Leonid Breydo | University of South Florida (original) (raw)

Papers by Leonid Breydo

Biochemistry, Jan 6, 2015

We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the fol... more We examined the effects of water-soluble polymers of various degrees of hydrophobicity on the folding and aggregation of proteins. The polymers we chose were polyethylene glycol (PEG) and UCON (1:1 copolymer of ethylene glycol and propylene glycol). The presence of additional methyl groups in UCON makes it more hydrophobic than PEG. Our earlier analysis revealed that similarly sized PEG and UCON produced different changes in the solvent properties of water in their solutions and induced morphologically different α-synuclein aggregates [Ferreira, L. A., et al. (2015) Role of solvent properties of aqueous media in macromolecular crowding effects. J. Biomol. Struct. Dyn., in press]. To improve our understanding of molecular mechanisms defining behavior of proteins in a crowded environment, we tested the effects of these polymers on secondary and tertiary structure and aromatic residue solvent accessibility of 10 proteins [five folded proteins, two hybrid proteins; i.e., protein contain...

Molecular neurobiology, Jan 2, 2015

Protein aggregation is involved in a variety of diseases. Alteration of the aggregation pathway, ... more Protein aggregation is involved in a variety of diseases. Alteration of the aggregation pathway, either to produce less toxic structures or to increase aggregate clearance, is a promising therapeutic route. Both active and passive immunization has been used for this purpose. However, the mechanism of action of antibodies on protein aggregates is not completely clear especially given poor ability of antibodies to cross blood-brain barrier. Here, we have shown that antibodies can interfere with protein aggregation at substoichiometric concentrations (as low as 1:1000 antibody to protein ratio). This is an indication that antibodies interact with aggregation intermediates in chaperone-like manner altering the aggregation pathways at very low antibody levels. This observation supports earlier suggestions that antibodies can inhibit aggregation by interaction with low abundance aggregation intermediates.

Molecules (Basel, Switzerland), 2015

Macromolecular crowding is known to affect protein folding, binding of small molecules, interacti... more Macromolecular crowding is known to affect protein folding, binding of small molecules, interaction with nucleic acids, enzymatic activity, protein-protein interactions, and protein aggregation. Although for a long time it was believed that the major mechanism of the action of crowded environments on structure, folding, thermodynamics, and function of a protein can be described in terms of the excluded volume effects, it is getting clear now that other factors originating from the presence of high concentrations of "inert" macromolecules in crowded solution should definitely be taken into account to draw a more complete picture of a protein in a crowded milieu. This review shows that in addition to the excluded volume effects important players of the crowded environments are viscosity, perturbed diffusion, direct physical interactions between the crowding agents and proteins, soft interactions, and, most importantly, the effects of crowders on solvent properties.

Biochemistry, Jan 23, 2007

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttr... more In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with an N-myristoylamidomaleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) i...

Biochemistry, 2007

Amyloid plaques are hallmark neuropathological lesions in Alzheimer's disease, which consist of a... more Amyloid plaques are hallmark neuropathological lesions in Alzheimer's disease, which consist of abnormally aggregated A protein. Multiple A aggregated species have been identified, and neurotoxicity appears to be correlated with the amount of nonfibrillar oligomers. Therefore, selective inhibition of A oligomer formation has emerged as an attractive means of therapeutic intervention. To investigate whether small molecules can modulate aggregation to achieve selective inhibition of neurotoxic amyloid oligomers, A aggregation was assayed in vitro in the presence of methylene blue, using immunoreactivity with the prefibrillar oligomer-specific antibody A11, transmission electron microscopy, and turbidity assays. Methylene blue inhibited oligomerization when used at substoichiometric concentrations relative to that of the A monomer. Inhibition of A oligomerization was achieved concomitant with promotion of fibrillization, suggesting that oligomer and fibril formation are distinct and competing pathways. Methylene blue-mediated promotion of fiber formation occurred via a dose-dependent decrease in the lag time and an increase in the fibrillization rate, consistent with promotion of both filament nucleation and elongation. Addition of methylene blue to preformed oligomers resulted in oligomer loss and promotion of fibrillization. The data show that A oligomer formation is inhibited by promoting fibril formation, which suggests that the relative pathological significance of oligomers and fibrils may be tested in vivo using methylene blue. If A oligomers represent the primary pathogenic species, then inhibition of this highly toxic species via promotion of formation of less toxic aggregates may be therapeutically useful.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2014

