

Generation of specific deoxynojirimycin-type inhibitors of the non-lysosomal glucosylceramidase.

The existence of a non-lysosomal glucosylceramidase in human cells has been documented (van Weely, S., Brandsma, M., Strijland, A., Tager, J. M., and Aerts, J. M. F. G. (1993) Biochim. Biophys. Acta 1181, 55-62). Hypothetically, the activity of this enzyme, which is localized near the cell surface, may influence ceramide-mediated signaling processes. To obtain insight in the physiological importance of the non-lysosomal glucosylceramidase, the availability of specific inhibitors would be helpful. Here we report on the generation of hydrophobic deoxynojirimycin (DNM) derivatives that potently inhibit the enzyme. The inhibitors were designed on the basis of the known features of the non-lysosomal glucosylceramidase and consist of a DNM moiety, an N-alkyl spacer, and a large hydrophobic group that promotes insertion in membranes. In particular, N-(5-adamantane-1-yl-methoxy)pentyl)-DNM is a very powerful inhibitor of the non-lysosomal glucosylceramidase at nanomolar concentrations. At such concentrations, the lysosomal glucocerebrosidase and alpha-glucosidase, the glucosylceramide synthase, and the N-linked glycan-trimming alpha-glucosidases of the endoplasmic reticulum are not affected.