| 1. |
- Dahlman, Anna, et al.
(författare)
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Thrombin-derived C-terminal peptide reduces Candida-induced inflammation and infection in vitro and in vivo
- 2021
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804. ; 65:11
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Tidskriftsartikel (refereegranskat)abstract
- Infections due to the opportunistic fungus Candida have been on the rise in the last decades, especially in immunocompromised individuals and hospital settings. Unfortunately, the treatments available today are limited. Thrombin-derived C-terminal peptide (TCP-25) is an antimicrobial peptide (AMP) with antibacterial and immunomodulatory effects. In this work, we, for the first time, demonstrate the ability of TCP-25 ability to counteract Candida in vitro and in vivo. Using a combination of viable count assay (VCA), radial diffusion assay (RDA), and fluorescence and transmission electron microscopy analyses, TCP-25 was found to exert a direct fungicidal activity. An inhibitory activity of TCP-25 on NF-kB activation induced by both zymosan alone and heat-killed C. albicans was demonstrated in vitro using THP-1 cells, and in vivo using NF-kB reporter mice. Moreover, the immunomodulatory property of TCP-25 was further substantiated in vitro by analyzing cytokine responses in human blood stimulated with zymosan, and in vivo employing a zymosan-induced peritonitis model in C57BL/6 mice. The therapeutic potential of TCP-25 was demonstrated in mice infected with luminescent C. albicans. Finally, the binding between TCP-25 and zymosan was investigated using circular dichroism spectroscopy and intrinsic fluorescence analysis. Taken together, our results show that TCP-25 has a dual function by inhibiting Candida as well as the associated zymosan-induced inflammation. The latter function is accompanied by a change in secondary structure upon binding to zymosan. TCP-25, therefore, shows promise as a novel drug candidate against Candida infections.
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| 2. |
- Dalla-Riva, Jonathan, et al.
(författare)
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Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle.
- 2013
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 54:5, s. 1275-1282
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Tidskriftsartikel (refereegranskat)abstract
- Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined if nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and if a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain specific peptides of apoA-I showed that the lipid-free C-terminal 190-243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190-243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. JLR Discoidal HDL and the 190-243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle and the C-terminal α-helical content of apoA-I may be an important determinant of this effect.
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| 3. |
- Dalla-Riva, Jonathan, et al.
(författare)
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Structural and Functional Analysis of the ApolipoproteinA-I A164S Variant
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:11, s. e0143915-e0143915
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Tidskriftsartikel (refereegranskat)abstract
- There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature ' s own anti-infective principles.
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| 4. |
- den Hartigh, Laura J., et al.
(författare)
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Postprandial apoE Isoform and Conformational Changes Associated with VLDL Lipolysis Products Modulate Monocyte Inflammation
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:11
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. Methods and Results: We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112 -> Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNF alpha secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. Conclusion: Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.
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| 5. |
- Hartman, Erik, et al.
(författare)
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Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Wound infection is a common and serious medical condition with an unmet need for improved diagnostic tools. A peptidomic approach, aided by mass spectrometry and bioinformatics, could provide novel means of identifying new peptide biomarkers for wound healing and infection assessment. Wound fluid is suitable for peptidomic analysis since it is both intimately tied to the wound environment and is readily available. In this study we investigate the peptidomes of wound fluids derived from surgical drainages following mastectomy and from wound dressings following facial skin grafting. By applying sorting algorithms and open source third party software to peptidomic label free tandem mass spectrometry data we provide an unbiased general methodology for analyzing and differentiating between peptidomes. We show that the wound fluid peptidomes of patients are highly individualized. However, differences emerge when grouping the patients depending on wound type. Furthermore, the abundance of peptides originating from documented antimicrobial regions of hemoglobin in infected wounds may contribute to an antimicrobial wound environment, as determined by in silico analysis. We validate our findings by compiling literature on peptide biomarkers and peptides of physiological significance and cross checking the results against our dataset, demonstrating that well-documented peptides of immunological significance are abundant in infected wounds, and originate from certain distinct regions in proteins such as hemoglobin and fibrinogen. Ultimately, we have demonstrated the power using sorting algorithms and open source software to help yield insights and visualize peptidomic data.
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| 6. |
- Hartman, Erik, et al.
(författare)
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Peptide clustering enhances large-scale analyses and reveals proteolytic signatures in mass spectrometry data
- 2024
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Ingår i: Nature Communications. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Recent advances in mass spectrometry-based peptidomics have catalyzed the identification and quantification of thousands of endogenous peptides across diverse biological systems. However, the vast peptidomic landscape generated by proteolytic processing poses several challenges for downstream analyses and limits the comparability of clinical samples. Here, we present an algorithm that aggregates peptides into peptide clusters, reducing the dimensionality of peptidomics data, improving the definition of protease cut sites, enhancing inter-sample comparability, and enabling the implementation of large-scale data analysis methods akin to those employed in other omics fields. We showcase the algorithm by performing large-scale quantitative analysis of wound fluid peptidomes of highly defined porcine wound infections and human clinical non-healing wounds. This revealed signature phenotype-specific peptide regions and proteolytic activity at the earliest stages of bacterial colonization. We validated the method on the urinary peptidome of type 1 diabetics which revealed potential subgroups and improved classification accuracy.
