Publications by authors named "Michał Dadlez"

141 Publications

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Plant J 2021 12 16;108(5):1400-1421. Epub 2021 Oct 16.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5a, 02-106, Warsaw, Poland.

Lipid anchors are common post-translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C-termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP-Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the structural basis of plant REP-Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule-specific, and in other cases they are common for a subset of Rabs. In particular, the C-terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C-terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.
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http://dx.doi.org/10.1111/tpj.15519DOI Listing
December 2021

ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling.

J Cell Sci 2021 10 21;134(20). Epub 2021 Oct 21.

Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin, Germany.

Sortilin is a neuronal receptor for apolipoprotein E (apoE). Sortilin-dependent uptake of lipidated apoE promotes conversion of polyunsaturated fatty acids (PUFA) into neuromodulators that induce anti-inflammatory gene expression in the brain. This neuroprotective pathway works with the apoE3 variant but is lost with the apoE4 variant, the main risk factor for Alzheimer's disease (AD). Here, we elucidated steps in cellular handling of lipids through sortilin, and why they are disrupted by apoE4. Combining unbiased proteome screens with analyses in mouse models, we uncover interaction of sortilin with fatty acid-binding protein 7 (FABP7), the intracellular carrier for PUFA in the brain. In the presence of apoE3, sortilin promotes functional expression of FABP7 and its ability to elicit lipid-dependent gene transcription. By contrast, apoE4 binding blocks sortilin-mediated sorting, causing catabolism of FABP7 and impairing lipid signaling. Reduced FABP7 levels in the brain of AD patients expressing apoE4 substantiate the relevance of these interactions for neuronal lipid homeostasis. Taken together, we document interaction of sortilin with mediators of extracellular and intracellular lipid transport that provides a mechanistic explanation for loss of a neuroprotective lipid metabolism in AD.
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http://dx.doi.org/10.1242/jcs.258894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8572006PMC
October 2021

Trimethylamine N-Oxide, a Gut Microbiota-Derived Metabolite, Is Associated with Cardiovascular Risk in Psoriasis: A Cross-Sectional Pilot Study.

Dermatol Ther (Heidelb) 2021 Aug 13;11(4):1277-1289. Epub 2021 May 13.

Department of Dermatology, Medical University of Warsaw, Koszykowa 82a, 02-008, Warsaw, Poland.

Introduction: Trimethylamine N-oxide (TMAO), a gut microbiota metabolite from dietary phosphatidylcholine, is involved in the pathogenesis of atherosclerosis and cardiovascular diseases. Psoriasis is associated with increased cardiovascular risk that is not captured by traditional biomarkers. The aim of the present study was to assess TMAO concentration in psoriasis and evaluate the relationship between TMAO and cardiovascular risk in psoriatic patients.

Methods: In 72 patients with psoriasis and 40 age- and sex-matched non-psoriatic controls, we evaluated fasting plasma TMAO, measured by high-performance liquid chromatography, and cardiovascular risk assessed by various scoring systems such as Framingham, QRISK2, AHA/ACC, and Reynolds risk scores.

Results: In patients with psoriasis, TMAO concentration was significantly higher than in the control group (195.68 [133.54-332.58] ng/ml versus 126.06 [84.29-156.88] ng/ml, respectively; p < 0.001). Plasma TMAO concentration was significantly correlated with age, total cholesterol, triglycerides, systolic and diastolic blood pressure. Furthermore, the receiver-operating characteristic (ROC) and multiple regression analysis showed that TMAO is an independent predictor of cardiovascular risk.

Conclusion: TMAO is a valuable candidate for biomarker and a translational link between dysbiosis and atherosclerosis in psoriasis.
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http://dx.doi.org/10.1007/s13555-021-00547-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322249PMC
August 2021

A model of full-length RAGE in complex with S100B.

Structure 2021 09 21;29(9):989-1002.e6. Epub 2021 Apr 21.

Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5a, 02-109 Warsaw, Poland.

The receptor for advanced glycation end products (RAGE) is an immunoglobulin-type multiligand transmembrane protein expressed in numerous cell types, including the central nervous system cells. RAGE interaction with S100B, released during brain tissue damage, leads to RAGE upregulation and initialization of a spiral proinflammatory associated with different neural disorders. Here, we present the structural characterization of the hetero-oligomeric complex of the full-length RAGE with S100B, obtained by a combination of mass spectrometry-based methods and molecular modeling. We predict that RAGE functions as a tightly packed tetramer exposing a positively charged surface formed by V domains for S100B binding. Based on HDX results we demonstrate an allosteric coupling of the distal extracellular V domains and the transmembrane region, indicating a possible mechanism of signal transmission by RAGE across the membrane. Our model provides an insight into RAGE-ligand interactions, providing a basis for the rational design of the therapeutic modifiers of its activity.
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http://dx.doi.org/10.1016/j.str.2021.04.002DOI Listing
September 2021

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication.

