Schay G, Borka B, Kernya L, Bulyáki É, Kardos J, Fekete M, Fidy J

Without Binding ATP, Human Rad51 Does Not Form Helical Filaments on ssDNA

J Phys Chem B. 2016 Mar 10;120(9):2165-78

Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K(d) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role.

Jeszenői N, Bálint M, Horváth I, van der Spoel D, Hetényi C

Exploration of Interfacial Hydration Networks of Target-Ligand Complexes

J Chem Inf Model. 2016 Jan 25;56(1):148-58

Interfacial hydration strongly influences interactions between biomolecules. For example, drug-target complexes are often stabilized by hydration networks formed between hydrophilic residues and water molecules at the interface. Exhaustive exploration of hydration networks is challenging for experimental as well as theoretical methods due to high mobility of participating water molecules. In the present study, we introduced a tool for determination of the complete, void-free hydration structures of molecular interfaces. The tool was applied to 31 complexes including histone proteins, a HIV-1 protease, a G-protein-signaling modulator, and peptide ligands of various lengths. The complexes contained 344 experimentally determined water positions used for validation, and excellent agreement with these was obtained. High-level cooperation between interfacial water molecules was detected by a new approach based on the decomposition of hydration networks into static and dynamic network regions (subnets). Besides providing hydration structures at the atomic level, our results uncovered hitherto hidden networking fundaments of integrity and stability of complex biomolecular interfaces filling an important gap in the toolkit of drug design and structural biochemistry. The presence of continuous, static regions of the interfacial hydration network was found necessary also for stable complexes of histone proteins participating in chromatin assembly and epigenetic regulation.

Gógl G, Alexa A, Kiss B, Katona G, Kovács M, Bodor A, Reményi A, Nyitray L.


J Biol Chem. 2016 Jan 1;291(1):11-27

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

Biri B, Kiss B, Király R, Schlosser G, Láng O, Kőhidai L, Fésüs L, Nyitray L.

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2

Biochem J. 2016 Jan 1;473(1):31-42

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

Sajó R, Tőke O, Hajdú I, Jankovics H, Micsonai A, Dobó J, Kardos J, Vonderviszt F

Structural plasticity of the Salmonella FliS flagellar export chaperone

FEBS Lett. 2016 Apr;590(8):1103-13

The Salmonella FliS flagellar export chaperone is a highly α-helical protein. Proteolytic experiments suggest that FliS has a compact core. However, the calorimetric melting profile of FliS does not show any melting transition in the 25-110 °C temperature range. Circular dichroism measurements reveal that FliS is losing its helical structure over a broad temperature range upon heating. These observations indicate that FliS unfolds in a noncooperative way and its native state shows features reminiscent of the molten globule state of proteins possessing substantial structural plasticity. As FliS has several binding partners within the cell, conformational adaptability seems to be an essential requirement to fulfill its multiple roles.

Zsakai A, Karkus Z, Utczas K, Biri B, Sievert LL, Bodzsar EB.

Body fatness and endogenous sex hormones in the menopausal transition

Maturitas. 2016 May;87:18-26

Age at the final menstrual period is of clinical and public health interest because the age at which natural menopause occurs may be a marker of ageing and health, and in general the menopausal transition increases the risk of many diseases, e.g. redistribution in the pattern of adiposity during the menopausal transition may increase risk of metabolic disease. The purpose of this research was to study the relationship between the menopausal status and body fatness.

A random sample of 1932 Hungarian women was studied. Body composition was estimated by body impedance analysis. In a subsample free estradiol and progesterone levels in saliva were quantified.

Body fat mass increased until the late 50s and then had a decrease through senescence. Premenopausal women who were much older than the median age at menopause had a higher amount of fat than their postmenopausal age-peers, while postmenopausal women, whose menopause occurred much earlier than the median age at menopause, had less fat than their premenopausal age-peers. The body fat mass in premenopausal women with low levels of sex hormones was always below the age-median value of the menopausal status subgroups, while the body fat mass of postmenopausal women with high levels of sex hormone levels was above the age-median values.

The analysis of body fatness in the menopausal transition revealed that (1) the rate of reproductive ageing and the body fat pattern were significantly related, and (2) body fat mass of women with unexpected levels of sex hormones was related more to their hormonal levels than to their menopausal status or their age. Thus future epidemiological screenings of women exposed to higher levels of menopause-related health risks should be expanded beyond the estimation of menopausal status based only on menstrual history to include sex hormone level assessment, as well as body composition analysis.

