Probiotic and technological characterization of selected Lactobacillus strains isolated from different egyptian cheeses.

BACKGROUND: Fresh milk and natural environmental conditions are used to produce traditional cheeses. Such cheeses are produced by dozens of different types of microbes. Non-starter lactobacilli are the most responsible genus of lactic acid bacteria exhibiting key technological and health promoting traits. The purpose of this study is to isolate Lactobacillus bacteria from conventional Egyptian cheeses and analyse their probiotic potential and technological properties. RESULTS: Lactobacillus isolates (33 isolates) were isolated from different Egyptian cheeses. Our results revealed that 18.18% of the isolates were fast-acidifying, 30.3% were medium-acidifying and 51.5% were slow-acidifying isolates. The results of autolytic activity showed that 24.3% of the isolates were good autolysis, 33.3% were fair autolysis, while 42.4% were poor autolysis. Fifteen isolates produced exopolysaccharides, while 9 isolates exhibited antimicrobial activities against Lactobacillus bulgaricus 340. All the isolates were resistant to pH 3 for 3 h except isolate No. 15 (MR4). The growth rate of the isolates ranged from 42.25 to 85.25% at 0.3% bile salts after 3 h of incubation. The surviving percentage of the Lactobacillus isolates decreased with increasing incubation time or the percentage of bile salts greater than 0.3%. All the isolates grew after incubation in artificial gastric and intestinal fluids. The auto-aggregation of 15 isolates ranged from 43.13 to 72.77%. Lacticaseibacillus paracasei BD3, Lactiplantibacillus plantarum BR4 and Limosilactobacillus fermentum MR2 were sensitive to the majority of the tested antibiotics and showed good bile salt hydrolase activity. CONCLUSION: L. paracasei BD3, L. plantarum BR4 and L. fermentum MR2 were isolated from Egyptian cheeses and showed probiotic and technological characterization, which are valuable for their practical application as starters, adjunct and protective cultures in cheese making.

Combined microwave processing and enzymatic proteolysis of bovine whey proteins: the impact on bovine ß-lactoglobulin allergenicity.

The main aim of this study was to develop a continuous microwave treatment system of whey proteins and then apply this process at 37 °C, 50 °C, 65 °C and 70 °C to achieve pepsinolysis and produce extensively hydrolysed bovine whey protein hydrolysates with low allergenic properties. The microwave process was compared to a conventional thermal treatment with similar temperature set points. Both processes were deeply analysed in terms of the thermal kinetics and operating conditions. The pepsin hydrolysates obtained by the continuous microwave treatment and conventional heating were characterized by SDS-PAGE and RP-HPLC. The allergenicity of the whey protein hydrolysates was explored using a human IgE sensitized rat basophil leukaemia cell assay. The results indicate that extensively hydrolysed whey protein hydrolysates were obtained by microwave only at 65 °C and in a shorter time compared with the conventional thermal treatment. In the same temperature conditions under conventional heating, ß-lactoglobulin was resistant to pepsinolysis, and 37% of it remained intact. As demonstrated by an in vitro degranulation assay using specific human IgE-sensitized rat basophils, the extensively hydrolysed whey protein obtained by microwave showed maximum degranulation values of 6.53% compared to those of the native whey protein isolate (45.97%) and hence elicited no more allergenic reactions in basophils. This work emphasizes the potential industrial use of microwave heating specific to milk protein processing to reduce their allergenicity and improve their end-use properties.

Inhibition of Staphylococcus aureus in vitro by bacteriocinogenic Lactococcus lactis KTH0-1S isolated from Thai fermented shrimp (Kung-som) and safety evaluation.

Lactococcus lactis KTH0-1S isolated from Thai traditional fermented shrimp (Kung-som) is able to produce heat-stable bacteriocin and inhibits food spoilage bacteria and food-borne pathogens. The inhibitory effect of bacteriocin remained intact after treatment with different pHs and after heating, but was sensitive to some proteolytic enzymes. Addition of bacteriocin KTH0-1S to Staphylococcus aureus cultures decreased viable cell counts by 2.8 log CFU/ml, demonstrating a bactericidal mode of action. Furthermore, the growth of S. aureus decreased significantly after 12-h co-cultivation with bacteriocinogenic strain. The molecular mass of bacteriocin KTH0-1S was found to be 3.346 kDa after ammonium sulfate precipitation, reversed phase (C8 Sep-Pak), cation-exchange chromatography, RP-HPLC on C8 column and mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z using PCR amplification and sequencing. The majority of tested virulence factors were absent, confirming the safety. Evidenced inhibitory effect of this strain, the absence of virulence factors creates the possibility for its application as protective culture to inhibit pathogenic bacteria in the several fermented seafood products.