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| 7. |
- Holdbrook, Daniel A., et al.
(författare)
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Influence of pH on the activity of thrombin-derived antimicrobial peptides
- 2018
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1860:11, s. 2374-2384
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Tidskriftsartikel (refereegranskat)abstract
- The wound environment is characterized by physiological pH changes. Proteolysis of thrombin by wound-derived proteases, such as neutrophil elastase, generates antimicrobial thrombin-derived C-terminal peptides (TCPs), such as HVF18 (HVFRLKKWIQKVIDQFGE). Presence of such TCPs in human wound fluids in vivo, as well as the occurrence of an evolutionarily conserved His residue in the primary amino acid sequence of TCPs, prompted us to investigate the pH-dependent antibacterial action of HVF18, as well as of the prototypic GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE). We show that protonation of this His residue at pH 5.5 increases the antibacterial activity of both TCPs against Gram-negative Escherichia coli by membrane disruption. Physiological salt level (150 mM NaCl) augments antibacterial activity of GKY25 but diminishes for the shorter HVF18. Replacing His with Leu or Ser in GKY25 abolishes the His protonation-dependent increase in antibacterial activity at pH 5.5, whereas substitution with Lys maintains activity at neutral (pH 7.4) and acidic pH. Interestingly, both TCPs display decreased binding affinities to human CD14 with decreasing pH, suggesting a likely switch in mode-of-action, from anti-inflammatory at neutral pH to antibacterial at acidic pH. Together, the results demonstrate that apart from structural prerequisites such as peptide length, charge, and hydrophobicity, the evolutionarily conserved His residue of TCPs influences their antibacterial effects and reveals a previously unknown aspect of TCPs biological action.
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| 8. |
- Lagerstedt, Jens, et al.
(författare)
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EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels.
- 2013
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Ingår i: Contrast Media & Molecular Imaging. - : Wiley. - 1555-4317 .- 1555-4309. ; 8:3, s. 252-264
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Tidskriftsartikel (refereegranskat)abstract
- We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe. Copyright © 2013 John Wiley & Sons, Ltd.
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| 9. |
- Lindahl, Maria, et al.
(författare)
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ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.
- 2015
-
Ingår i: Journal of Lipid Research. - 1539-7262. ; 56:12, s. 2248-2259
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Tidskriftsartikel (refereegranskat)abstract
- ApoA-I, the main protein component of high-density lipoprotein (HDL), is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of β-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA-1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-β-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/PKA signaling pathway.
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| 10. |
- Liu, Yang, et al.
(författare)
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Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing
- 2023
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Ingår i: Acta Biomaterialia. - : Elsevier BV. - 1878-7568 .- 1742-7061. ; 162, s. 164-181
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Tidskriftsartikel (refereegranskat)abstract
- Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.
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| 11. |
- Oda, Michael N., et al.
(författare)
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The secondary structure of apolipoprotein A-I on 9.6-nm reconstituted high-density lipoprotein determined by EPR spectroscopy
- 2013
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:14, s. 3416-3424
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Tidskriftsartikel (refereegranskat)abstract
- ApolipoproteinA-I (ApoA-I) is the major protein component of high-density lipoprotein (HDL), and is critical for maintenance of cholesterol homeostasis. During reverse cholesterol transport, HDL transitions between an array of subclasses, differing in size and composition. This process requires ApoA-I to adapt to changes in the shape of the HDL particle, transiting from an apolipoprotein to a myriad of HDL subclass-specific conformations. Changes in ApoA-I structure cause alterations in HDL-specific enzyme and receptor-binding properties, and thereby direct the HDL particle through the reverse cholesterol transport pathway. In this study, we used site-directed spin label spectroscopy to examine the conformational details of the ApoA-I central domain on HDL. The motional dynamics and accessibility to hydrophobic/hydrophilic relaxation agents of ApoA-I residues99-163 on 9.6-nm reconstituted HDL was analyzed by EPR. In previous analyses, we examined residues6-98 and 164-238 (of ApoA-I's 243 residues), and combining these findings with the current results, we have generated a full-length map of the backbone structure of reconstituted HDL-associated ApoA-I. Remarkably, given that the majority of ApoA-I's length is composed of amphipathic helices, we have identified nonhelical residues, specifically the presence of a -strand (residues149-157). The significance of these nonhelical residues is discussed, along with the other features, in the context of ApoA-I function in contrast to recent models derived by other methods.
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| 12. |
- Petrlova, Jitka, et al.
(författare)
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Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:21, s. E4213-E4222
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Tidskriftsartikel (refereegranskat)abstract
- Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation.
