Superanionic Solvent-Free Liquid Enzymes Exhibit Enhanced Structures and Activities.

The surface of a carboxylate-enriched octuple mutant of Bacillus subtilis lipase A (8M) is chemically anionized to produce core (8M)-shell (cationic polymer surfactants) bionanoconjugates in protein liquid form, which are termed anion-type biofluids. The resultant lipase biofluids exhibit a 2.5-fold increase in hydrolytic activity when compared with analogous lipase biofluids based on anionic polymer surfactants. In addition, the applicability of the anion-type biofluid using Myoglobin (Mb) that is well studied in anion-type solvent-free liquid proteins is evaluated. Although anionization resulted in the complete unfolding of Mb, the active α-helix level is partially recovered in the anion-type biofluids, and the effect is accentuated in the cation-type Mb biofluids. These highly active anion-type solvent-free liquid enzymes exhibit increased thermal stability and provide a new direction in solvent-free liquid protein research.

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity.

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition.

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson's disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.

Solution Structure of the Cutibacterium acnes-Specific Protein RoxP and Insights Into Its Antioxidant Activity.

Cutibacterium acnes is a predominant bacterium on human skin and is generally regarded as commensal. Recently, the abundantly secreted protein produced by C. acnes, RoxP, was shown to alleviate radical-induced cell damage, presumably via antioxidant activity, which could potentially be harnessed to fortify skin barrier function. The aim of this study was to determine the structure of RoxP and elucidate the mechanisms behind its antioxidative effect. Here, we present the solution structure of RoxP revealing a compact immunoglobulin-like domain containing a long flexible loop which, in concert with the core domain, forms a positively charged groove that could function as a binding site for cofactors or substrates. Although RoxP shares structural features with cell-adhesion proteins, we show that it does not appear to be responsible for adhesion of C. acnes bacteria to human keratinocytes. We identify two tyrosine-containing stretches located in the flexible loop of RoxP, which appear to be responsible for the antioxidant activity of RoxP.

Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages.

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

PAK Kinases Target Sortilin and Modulate Its Sorting.

The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.

Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State.

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.

CD4 T cells react to local increase of α-synuclein in a pathology-associated variant-dependent manner and modify brain microglia in absence of brain pathology.

We have previously shown that immunological processes in the brain during α-synuclein-induced neurodegeneration vary depending on the presence or absence of cell death. This suggests that the immune system is able to react differently to the different stages of α-synuclein pathology. However, it was unclear whether these immune changes were governed by brain processes or by a direct immune response to α-synuclein modifications. We have herein locally increased the peripheral concentration of α-synuclein or its pathology-associated variants, nitrated or fibrillar, to characterize the modulation of the CD4 T cell pool by α-synuclein and brain microglia in the absence of any α-synuclein brain pathology. We observed that α-synuclein changed the CD4:CD8 ratio by contracting the CD3+CD4+ T cell pool and reducing the pool of memory Regulatory T cells (Treg). Nitrated α-synuclein induced the expansion of both the CD3+CD4+ and CD3+CD4- T cells, while fibrils increased the percentage of Foxp3+ Treg cells and induced anti-α-synuclein antibodies. Furthermore, the activation pattern of CD3+CD4+ T cells was modulated in a variant-dependent manner; while nitrated and fibrillar α-synuclein expanded the fraction of activated Treg, all three α-synuclein variants reduced the expression levels of STAT3, CD25 and CD127 on CD3+CD4+ T cells. Additionally, while monomeric α-synuclein increased CD103 expression, the fibrils decreased it, and CCR6 expression was decreased by nitrated and fibrillar α-synuclein, indicating that α-synuclein variants affect the homing and tolerance capacities of CD3+CD4+ T cells. Indeed, this correlated with changes in brain microglia phenotype, as determined by FACS analysis, in an α-synuclein variant-specific manner and coincided in time with CD4+ T cell infiltration into brain parenchyma. We have shown that the peripheral immune system is able to sense and react specifically to changes in the local concentration and structure of α-synuclein, which results in variant-specific T cell migration into the brain. This may have a specific repercussion for brain microglia.

Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.

Latency transition of plasminogen activator inhibitor type 1 is evolutionarily conserved.

Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central ß-sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI-1 variants undergo latency transition with a similar rate. Since the functional stability of PAI-1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.

The effects of hypochlorous acid and neutrophil proteases on the structure and function of extracellular superoxide dismutase.

Extracellular superoxide dismutase (EC-SOD) is expressed by both macrophages and neutrophils and is known to influence the inflammatory response. Upon activation, neutrophils generate hypochlorous acid (HOCl) and secrete proteases to combat invading microorganisms. This produces a hostile environment in which enzymatic activity in general is challenged. In this study, we show that EC-SOD exposed to physiologically relevant concentrations of HOCl remains enzymatically active and retains the heparin-binding capacity, although HOCl exposure established oxidative modification of the N-terminal region (Met32) and the formation of an intermolecular cross-link in a fraction of the molecules. The cross-linking was also induced by activated neutrophils. Moreover, we show that the neutrophil-derived proteases human neutrophil elastase and cathepsin G cleaved the N-terminal region of EC-SOD irrespective of HOCl oxidation. Although the cleavage by elastase did not affect the quaternary structure, the cleavage by cathepsin G dissociated the molecule to produce EC-SOD monomers. The present data suggest that EC-SOD is stable and active at the site of inflammation and that neutrophils have the capacity to modulate the biodistribution of the protein by generating EC-SOD monomers that can diffuse into tissue.