Györffy BA, Gulyássy P, Gellén B, Völgyi K, Madarasi D, Kis V, Ozohanics O, Papp I, Kovács P, Lubec G, Dobolyi Á, Kardos J, Drahos L, Juhász G, Kékesi KA

Widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats

Brain Behav Immun. 2016 Aug;56:289-309

An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.

Thangaraju K, Biri B, Schlosser G, Kiss B, Nyitray L, Fésüs L, Király R

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Anal Biochem. 2016 Jul 15;505:36-42

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca(2+)-dependent transamidase activity forming protease-resistant N(ε)-(γ-glutamyl) lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet, mainly because of the lack of a protein-based method for its characterization. Here we report the development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarization of a protein substrate previously formed by crosslinking fluorescently labeled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds that influence isopeptidase activity of TG2.

Gyimesi M, Harami GM, Kocsis ZS, Kovács M.

Recent adaptations of fluorescence techniques for the determination of mechanistic parameters of helicases and translocases

Methods. 2016 Oct 1;108:24-39

Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their physiological behavior and various cellular functions. Here we describe how recent adaptations of fluorescence-based solution kinetic methods can be used to determine practically all important mechanistic parameters of NA-based motor proteins. We outline practically useful analysis procedures for equilibrium, steady-state and transient kinetic data. This analysis can be used to quantitatively characterize the enzymatic steps of the NTP hydrolytic cycle, the binding site size, stoichiometry and energetics of protein-NA interactions, the rate and processivity of translocation along and unwinding of NA strands, and the mechanochemical coupling between these processes. The described methods yield insights into the functional role of the enzymes, and also greatly aid the design and interpretation of single-molecule experiments as well as the engineering of enzymatic properties for biotechnological applications.

Mészáros B, Zeke A, Reményi A, Simon I, Dosztányi Z

Systematic analysis of somatic mutations driving cancer: uncovering functional protein regions in disease development

Biol Direct. 2016 May 5;11:23

Recent advances in sequencing technologies enable the large-scale identification of genes that are affected by various genetic alterations in cancer. However, understanding tumor development requires insights into how these changes cause altered protein function and impaired network regulation in general and/or in specific cancer types.

In this work we present a novel method called iSiMPRe that identifies regions that are significantly enriched in somatic mutations and short in-frame insertions or deletions (indels). Applying this unbiased method to the complete human proteome, by using data enriched through various cancer genome projects, we identified around 500 protein regions which could be linked to one or more of 27 distinct cancer types. These regions covered the majority of known cancer genes, surprisingly even tumor suppressors. Additionally, iSiMPRe also identified novel genes and regions that have not yet been associated with cancer.

While local somatic mutations correspond to only a subset of genetic variations that can lead to cancer, our systematic analyses revealed that they represent an accompanying feature of most cancer driver genes regardless of the primary mechanism by which they are perturbed during tumorigenesis. These results indicate that the accumulation of local somatic mutations can be used to pinpoint genes responsible for cancer formation and can also help to understand the effect of cancer mutations at the level of functional modules in a broad range of cancer driver genes.

This article was reviewed by Sándor Pongor, Michael Gromiha and Zoltán Gáspári.

Noda S, So M, Adachi M, Kardos J, Akazawa-Ogawa Y, Hagihara Y, Goto Y

Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain-Dominated Architecture of Amyloid Fibrils

Biochemistry. 2016 Jul 19;55(28):3937-48

Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation.

Gellén B, Völgyi K, Györffy BA, Darula Z, Hunyadi-Gulyás É, Baracskay P, Czurkó A, Hernádi I, Juhász G, Dobolyi Á, Kékesi KA.

Proteomic investigation of the prefrontal cortex in the rat clomipramine model of depression

J Proteomics. 2017 Feb 5;153:53-64

Neonatal rodents chronically treated with the tricyclic antidepressant clomipramine show depression-like behavior, which persists throughout adulthood. Therefore, this animal model is suitable to investigate the pathomechanism of depression, which is still largely unknown at the molecular level beyond monoaminergic dysfunctions. Here, we describe protein level changes in the prefrontal cortex of neonatally clomipramine-treated adult rats correlating with behavioral abnormalities. Clomipramine was administered to rat pups twice daily between postnatal days 8-21, while controls received saline injections. Behavioral tests were performed on 3months old rats. The proteomic study was conducted using two-dimensional differential gel electrophoresis. We have identified 32 proteins by mass spectrometry analysis of the significantly altered protein spots. The changed proteins are related to several biological functions, such as inflammation, transcription, cell metabolism and cytoskeleton organization. Among the altered proteins, the level of macrophage migration inhibitory factor showed the largest alteration, which was confirmed with Western blot. Macrophage migration inhibitory factor showed widespread distribution and was predominantly expressed in astrocytes in the forebrain of rats which were described using immunohistochemistry. We conclude that neonatal clomipramine exposure induces sustained modification in the proteome, which may form the molecular basis of the observed depression-like behavior in adult rats.