Elife 2021 03 11;10. Epub 2021 Mar 11.

Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

The duplication and ninefold symmetry of the centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.
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http://dx.doi.org/10.7554/eLife.57241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009671PMC
March 2021

Clinical Implications of Intestinal Barrier Damage in Psoriasis.

J Inflamm Res 2021 27;14:237-243. Epub 2021 Jan 27.

Department of Dermatology, Medical University of Warsaw, Warsaw, Poland.

Background: An increasing amount of evidence suggests an association between increased intestinal permeability and the pathogenesis of chronic inflammatory diseases. However, the clinical significance of gut barrier dysfunction in psoriasis remains to be established.

Objective: To evaluate whether there are differences in disease activity, the severity of gastrointestinal symptoms and the blood concentration of bacterial metabolites in psoriatic patients with a normal and altered intestinal barrier.

Patients And Methods: Gut barrier integrity was assessed with the serum concentrations of claudin-3, a modulator of intestinal tight junctions and an intestinal fatty acid-binding protein, a marker of enterocyte damage. Gastrointestinal symptoms were evaluated with a validated questionnaire. The concentration of trimethylamine N-oxide (TMAO), a gut microbiota-associated metabolite, was measured with high-performance liquid chromatography.

Results: One hundred and fourteen patients with psoriasis were finally enrolled in the study - 68 with an altered gut barrier and 46 with a properly functioning intestinal barrier. Patients with an altered gut barrier showed a significantly higher score in the Gastrointestinal Symptom Rating Scale (3.20 vs 1.46, p<0.001). Moreover, patients with psoriasis and a disrupted intestinal barrier demonstrated a higher disease activity (PASI: 19.7 vs 10.3, p<0.001) and systemic inflammatory parameters (neutrophil-to-lymphocyte ratio: 2.86 vs 1.71, p<0.001; C-reactive protein 3.76 vs 1.92; p<0.05). The marker of bacterial translocation was significantly higher in psoriatic patients with damaged gut integrity (TMAO: 375.7±51.9 vs 119.4±27.5 ng/mL; p<0.05).

Conclusion: The altered gut barrier in psoriasis is associated with gastrointestinal symptoms, systemic inflammatory profile and the increased blood concentration of gut microbiota-derived metabolite - TMAO. Intestinal barrier modulation represents a new promising therapeutic approach.
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http://dx.doi.org/10.2147/JIR.S292544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851376PMC
January 2021

Multi-omic signatures of atherogenic dyslipidaemia: pre-clinical target identification and validation in humans.

J Transl Med 2021 01 6;19(1). Epub 2021 Jan 6.

Department of Biochemistry, Faculty of Medicine, Medical University of Gdansk, 1 Debinki St, 80-211, Gdansk, Poland.

Background: Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH).

Methods: Liquid chromatography/mass spectrometry (LC/MS) and immunoassays were used to find out blood alterations at metabolite and protein levels in dyslipidaemic ApoE/LDLR mice and in FH patients to evaluate their human relevance.

Results: We identified 15 metabolites (inhibitors and substrates of nitric oxide synthase (NOS), low-molecular-weight antioxidants (glutamine, taurine), homocysteine, methionine, 1-methylnicotinamide, alanine and hydroxyproline) and 9 proteins (C-reactive protein, proprotein convertase subtilisin/kexin type 9, apolipoprotein C-III, soluble intercellular adhesion molecule-1, angiotensinogen, paraoxonase-1, fetuin-B, vitamin K-dependent protein S and biglycan) that differentiated FH patients from healthy controls. Most of these changes were consistently found in dyslipidaemic mice and were further amplified if mice were fed an atherogenic (Western or low-carbohydrate, high-protein) diet.

Conclusions: The alterations highlighted the involvement of an immune-inflammatory response system, oxidative stress, hyper-coagulation and impairment in the vascular function/regenerative capacity in response to dyslipidaemia that may also be directly engaged in development of atherosclerosis. Our study further identified potential biomarkers for an increased risk of atherosclerosis that may aid in clinical diagnosis or in the personalized treatment.
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http://dx.doi.org/10.1186/s12967-020-02663-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789501PMC
January 2021

Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Open Biol 2020 12 23;10(12):200343. Epub 2020 Dec 23.

Biological Research Centre, Institute of Biochemistry, MTA Lendület Laboratory of Cell Cycle Regulation, Szeged, H-6726, Hungary.

