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Babenko V.,University of Warsaw | Surmacz-Chwedoruk W.,Institute of Biotechnology and Antibiotics | Dzwolak W.,University of Warsaw
Langmuir | Year: 2015

Formation of amyloid fibrils is often facilitated in the presence of specific charge-compensating ions. Dissolved sodium chloride is known to accelerate insulin fibrillation at low pH that has been attributed to the shielding of electrostatic repulsion between positively charged insulin molecules by chloride ions. However, the subsequent fate of Cl- anions; that is, possible entrapment within elongating fibrils or escape into the bulk solvent, remains unclear. Here, we show that, while the presence of NaCl at the onset of insulin aggregation induces structural variants of amyloid with distinct fingerprint infrared features, a delayed addition of salt to fibrils that have been already formed in its absence and under quiescent conditions triggers a "condensation effect": amyloid superstructures with strong chiroptical properties are formed. Chloride ions appear to stabilize these superstructures in a manner similar to stabilization of DNA condensates by polyvalent cations. The concentration of residual chloride ions trapped within bovine insulin fibrils grown in 0.1 M NaCl, at pD 1.9, and rinsed extensively with water afterward is less than 1 anion per 16 insulin monomers (as estimated using ion chromatography) implying absence of defined solvent-sequestered nesting sites for chloride counterions. Our results have been discussed in the context of mechanisms of insulin aggregation. © 2015 American Chemical Society. Source

Dzwolak W.,University of Warsaw | Surmacz-Chwedoruk W.,Institute of Biotechnology and Antibiotics | Surmacz-Chwedoruk W.,Polish Academy of Sciences | Babenko V.,University of Warsaw
Langmuir | Year: 2013

Formation of amyloid fibrils is often associated with intriguing far-from-equilibrium phenomena such as conformational memory effects or flow-driven self-assembly. Insulin is a model amyloidogenic polypeptide forming distinct structural variants of fibrils, which self-propagate through seeding. According to infrared absorption, fibrils from bovine insulin ([BI]) and LysB31-ArgB32 human insulin analogue ([KR]) cross-seed each other and imprint distinct structural features in daughter fibrils. In the absence of preformed [KR] amyloid seeds, bovine insulin agitated at 60 C converts into chiral amyloid superstructures exhibiting negative extrinsic Cotton effect in bound thioflavin T. However, when agitated bovine insulin is simultaneously cross-seeded with [KR] amyloid, daughter fibrils reveal a positive extrinsic Cotton effect. Our study indicates that dramatic changes in global properties of amyloid superstructures may emerge from subtle conformational-level variations in single fibrils (e.g., alignment and twist of β-strands) that are encoded by memory effects. © 2012 American Chemical Society. Source

Surmacz-Chwedoruk W.,Institute of Biotechnology and Antibiotics | Surmacz-Chwedoruk W.,Polish Academy of Sciences | Nieznanska H.,Nencki Institute of Experimental Biology | Wojcik S.,University of Warsaw | Dzwolak W.,University of Warsaw
Biochemistry | Year: 2012

The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, determines the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant LysB31- ArgB32 human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the infrared amide I′ band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption maximum, and even fine fingerprint features of the amide I′ band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the LysB31-ArgB32 analog of human insulin lie outside of the polypeptides critical amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermolecular salt bridges, and the susceptibility to dissection and denaturation with dimethyl sulfoxide (DMSO). All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Analysis of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis. © 2012 American Chemical Society. Source

Saczynska V.,Institute of Biotechnology and Antibiotics
Acta Biochimica Polonica | Year: 2014

Recombinant subunit vaccines based on hemagglutinin proteins produced in bacteria (bacterial HAs) are promising candidates for enhancing the supply of vaccines against influenza, especially for a pandemic. Over 20 years after the failure to obtain the antigen with native HA characteristics in the early 1980's, there are increasing data on successful production of HA proteins in bacteria. The vast majority of bacterial HAs have been based on the HA1 subunit of HA expressed separately or as a component of conjugate vaccines, but those based on the ectodomain and the HA2 subunit have also been reported. The most of HAs have been efficiently expressed as insoluble aggregates called inclusion bodies. Refolded and purified proteins were extensively studied for structure, the ability to bind to sialic acid-containing receptors, antigenicity, immunogenicity and efficacy. The results from these studies contradict the view that glycosylation determines the correct structure of the hemagglutinin, as they proved that bacterial HAs can be valuable vaccine antigens when appropriate folding and purification methods are applied to rationally designed proteins. The best evidence for success in bacterial production of protective HA is that vaccines based on proprietary Toll-like Receptor (VaxInnate) and bacteriophage Qß-VLPs (Cytos Biotechnology) technologies have been advanced to clinical studies. Source

Lukawska M.,Institute of Biotechnology and Antibiotics | Wietrzyk J.,Institute of Immunology and Experimental Therapy | Opolski A.,Institute of Immunology and Experimental Therapy | Oszczapowicz J.,University of Warsaw | Oszczapowicz I.,Institute of Biotechnology and Antibiotics
Investigational New Drugs | Year: 2010

Summary: Oxazolinodaunorubicin, a new daunorubicin derivative with a modified daunosamine moiety, was synthesized. The biological properties of this derivative and the parent daunorubicin were compared. The results showed antiproliferative activity of the derivative with significantly lower toxicity (an LD50 value ca. 20 times higher than that of parent daunorubicin) and an ability to completely overcome the resistance of cancer cells to this drug in vitro. Cardiotoxicity determination using male mice treated with a single dose of 75% of the LD50 value indicated that the cardiotoxicity of new analog was much lower than that of the parent drug. Preliminary results in transplanted murine tumor models revealed that a single-dose injection of the tested compounds exhibited antitumor activity in P388 and L1210 leukemia and 16/C mammary adenocarcinoma bearing mice. © 2009 Springer Science+Business Media, LLC. Source

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