Structural and functional characterization of human and murine C5a anaphylatoxins.

Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8(Δ71-73), and of murine C5a and C5a-desArg are reported. Whereas A8(Δ71-73) adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae.

BACKGROUND: In recent years the generation of antibodies by recombinant methods, such as phage display technology, has increased the speed by which antibodies can be obtained. However, in some cases when recombinant antibodies have to be validated, expression in E. coli can be problematic. This primarily occurs when codon usage or protein folding of specific antibody fragments is incompatible with the E. coli translation and folding machinery, for instance when recombinant antibody formats that include the Fc-region are needed. In such cases other expression systems can be used, including the protozoan parasite Leishmania tarentolae (L. tarentolae). This novel host for recombinant protein expression has recently shown promising properties for the expression of single-chain antibody fragments. We have utilised the L. tarentolae T7-TR system to achieve expression and secretion of two scFvs fused to the Fc-region of rabbit immunoglobulin G (IgG). RESULTS: Based on the commercial vector pLEXSY_IE-blecherry4 (Jena Bioscience; Cat. No. EGE-255), we generated a vector containing the Fragment Crystallisable (Fc) region of rabbit IgG allowing insertions of single chain antibody fragments (scFvs) in frame via Ncol/Notl cloning (pMJ_LEXSY-rFc). For the expression of rabbit Fc-fusion scFvs (scFv-rFc) we cloned two scFvs binding to human vimentin (LOB7 scFv) and murine laminin (A10 scFv) respectively, into the modified vector. The LOB7-rFc and A10-rFc fusions expressed at levels up to 2.95 mg/L in L. tarentolae T7-TR. Both scFv-rFcs were purified from the culture supernatants using protein A affinity chromatography. Additionally, we expressed three different scFvs without the rFc regions using a similar expression cassette, obtaining yields up to 1.00 mg/L. CONCLUSIONS: To our knowledge, this is the first time that antibody fragments with intact Fc-region of immunoglobulin have been produced in L. tarentolae. Using the plasmid pMJ_LEXSY-rFc, L. tarentolae T7-TR can be applied as an efficient tool for expression of rFc fusion antibody fragments, allowing easy purification from the growth medium. This system provides an alternative in cases where antibody constructs express poorly in standard prokaryotic systems. Furthermore, in cases where bivalent Fc-fused antibody constructs are needed, using L. tarentolae for expression provides an efficient alternative to mammalian expression.

The role of higher-order protein structure in supporting binding by heteroclitic monoclonal antibodies: the monoclonal antibody KIM185 to CD18 also binds C4-binding protein.

Heteroclitic monoclonal antibodies are characterized by the ability to bind multiple epitopes with little or no similarity. Such antibodies have been reported earlier, but insight into to the molecular basis of this propensity is limited. Here we report that the KIM185 antibody to human CD18 reacts with the plasma protein C4b-binding protein (C4BP). This was revealed during affinity purification procedures where human serum was incubated with surfaces coated with monoclonal antibodies to CD18. Other monoclonal antibodies to CD18 (KIM127 and TS1/18) showed no such interaction with C4BP. We constructed a sandwich-type time-resolved immunofluorometric assay using KIM185 both as capture and developing antibody. By use of proteolytic fragments of KIM185 and recombinant deletion mutants of C4BP the interaction sites were mapped to the variable region of KIM185 and the oligomerization domain of C4BP, respectively. C4BP is a large oligomeric plasma protein that binds activated complement factor C4b and other endogenous ligands as well as microorganisms. By use of the recent crystallographic data on the structure of CD11c/CD18 and prediction of the secondary structure of the C4BP oligomerization domain, we show that epitopes bound by KIM185 in these proteins are unlikely to share any major structural similarity. However, both antigens may form oligomers that would enable avid binding by the antibody. Our report points to the astonishing ability of heteroclitic antibodies to accommodate the binding of multiple proteins with no or little structural similarity within the confined space of the variable regions.

Widespread abundance of functional bacterial amyloid in mycolata and other gram-positive bacteria.

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phyla Actinobacteria and Firmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the genera Streptomyces, Bacillus, and Nocardia were all coated with amyloids. FuBA was purified from Gordonia amarae (from the cell envelope) and Geodermatophilus obscurus, and they had the morphology, tinctorial properties, and beta-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope of G. amarae was visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants.

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for alpha-lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates.

A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobin-hemoglobin complex.

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.

Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein.

Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present study revealed that recombinant Hpr binds hemoglobin as efficiently as haptoglobin (Hp). However, in contrast to Hp, Hpr did not promote any high-affinity binding to the scavenger receptor CD163. Binding of hemoglobin to circulating native Hpr incorporated into the HDL fraction was indicated by hemoglobin-affinity precipitation of plasma Hpr together with apoL-I. In conclusion, plasma has 2 high-affinity hemoglobin-binding haptoglobins instead of one, but only Hp-hemoglobin complexes are efficiently recognized by CD163. Circulating Hpr-bound hemoglobin should therefore be taken into consideration when measuring "free" plasma hemoglobin. Furthermore, Hpr-bound hemoglobin might contribute to the biologic activity of the circulating apoL-I/Hpr-containing HDL particles.