Pálfy G, Kiss B, Nyitray L, Bodor A.

Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium-Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment.

Chembiochem. 2016 Oct 4;17(19):1829-1838

Dysregulation of Ca2+ -binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca2+ -bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca2+ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift-based secondary-structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow-timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca2+ -coordinating EF-hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca2+ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine-tuning of its protein-protein interactions inside the cell during Ca2+ signaling.

Herman BE, Szabó J, Bacsa I, Wölfling J, Schneider G, Bálint M, Hetényi C, Mernyák E, Szécsi M

Comparative investigation of the in vitro inhibitory potencies of 13-epimeric estrones and D-secoestrones towards 17β-hydroxysteroid dehydrogenase type 1

J Enzyme Inhib Med Chem. 2016;31(sup3):61-69

The inhibitory effects of 13-epimeric estrones, D-secooxime and D-secoalcohol estrone compounds on human placental 17β-hydroxysteroid dehydrogenase type 1 isozyme (17β-HSD1) were investigated. The transformation of estrone to 17β-estradiol was studied by an in vitro radiosubstrate incubation method. 13α-Estrone inhibited the enzyme activity effectively with an IC50 value of 1.2 μM, which indicates that enzyme affinity is similar to that of the natural estrone substrate. The 13β derivatives and the compounds bearing a 3-hydroxy group generally exerted stronger inhibition than the 13α and 3-ether counterparts. The 3-hydroxy-13β-D-secoalcohol and the 3-hydroxy-13α-D-secooxime displayed an outstanding cofactor dependence, i.e. more efficient inhibition in the presence of NADH than NADPH. The 3-hydroxy-13β-D-secooxime has an IC50 value of 0.070 μM and is one of the most effective 17β-HSD1 inhibitors reported to date in the literature.

Osteikoetxea-Molnár A, Szabó-Meleg E, Tóth EA, Oszvald Á, Izsépi E, Kremlitzka M, Biri B, Nyitray L, Bozó T, Németh P, Kellermayer M, Nyitrai M, Matko J.

The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes.

Cell Mol Life Sci. 2016 Dec;73(23):4531-4545

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca2+-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca2+ in regulation of TNT formation. Finally, we observed transport of various GM1/GM3+ vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.

Green B, Kovalev S, Asgekar V, Geloni G, Lehnert U, Golz T, Kuntzsch M, Bauer C, Hauser J, Voigtlaender J, Wustmann B, Koesterke I, Schwarz M, Freitag M, Arnold A, Teichert J, Justus M, Seidel W, Ilgner C, Awari N, Nicoletti D, Kaiser S, Laplace Y, Rajasekaran S, Zhang L, Winnerl S, Schneider H, Schay G, Lorincz I, Rauscher AA, Radu I, Mährlein S, Kim TH, Lee JS, Kampfrath T, Wall S, Heberle J, Malnasi-Csizmadia A, Steiger A, Müller AS, Helm M, Schramm U, Cowan T, Michel P, Cavalleri A et al.

High-Field High-Repetition-Rate Sources for the Coherent THz Control of Matter

Sci Rep. 2016 Feb 29;6:22256

Ultrashort flashes of THz light with low photon energies of a few meV, but strong electric or magnetic field transients have recently been employed to prepare various fascinating nonequilibrium states in matter. Here we present a new class of sources based on superradiant enhancement of radiation from relativistic electron bunches in a compact electron accelerator that we believe will revolutionize experiments in this field. Our prototype source generates high-field THz pulses at unprecedented quasi-continuous-wave repetition rates up to the MHz regime. We demonstrate parameters that exceed state-of-the-art laser-based sources by more than 2 orders of magnitude. The peak fields and the repetition rates are highly scalable and once fully operational this type of sources will routinely provide 1 MV/cm electric fields and 0.3 T magnetic fields at repetition rates of few 100 kHz. We benchmark the unique properties by performing a resonant coherent THz control experiment with few 10 fs resolution.