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.
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http://dx.doi.org/10.1098/rsob.200343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776573PMC
December 2020

Interaction interface in the C-terminal parts of centriole proteins Sas6 and Ana2.

Open Biol 2020 11 11;10(11):200221. Epub 2020 Nov 11.

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

The centriole is a ninefold symmetrical structure found at the core of centrosomes and, as a basal body, at the base of cilia, whose conserved duplication is regulated by Plk4 kinase. Plk4 phosphorylates a single serine residue at the N-terminus of Ana2 to promote Ana2's loading to the site of procentriole formation. Four conserved serines in Ana2's STAN motif are then phosphorylated by Plk4, enabling Sas6 recruitment. Crystallographic data indicate that the coiled-coil domain of Ana2 forms a tetramer but the structure of full-length Ana2 has not been solved. Here, we have employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to uncover the conformational dynamics of Ana2, revealing the high flexibility of this protein with one rigid region. To determine the elusive nature of the interaction surfaces between Ana2 and Sas6, we have confirmed complex formation between the phosphomimetic form of Ana2 (Ana2-4D) and Sas6 and . Analysis of this complex by HDX-MS identifies short critical regions required for this interaction, which lie in the C-terminal parts of both proteins. Mutational studies confirmed the relevance of these regions for the Ana2-Sas6 interaction. The Sas6 site required for Ana2 binding is distinct from the site required for Sas6 to bind Gorab and Sas6 is able to bind both these protein partners simultaneously.
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http://dx.doi.org/10.1098/rsob.200221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729032PMC
November 2020

Enhanced cardiac hypoxic injury in atherogenic dyslipidaemia results from alterations in the energy metabolism pattern.

Metabolism 2021 01 13;114:154400. Epub 2020 Oct 13.

Department of Biochemistry, Faculty of Medicine, Medical University of Gdansk, 1 Debinki St., 80-211 Gdansk, Poland. Electronic address:

Objective: Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury.

Methods: Dyslipidaemic apolipoprotein E and low-density lipoprotein receptor (ApoE/LDLR) double knockout (ApoE/LDLR) and wild-type C57BL/6 (WT) mice aged six months old were studied. Cardiac injury under reduced oxygen supply was evaluated by 5 min exposure to 5% oxygen in the breathing air under electrocardiogram (ECG) recording and with the assessment of troponin I release. To address the mechanisms LC/MS was used to analyse the cardiac proteome pattern or in vivo metabolism of stable isotope-labelled substrates and HPLC was applied to measure concentrations of cardiac high-energy phosphates. Furthermore, the effect of blocking fatty acid use with ranolazine on the substrate preference and cardiac hypoxic damage was studied in ApoE/LDLR mice.

Results: Hypoxia induced profound changes in ECG ST-segment and troponin I leakage in ApoE/LDLR mice but not in WT mice. The evaluation of the cardiac proteomic pattern revealed that ApoE/LDLR as compared with WT mice were characterised by coordinated increased expression of mitochondrial proteins, including enzymes of fatty acids' and branched-chain amino acids' oxidation, accompanied by decreased expression levels of glycolytic enzymes. These findings correlated with in vivo analysis, revealing a reduction in the entry of glucose and enhanced entry of leucine into the cardiac Krebs cycle, with the cardiac high-energy phosphates pool maintained. These changes were accompanied by the activation of molecular targets controlling mitochondrial metabolism. Ranolazine reversed the oxidative metabolic shift in ApoE/LDLR mice and reduced cardiac damage induced by hypoxia.

Conclusions: We suggest a novel mechanism for myocardial injury in dyslipidaemia that is consequent to an increased reliance on oxidative metabolism in the heart. The alterations in the metabolic pattern that we identified constitute an adaptive mechanism that facilitates maintenance of metabolic equilibrium and cardiac function under normoxia. However, this adaptation could account for myocardial injury even in a mild reduction of oxygen supply.
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http://dx.doi.org/10.1016/j.metabol.2020.154400DOI Listing
January 2021

Low Content of Cyclosporine A and Its Metabolites in the Colostrum of Post-Transplant Mothers.

Nutrients 2020 Sep 4;12(9). Epub 2020 Sep 4.

First Department of Obstetrics and Gynecology, Medical University of Warsaw, 02-015 Warsaw, Poland.