ß-Lactoglobulin: An efficient nanocarrier for advanced delivery systems.

Thanks to the progress of nanotechnology there are several agent-delivery systems that can be selected to achieve rapid and specific delivery of a wide variety of biologically active agents. Consequently, the manipulation and engineering of biopolymers has become one of the most exciting subjects for those who study delivery systems on the nanoscale. In this regard, both nanoparticle formation and a carrier role have been observed in the case of the globular milk whey protein, ß-lactoglobulin (ß-LG), setting it apart from many other proteins. To date, many efforts adopting different approaches have created ß-LG nanoparticles useful in forming delivery systems for various agents with specific targets. In this review, the potential of ß-LG to play the role of an efficient and diverse carrier protein, as well as its ability to form a well-targeted nano-scale delivery system is discussed.

Inhibition of Prion Propagation by 3,4-Dimethoxycinnamic Acid.

Neurodegenerative diseases are associated with accumulation of amyloid-type protein misfolding products. Prion protein (PrP) is known for its ability to aggregate into soluble oligomers that in turn associate into amyloid fibrils. Preventing the formation of these infective and neurotoxic entities represents a viable strategy to control prion diseases. Numerous attempts to find dietary compounds with anti-prion properties have been made; however, the most promising agent found so far was curcumin, which is poorly soluble and merely bioavailable. In the present work, we identify 3,4-dimethoxycinnamic acid (DMCA) which is a bioavailable coffee component as a perspective anti-prion compound. 3,4-Dimethoxycinnamic acid was found to bind potently to prion protein with a Kd of 405 nM. An in vitro study of DMCA effect on PrP oligomerization and fibrillization was undertaken using isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and circular dichroism (CD) methodologies. We demonstrated that DMCA affects PrP oligomer formation reducing the oligomer content by 30-40%, and enhancing SH-SY5Y cell viability treated with prion oligomers. Molecular docking studies allowed to suggest a site where DMCA is able to bind stabilizing PrP tertiary structure. We suggest that DMCA is a perspective dietary compound for prophylaxis of neurodegenerative diseases that needs further research. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluation of marine bacteriocinogenic enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria.

Use of lactic acid bacteria (LAB) as probiotics may provide an alternative to the use of antibiotics in aquaculture. LAB strains isolated from wild fish viscera and skin were evaluated for bacteriocin production and safety aspects (lack of antibiotic resistance, production of virulence factors). 16S rRNA gene sequences revealed the presence of Enterococcus faecium (13 isolates) and Lactococcus lactis (3 isolates) from fish samples. Pulsed-field gel electrophoresis analyses of the 13 enterococci isolates showed that they were all clustered, with greater than 95% similarity. However, RAPD analysis revealed significant molecular diversity between enterococci strains. Six enterococci strains were chosen and evaluated for their antibacterial activities. These strains produced a bacteriocin-like substance and exhibited a broad spectrum of inhibition against pathogenic bacteria isolated from diseased fish, including Streptococcus parauberis, Vagococcus spp., and Carnobacterium maltaromaticum, and in particular against the Gram-negative bacteria Flavobacterium frigidarium, Vibrio pectenicida, V. penaeicida, and Photobacterium damselae. The inhibition activity towards bacterial indicator strains was at a maximum when bacteria were grown at 37°C. However, bacteriocin production was observed at 15°C after 12 h of incubation. Only structural genes of enterocins A and B were detected by PCR in the 6 enterococci strains, suggesting the production of these enterocins. In addition, these strains did not harbor any virulence factors or any significant antibiotic resistance, and they tolerated bile. Our results suggest that enterococci are an important part of the bacterial flora of fish and that some strains have the potential to be used as probiotics.

Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (ß-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Improving safety of salami by application of bacteriocins produced by an autochthonous Lactobacillus curvatus isolate.