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| 13. |
- Petrlova, Jitka, et al.
(författare)
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Conformational and aggregation properties of the 1-93 fragment of apolipoprotein A-I
- 2014
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 23:11, s. 71-1559
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Tidskriftsartikel (refereegranskat)abstract
- Several disease-linked mutations of apolipoprotein A-I, the major protein in high-density lipoprotein (HDL), are known to be amyloidogenic, and the fibrils often contain N-terminal fragments of the protein. Here, we present a combined computational and experimental study of the fibril-associated disordered 1-93 fragment of this protein, in wild-type and mutated (G26R, S36A, K40L, W50R) forms. In atomic-level Monte Carlo simulations of the free monomer, validated by circular dichroism spectroscopy, we observe changes in the position-dependent β-strand probability induced by mutations. We find that these conformational shifts match well with the effects of these mutations in thioflavin T fluorescence and transmission electron microscopy experiments. Together, our results point to molecular mechanisms that may have a key role in disease-linked aggregation of apolipoprotein A-I.
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| 14. |
- Petrlova, Jitka, et al.
(författare)
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Molecular crowding impacts the structure of apolipoprotein A-I with potential implications on in vivo metabolism and function
- 2016
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Ingår i: Biopolymers. - : Wiley. - 0006-3525. ; 105:10, s. 683-692
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Tidskriftsartikel (refereegranskat)abstract
- The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin–spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed.
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| 15. |
- Petrlova, Jitka, et al.
(författare)
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SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide
- 2022
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 596:19, s. 2566-2575
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein–LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.
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| 16. |
- Petrlova, Jitka, et al.
(författare)
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Secondary Structure Changes in ApoA-I Milano (R173C) Are Not Accompanied by a Decrease in Protein Stability or Solubility.
- 2014
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL) and a principal mediator of the reverse cholesterol transfer pathway. Variants of apoA-I have been shown to be associated with hereditary amyloidosis. We previously characterized the G26R and L178H variants that both possess decreased stability and increased fibril formation propensity. Here we investigate the Milano variant of apoAI (R173C; apoAI-M), which despite association with low plasma levels of HDL leads to low prevalence of cardiovascular disease in carriers of this mutation. The R173C substitution is located to a region (residues 170 to 178) that contains several fibrillogenic apoA-I variants, including the L178H variant, and therefore we investigated a potential fibrillogenic property of the apoAI-M protein. Despite the fact that apoAI-M shared several features with the L178H variant regarding increased helical content and low degree of ThT binding during prolonged incubation in physiological buffer, our electron microscopy analysis revealed no formation of fibrils. These results suggest that mutations inducing secondary structural changes may be beneficial in cases where fibril formation does not occur.
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| 17. |
- Petrlova, Jitka, et al.
(författare)
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Selective protein aggregation confines and inhibits endotoxins in wounds : Linking host defense to amyloid formation
- 2023
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 26:10
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.
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| 18. |
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| 19. |
- Petrlova, Jitka, et al.
(författare)
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The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils.
- 2012
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 53:3, s. 390-398
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Tidskriftsartikel (refereegranskat)abstract
- A number of amyloidogenic variants of apolipoprotein A-I (apoA-I) have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ~90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposit. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to WT protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37oC with a t1/2 of about 12 days. Upon prolonged incubation the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. JLR These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.
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| 20. |
- Petrlova, Jitka, et al.
(författare)
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Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products
- 2020
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 295:11, s. 3417-3430
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.
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| 21. |
- Petrlova, Jitka, et al.
(författare)
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Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products
- 2020
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Ingår i: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 295:11, s. 3417-3430
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.
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| 22. |
- Petruk, Ganna, et al.
(författare)
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Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25.
- 2020
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.
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| 23. |
- Petruk, Ganna, et al.
(författare)
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SARS-CoV-2 Spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity
- 2020
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Ingår i: Journal of Molecular Cell Biology. - : Oxford University Press (OUP). - 1759-4685. ; 12:12, s. 916-932
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Tidskriftsartikel (refereegranskat)abstract
- There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.
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| 24. |
- Petruk, Ganna, et al.
(författare)
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Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs
- 2023
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Ingår i: Nature Communications. - : Springer. - 2041-1723. ; 14, s. 1-20
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Tidskriftsartikel (refereegranskat)abstract
- There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
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| 25. |
- Petruk, Ganna, et al.
(författare)
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The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins
- 2021
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Ingår i: Journal of Lipid Research. - 0022-2275. ; 62
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Tidskriftsartikel (refereegranskat)abstract
- ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxin components such as lipopolysaccharide (LPS) and lipid A, yielding similar increases in the apoE α-helical content. Moreover, high-molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with lipid A. Together, our results demonstrate the ability of apoE to kill Gram-negative bacteria, interact with their endotoxins, which leads to the structural changes in apoE and the formation of aggregate-like complexes.
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