The rate of post-transplant mothers who breastfeed while on immunosuppression is progressively increasing. Data on breastfeeding while on cyclosporine-based regimens are limited. Therefore, we assessed the amount of cyclosporine and its metabolites that might be ingested by a breastfed infant by measuring the concentration of cyclosporine and its metabolites in the colostrum of seven post-transplant mothers. The mean concentration of cyclosporine in the colostrum was 22.40 ± 9.43 mcg/L, and the estimated mean daily dose of the drug was 1049.22 ± 397.41 ng/kg/24 h. Only three metabolites (AM1, DHCsA, and THCsA) had mean colostrum amounts comparable to or higher than cyclosporine itself, with the daily doses being 468.51 ± 80.37, 2757.79 ± 1926.11, and 1044.76 ± 948.56 ng/kg/24 h, respectively. Our results indicate a low transfer of cyclosporine and its metabolites into the colostrum in the first two days postpartum and confirm the emerging change to the policy on breastfeeding among post-transplant mothers. A full assessment of the safety of immunosuppressant exposure via breastmilk will require further studies with long-term follow-ups of breastfed children.
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http://dx.doi.org/10.3390/nu12092713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551077PMC
September 2020

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations.

Int J Mol Sci 2020 Jul 24;21(15). Epub 2020 Jul 24.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106 Warsaw, Poland.

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH-NPAT and YARP-NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple α -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single α -helix that makes multiple contacts with α -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.
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http://dx.doi.org/10.3390/ijms21155268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432317PMC
July 2020

HaDeX: an R package and web-server for analysis of data from hydrogen-deuterium exchange mass spectrometry experiments.

Bioinformatics 2020 08;36(16):4516-4518

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw 02-106, Poland.

Motivation: Hydrogen-deuterium mass spectrometry (HDX-MS) is a rapidly developing technique for monitoring dynamics and interactions of proteins. The development of new devices has to be followed with new software suites addressing emerging standards in data analysis.

Results: We propose HaDeX, a novel tool for processing, analysis and visualization of HDX-MS experiments. HaDeX supports a reproducible analytical process, including data exploration, quality control and generation of publication-quality figures.

Availability And Implementation: HaDeX is available primarily as a web-server (http://mslab-ibb.pl/shiny/HaDeX/), but its all functionalities are also accessible as the R package (https://CRAN.R-project.org/package=HaDeX) and standalone software (https://sourceforge.net/projects/HaDeX/).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575049PMC
August 2020

Mitochondrial protein biogenesis in the synapse is supported by local translation.

EMBO Rep 2020 08 18;21(8):e48882. Epub 2020 Jun 18.

Laboratory of Molecular Basis of Synaptic Plasticity, Centre of New Technologies, University of Warsaw, Warsaw, Poland.

Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions.
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http://dx.doi.org/10.15252/embr.201948882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403725PMC
August 2020

Higher Concentrations of Cyclosporine Metabolites in Liver Transplant Recipients With a History of Viral and Bacterial Infections.

Transplant Proc 2020 Oct 31;52(8):2503-2506. Epub 2020 May 31.

Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, Warsaw, Poland.

Background: Infection remains a serious clinical problem in liver transplant (LTX) recipients. A higher risk of infection is connected with immunosuppression therapy. The aim of the study was to assess the relationships between infections' incidence and concentrations of cyclosporine (CsA) metabolites after LTX.

Methods: Forty-three liver transplant recipients receiving CsA were included in the study. With the use of liquid chromatography combined with tandem mass spectrometry, concentrations of CsA and its metabolites were measured: dihydroxylated cyclosporine metabolites (DiHCsA), trihydroxylated cyclosporine metabolites (TriHCsA), demethylcarboxylated cyclosporine metabolites (DemCarbCsA), AM1, AM9, and AM4N. The study protocol conformed with the Declaration of Helsinki.

Results: Patients with a history of Epstein-Barr virus (EBV) infection had higher DiHCsA, TriHCsA, DemCarbCsA, AM1/CsA, DiHCsA/CsA, TriHCsA/CsA i DemCarbCsA/CsA in comparison with group without such infection (P = .049, P = .037, P = .006, P = .018, P = .005, P = .027, and P = .026, respectively). LTX recipients with a history of all viral infections had higher DiHCsA, TriHCsA, DiHCsA/CsA, TriHCsA/CsA than patients without viral infections (P = .013, P = .021, P = .013, and P = .048, respectively). Multivariable analysis showed that AM1, DiHCsA, TriHCsA, DemCarbCsA, AM4N/CsA had positively influence on the incidence of all viral infections (β = 0.0302, P = .0328; β = 0.0699, P = .0453; β = 0.6781, P = .0382; β = 0.6767, P = .0414; and β = 0.8307, P = .0267, respectively). In multivariable analysis, patients with a history of all bacterial infections had higher AM1 and higher AM1/CsA in comparison with LTX recipients without such infections (β = 0.0118, P = .0279; and β = 0.0099, P = .036, respectively).