The aims of this study were to isolate LAB with anti-Listeria activity from salami samples, characterize the bacteriocin/s produced by selected isolates, semi-purify them and evaluate their effectiveness for the control of Listeria monocytogenes during manufacturing of salami in a pilot scale. Two isolates (differentiated by RAPD-PCR) presented activity against 22 out of 23 L. monocytogenes strains for bacteriocin MBSa2, while the bacteriocin MBSa3 inhibited all 23 strains in addition to several other Gram-positive bacteria for both antimicrobials and were identified as Lactobacillus curvatus based on 16S rRNA sequencing. A three-step purification procedure indicated that both strains produced the same two active peptides (4457.9 Da and 4360.1 Da), homlogous to sakacins P and X, respectively. Addition of the semi-purified bacteriocins produced by Lb. curvatus MBSa2 to the batter for production of salami, experimentally contaminated with L. monocytogenes (10(4)-10(5) CFU/g), caused 2 log and 1.5 log reductions in the counts of the pathogen in the product after 10 and 20 days respectively, highlighting the interest for application of these bacteriocins to improve safety of salami during its manufacture.

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions).

BACKGROUND: It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). METHODS: Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein-polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity. RESULTS: Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH. CONCLUSIONS: The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications. GENERAL SIGNIFICANCE: Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.

Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.

The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes.

Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera.

Nine lactic acid bacteria strains showing bacteriocin-like activity were isolated from various fresh fish viscera. The following species were identified based on 16S rDNA sequences: Enterococcus durans (7 isolates), Lactococcus lactis (1) and Enterococcus faecium (1). These strains were active against Listeria innocua and other LAB. Random amplified polymorphic DNA analyses showed four major patterns for the E. durans species. PCR analyses revealed a nisin gene in the genome of the Lc. lactis strain. Genes coding enterocins A, B and P were found in the genome of the E. faecium isolate. Enterocins A and B genes were also present in the genome of E. durans GM19. Hence, this is the first report describing E. durans strains producing enterocins A and B. Electrospray ionization mass spectrometry revealed that the purified bacteriocin produced by the E. durans GMT18 strain had an exact molecular mass of 6,316.89 Da. This bacteriocin was designated as durancin GMT18. Edman sequencing failed to proceed; suggesting that durancin GTM18 may contain terminal lanthionine residues. Overall, the results obtained revealed the presence of a variety of enterococci in Mediterranean fish viscera, as evidenced by their genetic profiles and abilities to produce different bacteriocins. These strains could be useful for food biopreservation or as probiotics.

Antimicrobial and antifungal activities of Lactobacillus curvatus strain isolated from homemade Azerbaijani cheese.

The aims of this study were to characterize inhibitory activity spectra, some probiotic properties and safety of Lactobacillus curvatus A61 for its future application in production of fermented foods. The studied strain was isolated from traditional homemade cheese manufactured in Azerbaijan. The cell-free supernatant of culture of Lb. curvatus A61 inhibited the growth of tested LAB, as well as of Listeria monocytogenes and Bacillus cereus strains. The strain presented antifungal activity and inhibited the growth of Cladosporium and Fusarium ssp. during co-cultivation on agar media. PCR amplification with specific primers revealed the presence of curvacin A encoding gene in Lb. curvatus A61. Bacteriocin produced by the studied strain was heat stable and active in a broad pH range, and in the presence of Triton X-20, Triton X-80, Triton X-100, ß-mercaptoethanol, Na-EDTA, SDS and NaCl. The mode of action of bacteriocin against selected indicator strains was found to be bacteriostatic. Lb. curvatus A61 was resistant to physiological concentrations of bile salts and showed high auto-aggregation ability, as well as co-aggregation ability with pathogenic L. monocytogenes strains. It was sensitive to chloramphenicol, penicillin, tetracycline, ciprofloxacin and vancomycin, but resistant to ampicillin and gentamicin.

Impact of Enterococcus italicus ONU547 on the growth and acclimatization of micropropagated Rubus fruticosus L. and Paulownia tomentosa Steud. plants to ex vitro conditions.