a b s t r a c t 9 resulting from either loss-of-function or gain-of-function mutations in the 49 ... more a b s t r a c t 9 resulting from either loss-of-function or gain-of-function mutations in the 49 human erythroid specific gene, ALAS2, can lead to X-linked sideroblastic 50 anemia and X-linked dominant protoporphyria, respectively . 51 Murine erythroid ALAS (henceforth abbreviated as mALAS2) is a 52 homodimer with a molecular weight of 112 kDa [5]. Each of its two 53 active sites is located at the dimeric interface, and is composed of 54 amino acids from the individual monomers [6]. Pyridoxal-5′phosphate 55 (PLP), which serves as a cofactor during catalysis, is covalently attached 56 to an active site lysine (K313 in mALAS2), forming an internal aldimine 57 [7]. The absorbance spectrum of mALAS2 in the absence of substrates 58 displays maxima at 330 and 420 nm, representing different tautomeric 59 species of the internal aldimine [8]. Previously, we assigned the 420 nm-60 absorbance maximum to a ketoenamine species, and we utilized fluo-61 rescence spectroscopy to assign the 330 nm maximum to a substituted 62 aldamine [8]. Moreover, positive dichroic bands with maxima at ap-63 proximately 330 and 420 nm are detected in the circular dichroism 64 (CD) spectrum of mALAS2 [9,10], and since PLP by itself is not a chiral 65 molecule [10], the two maxima indicate an equilibrium between two 66 populations of internal aldimine species with different chiral active 67 site environments. 68 The catalytic activity of mALAS2, monitored under steady and pre-69 steady state conditions, exhibits dependence on pH [8,11]. Specifically, 70 an increase in alkalinity is accompanied by a decrease in the k cat value 71 [8] and a decrease in the pre-steady state rate formation of the second 72 quinonoid intermediate [11]. Temperature also affects the catalytic 73 turnover of mALAS2. In fact, when the temperature changes from 20 to 74 37°C, the k cat value is increased from 0.02 s −1 [12] to 0.6 s −1 , as deter-75 mined using a continuous, coupled, enzymatic assay [13]. The tempera-76 ture at which 50% of the enzymatic activity of mALAS2 is lost was Biochimica et Biophysica Acta xxx (2014) xxx-xxx ⁎ Please cite this article as: B.M. Stojanovski, et al., Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinat..., Biochim. Biophys. Acta (2014), http://dx.

Journal of Biomolecular Structure and Dynamics, 2015

Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volum... more Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volume effect is not the only factor affecting the behavior of biomolecules in a crowded environment. The observed inconsistencies are commonly explained by the so-called soft interactions, such as electrostatic, hydrophobic, and van der Waals interactions, between the crowding agent and the protein, in addition to the hard nonspecific steric interactions. We suggest that the changes in the solvent properties of aqueous media induced by the crowding agents may be the root of these "soft" interactions. To check this hypothesis, the solvatochromic comparison method was used to determine the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions of different polymers (dextran, poly(ethylene glycol), Ficoll, Ucon, and polyvinylpyrrolidone) with the polymer concentration up to 40% typically used as crowding agents. Polymer-induced changes in these features were found to be polymer type and concentration specific, and, in case of polyethylene glycol (PEG), molecular mass specific. Similarly sized polymers PEG and Ucon producing different changes in the solvent properties of water in their solutions induced morphologically different α-synuclein aggregates. It is shown that the crowding effects of some polymers on protein refolding and stability reported in the literature can be quantitatively described in terms of the established solvent features of the media in these polymers solutions. These results indicate that the crowding agents do induce changes in solvent properties of aqueous media in crowded environment. Therefore, these changes should be taken into account for crowding effect analysis.

Methods in Molecular Biology, 2008

Neurobiology of Disease, 2014

Molecular Neurodegeneration, 2012

Background: It is well established that vaccination of humans and transgenic animals against fibr... more Background: It is well established that vaccination of humans and transgenic animals against fibrillar Aβ prevents amyloid accumulation in plaques and preserves cognitive function in transgenic mouse models. However, autoimmune side effects have halted the development of vaccines based on full length human Aβ. Further development of an effective vaccine depends on overcoming these side effects while maintaining an effective immune response.

Protein Science, 2005

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-2... more In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP Sc produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive.