Conclusion: In liver transplant recipients with a history of viral or bacterial infections higher concentrations of CsA metabolites were found. Possibly CsA metabolites could be used to assess the risk of infection in patients after liver transplantation. It should be confirmed in further investigations.
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http://dx.doi.org/10.1016/j.transproceed.2020.03.039DOI Listing
October 2020

Vimentin S-glutathionylation at Cys328 inhibits filament elongation and induces severing of mature filaments in vitro.

FEBS J 2020 12 21;287(24):5304-5322. Epub 2020 Apr 21.

Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Vimentin intermediate filaments are a significant component of the cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and disassemble and thus participate in the dynamic processes of the cell. Post-translational modifications (PTMs) such as protein phosphorylation regulate the multiphasic association of vimentin from soluble complexes to insoluble filaments and the reverse processes. The thiol side chain of the single vimentin cysteine at position 328 (Cys328) is a direct target of oxidative modifications inside cells. Here, we used atomic force microscopy, electron microscopy and a novel hydrogen-deuterium exchange mass spectrometry (HDex-MS) procedure to investigate the structural consequences of S-nitrosylation and S-glutathionylation of Cys328 for in vitro oligomerisation of human vimentin. Neither modification affects the lateral association of tetramers to unit-length filaments (ULF). However, S-glutathionylation of Cys328 blocks the longitudinal assembly of ULF into extended filaments. S-nitrosylation of Cys328 does not hinder but slows down the elongation. Likewise, S-glutathionylation of preformed vimentin filaments causes their extensive fragmentation to smaller oligomeric species. Chemical reduction of the S-glutathionylated Cys328 thiols induces reassembly of the small fragments into extended filaments. In conclusion, our in vitro results suggest S-glutathionylation as a candidate PTM for an efficient molecular switch in the dynamic rearrangements of vimentin intermediate filaments, observed in vivo, in response to changes in cellular redox status. Finally, we demonstrate that HDex-MS is a powerful method for probing the kinetics of vimentin filament formation and filament disassembly induced by PTMs.
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http://dx.doi.org/10.1111/febs.15321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818121PMC
December 2020

Formation of highly stable multinuclear AgS clusters in zinc fingers disrupts their structure and function.

Chem Commun (Camb) 2020 Jan 8;56(9):1329-1332. Epub 2020 Jan 8.

Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.

Silver (Ag(i)) binding to consensus zinc fingers (ZFs) causes Zn(ii) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgS clusters. A compact eight-membered AgS structure formed by a CCCC ZF is the first cluster example reported for a single biological molecule. Ag(i)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.
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http://dx.doi.org/10.1039/c9cc09418kDOI Listing
January 2020

Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains.

Sci Rep 2019 12 30;9(1):20332. Epub 2019 Dec 30.

Institute of Biochemistry and Biophysics, PAN, Pawinskiego 5a, 02-109, Warsaw, Poland.

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen-deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.
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http://dx.doi.org/10.1038/s41598-019-56993-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937306PMC
December 2019

Composition and processing activity of a semi-recombinant holo U7 snRNP.

Nucleic Acids Res 2020 02;48(3):1508-1530

Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3' end processing of histone pre-mRNAs.
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http://dx.doi.org/10.1093/nar/gkz1148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026596PMC
February 2020

Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms.

Plant Physiol 2020 02 7;182(2):1142-1160. Epub 2019 Nov 7.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis () gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing ::--- or ::--- in the - knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins.
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http://dx.doi.org/10.1104/pp.19.00900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997710PMC
February 2020

Identification of glycated and acetylated lysine residues in human α2-antiplasmin.

Biochem Biophys Res Commun 2020 01 23;521(1):19-23. Epub 2019 Oct 23.

Institute of Cardiology, Jagiellonian University Medical College, Cracow, Poland; John Paul II Hospital, Cracow, Poland.

Background: The post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.

Aim: We sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.

Methods: We analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.

Results: Eleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.

Conclusion: Human α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.144DOI Listing
January 2020

Dose-adjusted and dose/kg-adjusted concentrations of mycophenolic acid precursors reflect metabolic ratios of their metabolites in contrast with tacrolimus and cyclosporine.

Biosci Rep 2019 09 13;39(9). Epub 2019 Sep 13.

Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, Poland.