Peragovics A, Simon Z, Málnási-Csizmadia A, Bender A

Modeling Polypharmacological Profiles by Affinity Fingerprinting

Curr Pharm Des. 2016;22(46):6885-6894

Single target based approaches often proved to be unsuccessful in complex multigenic diseases such as cancer or schizophrenia. Multi-target drugs can be more efficacious in this regard by modulating multiple processes in the organism. According to the theory of polypharmacology, bioactive molecules possess characteristic interaction patterns that are responsible for their effects and side-effects and getting acquainted with this typical profile is increasingly desired to promote pharmaceutical research and development. There is a novel way of approaching polypharmacology that takes into account the interaction of molecules to a set of proteins that are not necessarily known biological targets of the compounds. Applying a carefully selected panel of proteins that can model the possible interactions a molecule can exert when administered to a human body, holds out a promise of biological activity prediction. This review aims to summarize a number of such bioactivity profiling-based approaches set up recently and present their application areas within the drug discovery field.

Várkuti BH, Képiró M, Horváth IÁ, Végner L, Ráti S, Zsigmond Á, Hegyi G, Lenkei Z, Varga M, Málnási-Csizmadia A

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative.

Sci Rep. 2016 May 31;6:26141.

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

Pilely K, Rosbjerg A, Genster N, Gal P, Pál G, Halvorsen B, Holm S, Aukrust P, Bakke SS, Sporsheim B, Nervik I, Niyonzima N, Bartels ED, Stahl GL, Mollnes TE, Espevik T, Garred P

Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL - implications for the progression of atherosclerosis

J Immunol. 2016 Jun 15;196(12):5064-74.

Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.

Oroszlán G, Kortvely E, Szakács D, Kocsis A, Dammeier S, Zeck A, Ueffing M, Závodszky P, Pál G, Gál P, Dobó J

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

J Immunol. 2016 Jan 15;196(2):857-65.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼ 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

Kiss B, Kalmár L, Nyitray L, Pál G

Structural determinants governing S100A4-induced isoform-selective disassembly of non-muscle myosin II filaments.

FEBS J. 2016 Jun;283(11):2164-80.

The Ca(2+) -binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA (NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 (NMIIA numbering), the NMIIA/NMIIC-specific alanine provides about 60-fold higher affinity than the NMIIB-specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α-helical binding segments. This communication is completely abolished by the Ala-to-Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C-terminal coiled-coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform-selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4-dependent and -independent regulations, respectively.

Dobó J, Szakács D, Oroszlán G, Kortvely E, Kiss B, Boros E, Szász R, Závodszky P, Gál P, Pál G

MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

Sci Rep. 2016 Aug 18;6:31877

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

Dobó J, Pál G, Cervenak L, Gál P

The emerging roles of mannose-binding lectin-associated serine proteases (MASPs) in the lectin pathway of complement and beyond

Immunol Rev. 2016 Nov;274(1):98-111

Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are the enzymatic constituents of the lectin pathway of the complement system. They are complexed with large pattern recognition molecules (PRMs) such as MBL, other collectins, and ficolins. The main function of two of the three MASPs has crystallized lately: MASP-1 autoactivates first, then it activates MASP-2, and finally both participate in the formation of the C4b2a convertase. In addition to this, both enzymes are involved in several other processes which are subject to intense research nowadays. Notably, MASP-1, as a promiscuous enzyme, has been implicated in the coagulation cascade, in the kinin generating contact system, and in cellular activation through protease-activated receptor (PAR) cleavage on endothelial cells. The third protease MASP-3 has emerged recently as the protease responsible for pro-factor D activation in resting blood, providing a fundamental link between two complement pathways. At present all three MASPs have at least one well-defined role and several other possible functions were implicated. Defect or more likely over-activation of MASPs may culminate into diseases such as ischemia-reperfusion injury (IRI); hence, MASPs are all potential targets of drug development.

Lin Y, Kardos J, Imai M, Ikenoue T, Kinoshita M, Sugiki T, Ishimori K, Goto Y, Lee YH

Amorphous aggregation of cytochrome c with inherently low amyloidogenicity is characterized by the metastability of supersaturation and the phase diagram.

Langmuir. 2016 Mar 1;32(8):2010-22.

Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.