Protein Science, 2006

Despite possessing a common cross-b core, amyloid fibrils are known to exhibit great variations i... more Despite possessing a common cross-b core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.

Neuroscience Research, 2011

Molecular Neurobiology, 2013

Epidemiological, population-based case-control, and experimental studies at the molecular, cellul... more Epidemiological, population-based case-control, and experimental studies at the molecular, cellular, and organism levels revealed that exposure to various environmental agents, including a number of structurally different agrochemicals, may contribute to the pathogenesis of Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) and several other neurodegenerative disorders. The role of genetic predisposition in PD has also been increasingly acknowledged, driven by the identification of a number of disease-related genes [e.g., α-synuclein, parkin, DJ-1, ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1), and nuclear receptor-related factor 1]. Therefore, the etiology of this multifactorial disease is likely to involve both genetic and environmental factors. Various neurotoxicants, including agrochemicals, have been shown to elevate the levels of α-synuclein expression in neurons and to promote aggregation of this protein in vivo. Many agrochemicals physically interact with α-synuclein and accelerate the fibrillation and aggregation rates of this protein in vitro. This review analyzes some of the aspects linking α-synuclein to PD, provides brief structural and functional descriptions of this important protein, and represents some data connecting exposure to agrochemicals with α-synuclein aggregation and PD pathogenesis.

Molecular BioSystems, 2013

In an aqueous two-phase system (ATPS), the partitioning of a protein is defined by the differenti... more In an aqueous two-phase system (ATPS), the partitioning of a protein is defined by the differential interactions of the protein with aqueous media in the two phases. Our study shows that partitioning of proteins in a set of ATPSs of different ionic compositions can be used to quantify structural differences between a-synuclein, its variants and several globular proteins. Since application of ATPSs implies the use of high concentrations of two polymers in water when a certain threshold concentration of the polymers is exceeded, and since these levels of polymer concentrations are similar to those commonly used to mimic the effects of macromolecular crowding on proteins, we used circular dichroism spectroscopy to evaluate the structural consequences of placing proteins in solutions with high polymer concentrations and various ionic compositions.

Molecular BioSystems, 2014

Sup35 protein (Sup35p), or eukaryotic peptide chain release factor GTP binding subunit (eRF3), is... more Sup35 protein (Sup35p), or eukaryotic peptide chain release factor GTP binding subunit (eRF3), is a wellknown yeast prion responsible for the characteristic [PSI + ] trait. N-and M-domains of this protein have been the foci of intensive research due to their importance for the prion formation. Sup35p C-terminal domain (Sup35pC) is essential for translation termination and cell viability. Deletion of Sup35pC was shown to lead to malformation of cells during mitosis. In this study we confirm that Sup35pC domain possesses high sequence and structural similarity to the eukaryotic translation elongation factor 1-a (eEF1A) from yeast and show that its sequence is conserved across different species including human.

Metallomics, 2011

Neurodegenerative diseases constitute a set of pathological conditions originating from the slow,... more Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible, and systematic cell loss within the various regions of the brain and/or the spinal cord. Depending on the affected region, the outcomes of the neurodegeneration are very broad and diverse, ranging from the problems with movements to dementia. Some neurodegenerative diseases are associated with protein misfolding and aggregation. Many proteins that misfold in human neurodegenerative diseases are intrinsically disordered; i.e., they lack a stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) functionally complement ordered proteins, being typically involved in regulation and signaling. There is accumulating evidence that altered metal homeostasis may be related to the progression of neurodegenerative diseases. This review examines the effects of metal ion binding on the aggregation pathways of IDPs found in neurodegenerative diseases.

Journal of the American Chemical Society, 2011

Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxi... more Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNAintercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 10 3 M −1 . Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allows construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA. Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. 1-9 Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. 2,8-10 For example, studies of DNA-damaging natural products such as the bleomycins, 11-16 the azinomycins, 17-19 acylfulvenes, 20 fecapentaenes, 21 resorcinols, 22 coumarins, 23 mitomycin C, 24,25 CC-1065, 26 duocarmycin, 26 aflatoxin B 1 , 27,28 pluramycin, 29 neocarzinostatin, 30 calicheamicin, 31-34 fasicularin, 35 and ecteinascidin 743 36 have provided a wealth of insight regarding the chemical and biological mechanisms underlying the cytostatic, cytotoxic, and mutagenic properties of DNA-damaging agents.