Background And Purpose: Therapeutic drug monitoring is a valuable tool supporting immunosuppressive therapy. Significant variation of immunosuppressive drug (ISD) concentrations during their use at similar doses is the basis of dose-normalization strategy. The strategy of dose-adjustment is proposed to identify variability in the rate of ISD metabolism. While the parent drug-to-metabolite ratio (metabolic ratio, MR) represents the rate of formation of individual metabolites. The present study was aimed at evaluation of associations between ISDs' metabolism rate expressed as dose-adjusted concentrations (C/D) and dose/kg-adjusted concentrations (C/D/kg) and MRs of individual metabolites of tacrolimus, cyclosporine A and MPA precursors.

Experimental Approach: 506 patients have participated: 284 males (56.13%) and 222 females (43.87%); 318 after kidney (62.85%) and 188 after liver transplantation; median age was 51.34 (39.32-59.95) years and median time after transplantation 78.92 (33.87-138.4) months.

Key Results: Generally, we have not observed significant relationships between dose-adjusted and dose/kg-adjusted concentrations and MRs of cyclosporine and tacrolimus. Significant correlations were found for: AM9/CsA and dMC-CsA/CsA in kidney transplant recipients and MIII/Tac, AM1/CsA and AM4N/CsA in liver transplant recipients. In contrast, MRs of mycophenolic acid (MPA) metabolites correlated significantly with MPA C/D and C/D/kg both in kidney and liver transplant recipients.

Conclusion And Implications: In conclusion, easily available and easy to use in clinical practice C/D and C/D/kg ratios cannot be considered as parameters directly reflecting the rate of generation of major metabolites of cyclosporine and tacrolimus both in liver and kidney transplant recipients.
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http://dx.doi.org/10.1042/BSR20182031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744583PMC
September 2019

Insight into the Structural Dynamics of the Lysenin During Prepore-to-Pore Transition Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Toxins (Basel) 2019 08 7;11(8). Epub 2019 Aug 7.

The Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland.

Lysenin is a pore-forming toxin of the aerolysin family, which is derived from coelomic fluid of the earthworm . Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the formation of a lytic β-barrel pore. The soluble monomer and transmembrane pore structures were recently described, but the underlying structural details of oligomerization remain unclear. To investigate the molecular mechanisms controlling the conformational rearrangements accompanying pore formation, we compared the hydrogen-deuterium exchange pattern between lysenin and its mutant lysenin. This mutation arrests lysenin oligomers in the prepore state at the membrane surface and does not affect the structural dynamics of the water-soluble form of lysenin. In contrast, membrane-bound lysenin exhibited increased structural stabilization, especially within the twisted β-sheet of the N-terminal domain. We demonstrated that the structural stabilization of the lysenin prepore started at the site of lysenin's initial interaction with the lipid membrane and was transmitted to the twisted β-sheet of the N-terminal domain, and that lysenin was arrested in this conformation. In lysenin, stabilization of these regions drove the conformational changes necessary for pore formation.
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http://dx.doi.org/10.3390/toxins11080462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722932PMC
August 2019

His6, His13, and His14 residues in Aβ 1-40 peptide significantly and specifically affect oligomeric equilibria.

Sci Rep 2019 07 1;9(1):9449. Epub 2019 Jul 1.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Oligomers of Aβ peptide are implicated as the most probable causative agent in Alzheimer's disease. However, their structural properties remain elusive due to the dynamic and heterogeneous character of oligomeric species coexisting in solution. Nevertheless, new approaches, mainly based on mass spectrometry, provide unique access to these different structural forms. Using these methods, we previously showed that the N-terminal, non-amyloidogenic region of Aβ is involved in the network of interactions specifically stabilizing oligomers. In the present study, we identified three histidine residues as active participants in this network. Detailed knowledge of the structural features that are potentially important for oligomer-mediated neurotoxicity is a prerequisite for the rational design of oligomerization modifiers.
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http://dx.doi.org/10.1038/s41598-019-45988-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602940PMC
July 2019

Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7.

EMBO Mol Med 2019 05;11(5)

Laboratory of Mitochondrial Biogenesis, Centre of New Technologies, University of Warsaw, Warsaw, Poland

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that COA7 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS We also found that pathogenic mutant versions of COA7 are imported slower than the wild-type protein, and mislocalized proteins are degraded in the cytosol by the proteasome. Interestingly, proteasome inhibition rescued both the mitochondrial localization of COA7 and complex IV activity in patient-derived fibroblasts. We propose proteasome inhibition as a novel therapeutic approach for a broad range of mitochondrial pathologies associated with the decreased levels of mitochondrial proteins.
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http://dx.doi.org/10.15252/emmm.201809561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505684PMC
May 2019

A structural model of the immune checkpoint CD160-HVEM complex derived from HDX-mass spectrometry and molecular modeling.

Oncotarget 2019 Jan 11;10(4):536-550. Epub 2019 Jan 11.