Clonal micropropagation is an effective method for plant reproduction, applicable in both scientific and industrial domains. However, a significant number of microclones are lost during the ex vitro acclimatization process. To address this, the introduction of beneficial microorganisms into the rhizosphere of micropropagated plants could have a positive effect on the survival rates and external characteristics of acclimatized plantlets. The aim of this study was to determine the protective and growth-promoting potential of Enterococcus italicus ONU547 and its effect on micropropagated plants during acclimatization. The antagonistic activity of the bacteria was determined using the agar block method. Lepidium sativum L. seeds were inoculated with bacterial suspensions at concentrations of 106, 107, and 108 CFU/ml. Subsequently, the roots of the microclones were treated with suspensions of 106 and 107 CFU/ml, and biometric characteristics were measured. The results demonstrated antagonistic properties against various phytopathogenic fungi, including Aspergillus niger, Cladosporium cladosporioides, Alternaria alternata, Alternaria tenuissima, Rhizoctonia cerealis, Penicillium expansum, and Paecilomyces variotii. Inoculation of L. sativum L. seeds resulted in improved germination rates, increased root numbers, and enhanced root and shoot lengths. Similarly, the effects of the studied bacteria on Rubus fruticosus L. and Paulownia tomentosa Steud. during the acclimatization stage led to higher survival rates, increased shoot lengths, greater node numbers, and larger leaf areas. A concentration of 107 CFU/ml was identified as optimal for inoculating the microclones. The findings indicate that E. italicus ONU547 holds promise for the inoculation of micropropagated plants during the acclimatization process. Further research is recommended to establish the specific interaction mechanisms between these bacteria and plants.

Modulation of cytotoxic amyloid fibrillation and mitochondrial damage of α-synuclein by catechols mediated conformational changes.

The interplay between α-synuclein (α-syn) and catechols plays a central role in Parkinson's disease. This may be related to the modulating effects of catechols on the various aspects of α-syn fibrillization. Some of these effects may be attributed to the membrane-binding properties of the protein. In this work, we compare the effect of some catechols, including dopamine, epinephrine, DOPAL, and levodopa in micromolar concentrations, on the in vitro cytotoxicity of α-syn fibrils on human neuroblastoma SH-SY5Y cells. The study was followed by comparing the interactions of resulting structures with rat brain mitochondria used as an in vitro biological model. The obtained results demonstrate that catechols-induced structures have lost their cytotoxicity mimicking apoptotic cell death mediated by α-syn aggregates in different proportions. Moreover, α-syn fibrils-induced mitochondrial dysfunction, evaluated by a range of biochemical assays, was modulated by catechols-modified α-syn oligomers in different manners, as levodopa and DOPAL demonstrated the maximal and minimal effects, respectively. The plausible mechanism causing the inhibition of α-syn cytotoxic fibrillization and mitochondrial dysfunction by catechols is discussed. Taken together, we propose that catechols can prevent the cytotoxic assembly of α-syn and its destructive effects on mitochondria at various stages, suggesting that decreased levels of catechols in dopaminergic neurons might accelerate the α-syn cytotoxicity and mitochondrial dysfunction implicating Parkinson's disease.

Stabilization challenges and aggregation in protein-based therapeutics in the pharmaceutical industry.

Protein-based therapeutics have revolutionized the pharmaceutical industry and become vital components in the development of future therapeutics. They offer several advantages over traditional small molecule drugs, including high affinity, potency and specificity, while demonstrating low toxicity and minimal adverse effects. However, the development and manufacturing processes of protein-based therapeutics presents challenges related to protein folding, purification, stability and immunogenicity that should be addressed. These proteins, like other biological molecules, are prone to chemical and physical instabilities. The stability of protein-based drugs throughout the entire manufacturing, storage and delivery process is essential. The occurrence of structural instability resulting from misfolding, unfolding, and modifications, as well as aggregation, poses a significant risk to the efficacy of these drugs, overshadowing their promising attributes. Gaining insight into structural alterations caused by aggregation and their impact on immunogenicity is vital for the advancement and refinement of protein therapeutics. Hence, in this review, we have discussed some features of protein aggregation during production, formulation and storage as well as stabilization strategies in protein engineering and computational methods to prevent aggregation.

Aggregation and structural changes of α(S1)-, ß- and κ-caseins induced by homocysteinylation.