University of Gdansk, Faculty of Chemistry, Department of Biomedical Chemistry, Gdansk, Poland.

CD160 is a T cell coinhibitory molecule that interacts with the herpes virus entry mediator (HVEM) on antigen-presenting cells to provide an inhibitory signal to T cells. To date, the structure of CD160 and its complex with HVEM are unknown. Here, we have identified the fragments of CD160 interacting with HVEM using ELISA tests, hydrogen/deuterium studies, affinity chromatography and mass spectrometry (MS). By combining hydrogen/deuterium exchange and mass spectrometry (HDX-MS) we obtained key information about the tertiary structure of CD160, predicting the 3D structure of the CD160-HVEM complex. Our results provide insights into the molecular architecture of this complex, serving as a useful basis for designing inhibitors for future immunotherapies.
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http://dx.doi.org/10.18632/oncotarget.26570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355189PMC
January 2019

Mass Spectrometry-Based Comprehensive Analysis of Pancreatic Cyst Fluids.

Biomed Res Int 2018 29;2018:7169595. Epub 2018 Nov 29.

Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Warsaw, Poland.

Pancreatic cyst fluids (PCFs) enriched in tumour-derived proteins are considered a potential source of new biomarkers. This study aimed to determine compositional and quantitative differences between the degradome and proteome of PCFs aspirated from different types of pancreatic cyst lesions (PCLs). 91 patients who underwent endoscopic ultrasound-fine needle aspiration under routine clinical diagnosis of PCLs were enrolled. Four cysts were malignant (CAs), and 87 were nonmalignant and consisted of 18 intraductal papillary mucinous neoplasms (IPMNs), 14 mucinous cystic neoplasms (MCNs), nine serous cystic neoplasms (SCNs), 29 pseudocysts (PCs), and 17 unclassified. Profiles of the <5 kDa fraction, the degradome, and the trypsin-digested proteome were analysed using an LTQ-Orbitrap Elite mass spectrometer coupled with a nanoACQUITY LC system. Qualitative analyses identified 796 and 366 proteins in degradome and proteome, respectively, and 689 (77%) and 285 (78%) of them were present in the Plasma Proteome Database. Gene Ontology analysis showed a significant overrepresentation of peptidases and peptidases inhibitors in both datasets. In the degradome fraction, quantitative values were obtained for 6996 peptides originating from 657 proteins. Of these, 2287 peptides were unique to a single type, and 515 peptides, derived from 126 proteins, were shared across cyst types. 32 peptides originating from 12 proteins had differential (adjusted -value ≤0.05, FC ≥1.5) abundance in at least one of the five cysts types. In proteome, relative expression was measured for 330 proteins. Of them, 33 proteins had significantly (adjusted -value ≤0.05, FC ≥1.5) altered abundance in at least one of the studied groups and 19 proteins appeared to be unique to a given cyst type. PCFs are dominated by blood proteins and proteolytic enzymes. Although differences in PCF peptide composition and abundance could aid classification of PCLs, the unpredictable inherent PCF proteolytic activity may limit the practical applications of PCF protein profiling.
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http://dx.doi.org/10.1155/2018/7169595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304507PMC
April 2019

The protein-binding N-terminal domain of human translation elongation factor 1Bβ possesses a dynamic α-helical structural organization.

Int J Biol Macromol 2019 Apr 24;126:899-907. Epub 2018 Dec 24.

Institute of Molecular Biology and Genetics, NAS of Ukraine, 150, Zabolotnogo St., 03680 Kyiv, Ukraine.

Translation elongation factor 1Bβ (eEF1Bβ) is a metazoan-specific protein involved into the macromolecular eEF1B complex, containing also eEF1Bα and eEF1Bγ subunits. Both eEF1Bα and eEF1Bβ ensure the guanine nucleotide exchange on eEF1A while eEF1Bγ is thought to have a structural role. The structures of the eEF1Bβ catalytic C-terminal domain and neighboring central acidic region are known while the structure of the protein-binding N-terminal domain remains unidentified which prevents clear understanding of architecture of the eEF1B complex. Here we show that the N-terminal domain comprising initial 77 amino acids of eEF1Bβ, eEF1Bβ(1-77), is a monomer in solution with increased hydrodynamic volume. This domain binds eEF1Bγ in equimolar ratio. The CD spectra reveal that the secondary structure of eEF1Bβ(1-77) consists predominantly of α-helices and a portion of disordered region. Very rapid hydrogen/deuterium exchange for all eEF1Bβ(1-77) peptides favors a flexible tertiary organization of eEF1Bβ(1-77). Computational modeling of eEF1Bβ(1-77) suggests several conformation states each composed of three α-helices connected by flexible linkers. Altogether, the data imply that the protein-binding domain of eEF1Bβ shows flexible spatial organization which may be needed for interaction with eEF1Bγ or other protein partners.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.12.220DOI Listing
April 2019

Cyclosporine Metabolites' Metabolic Ratios May Be Markers of Cardiovascular Disease in Kidney Transplant Recipients Treated with Cyclosporine A-Based Immunosuppression Regimens.

Cardiovasc Toxicol 2019 06;19(3):255-263

Department of Immunology, Transplant Medicine and Internal Diseases, Medical University of Warsaw, 59 Nowogrodzka St, Warsaw, Poland.

Cardiovascular disease (CVD) remains one of the primary causes of death after kidney transplantation (KTX). Cyclosporine (CsA) metabolites may play a role in CVD. Metabolic ratio (MR) may be considered a measure of intra-individual differences of CsA metabolism. The study was aimed at analysis of associations of CVD with indices of CsA metabolism: MRs and dose-adjusted CsA concentrations (C/D and C/D/kg). The study was performed in the Department of Immunology, Transplant Medicine, and Internal Diseases of the Medical University of Warsaw and involved 102 KTX recipients. Whole blood concentrations of cyclosporine A, AM1, AM9, AM4N, demethylcarboxylated (dMC-CsA), dihydroxylated (DiH-CsA), trihydroxylated (TriH-CsA) cyclosporine metabolites were determined by liquid chromatography coupled with tandem mass spectrometry. Lower AM9/CsA were observed in diabetics. Patients with coronary disease and/or myocardial infarction had lower dMC-CsA/CsA and higher AM4N/CsA. Supraventricular arrhythmia (SVA) was associated with higher AM1/CsA and AM4N/CsA. Hypertriglyceridemia (hTG) was associated with lower AM9/CsA, higher C/D and C/D/kg. Decrease of AM9/CsA and AM4N and higher D/C were associated with overweight/obesity. Systolic blood pressure (BP) positively correlated with dMC-CsA/CsA and negatively with C/D/kg. Diastolic BP correlated positively with AM1/CsA, dMC-CsA/CsA, DiH-CsA/CsA and TriH-CsA/CsA. We have demonstrated the association of coronary disease/myocardial infarction, SVA, hTG, overweight/obesity and elevated arterial BP with higher MRs of AM1, AM4N, dMC-CsA, DiH-CsA and TriH-CsA, and lower MRs of AM9, which may indicate deleterious and favourable effects of individual CsA metabolites on cardiovascular system and suggest engagement of specific enzymatic pathways.
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http://dx.doi.org/10.1007/s12012-018-9492-9DOI Listing
June 2019

Fine-tuning of the stability of β-strands by Y181 in perfringolysin O directs the prepore to pore transition.

Biochim Biophys Acta Biomembr 2019 01 21;1861(1):110-122. Epub 2018 Aug 21.

Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland; Institute of Genetics and Biotechnology, Department of Biology, Warsaw University, 1 Miecznikowa St., 02-185 Warsaw, Poland. Electronic address:

Perfringolysin O (PFO) is a toxic protein that forms β-barrel transmembrane pores upon binding to cholesterol-containing membranes. The formation of lytic pores requires conformational changes in PFO that lead to the conversion of water-soluble monomers into membrane-bound oligomers. Although the general outline of stepwise pore formation has been established, the underlying mechanistic details await clarification. To extend our understanding of the molecular mechanisms that control the pore formation, we compared the hydrogen-deuterium exchange patterns of PFO with its derivatives bearing mutations in the D3 domain. In the case of two of these mutations F318A, Y181A, known from previous work to lead to a decreased lytic activity, global destabilization of all protein domains was observed in their water-soluble forms. This was accompanied by local changes in D3 β-sheet, including unexpected stabilization of functionally important β1 strand in Y181A. In case of the double mutation (F318A/Y181A) that completely abolished the lytic activity, several local changes were retained, but the global destabilization effects of single mutations were reverted and hydrogen-deuterium exchange (HDX) pattern returned to PFO level. Strong structural perturbations were not observed in case of remaining variants in which other residues of the hydrophobic core of D3 domain were substituted by alanine. Our results indicate the existence in PFO of a well-tuned H-bonding network that maintains the stability of the D3 β-strands at appropriate level at each transformation step. F318 and Y181 moieties participate in this network and their role extends beyond their direct intermolecular interaction during oligomerization that was identified previously.
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http://dx.doi.org/10.1016/j.bbamem.2018.08.008DOI Listing
January 2019
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