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, ß- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one ß-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3µm. Homocysteinylation of α(S1)- and ß-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.

Chaperonins induce an amyloid-like transformation of ovine prion protein: the fundamental difference in action between eukaryotic TRiC and bacterial GroEL.

Molecular chaperones have been shown to be involved in the processes taking place during the pathogenesis of various amyloid neurodegenerative diseases. However, contradictory literature reports suggest that different molecular chaperones can either stimulate or prevent the formation of amyloid structures from distinct amyloidogenic proteins. In the present work, we concentrated on the effects caused by two molecular chaperonins, ovine TRiC and bacterial GroEL, on the aggregation and conformational state of ovine PrP. Both chaperonins were shown to bind native PrP and to produce amyloid-like forms of ovine PrP enriched with beta-structures but, while GroEL acted in an ATP-dependent manner, TRiC was shown to cause the same effect only in the absence of Mg-ATP (i.e. in the inactive form). In the presence of chaperonin GroEL, ovine PrP was shown to form micellar particles, approximately 100-200nm in diameter, which were observed both by dynamic light scattering assay and by electron microscopy. The content of these particles was significantly higher in the presence of Mg-ATP and, only under these conditions, GroEL produced amyloid-like species enriched with beta-structures. TRiC was shown to induce the formation of amyloid fibrils observed by electron microscopy, but only in the absence of Mg-ATP. This study suggests the important role of the cytosolic chaperonin TRiC in the propagation of amyloid structures in vivo during the development of amyloid diseases and the possible role of the bacterial chaperonin GroEL, located in the intestinal microflora, in the induction of these diseases.

N-homocysteinylation of ovine prion protein induces amyloid-like transformation.

Modification of protein lysyl residues by homocysteine (Hcy)-thiolactone generates proteins with altered structures and functions. It has been supposed to be one of the factors inducing protein condensation pathologies. To test a hypothesis that N-homocysteinylation may induce structural changes and in particular amyloidogenic conversion, ovine prion protein (PrP) was modified with Hcy-thiolactone and its physico-chemical properties were studied. N-Hcy-PrP formed insoluble multimers. Mass spectrometry analyses showed that at least K197 and K207 residues of PrP were the sites of N-homocysteinylation. Dynamic light scattering measurements revealed large aggregated N-Hcy-PrP particles of 1µm diameter. They were resistant to proteinase K digestion, and enhanced thioflavin T (ThT)-binding fluorescence, what is characteristic of amyloid structures. Infrared spectroscopy measurements showed increased content of beta-sheet in N-Hcy-PrP compared to unmodified PrP. Epifluorescence microscopy in the presence of ThT revealed cluster-like aggregates of N-Hcy-PrP. The collected data indicate that the N-homocysteinylation causes amyloidogenic transformation of PrP in vitro.

Thermal stability enhancement: Fundamental concepts of protein engineering strategies to manipulate the flexible structure.

Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.

Bivalent metal ions induce formation of α-synuclein fibril polymorphs with different cytotoxicities.

α-Synuclein (α-Syn) aggregates are key components of intracellular inclusion bodies characteristic of Parkinson's disease (PD) and other synucleinopathies. Metal ions have been considered as the important etiological factors in PD since their interactions with α-Syn alter the kinetics of fibrillation. In the present study, we have systematically explored the effects of Zn2+, Cu2+, Ca2+, and Mg2+ cations on α-Syn fibril formation. Specifically, we determined fibrillation kinetics, size, morphology, and secondary structure of the fibrils and their cytotoxic activity. While all cations accelerate fibrillation, we observed distinct effects of the different ions. For example, Zn2+ induced fibrillation by lower tlag and higher kapp and formation of shorter fibrils, while Ca2+ ions lead to formation of longer fibrils, as evidenced by dynamic light scattering and atomic force microscopy studies. Additionally, the morphology of formed fibrils was different. Circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopies revealed higher contents of ß-sheets in fibrils. Interestingly, cell viability studies indicated nontoxicity of α-Syn fibrils formed in the presence of Zn2+ ions, while the fibrils formed in the presence of Cu2+, Ca2+, and Mg2+ were cytotoxic. Our results revealed that α-Syn fibrils formed in the presence of different divalent cations have distinct structural and cytotoxic features.