Isolation of Amyloid-like Protein Aggregates (APA) from white bread and their characterisation.

High temperature, acidic pH, and physical agitation are commonly observed during cooking or industrial food processing, which are often considered as favorable conditions, at least for some proteins, to misfold and form amyloid-like protein aggregates (APA). The proteins in various bakery products generally experience high temperatures that might lead to the formation of APA. To test this hypothesis, the presence of APA in white bread was examined in this study. The APA isolated from white bread displayed typical characteristics of amyloids, like bathochromic shift in Congo red (CR) absorbance maxima, increased fluorescence of Thioflavin T (ThT) & 8-anilino-1-naphthalene sulfonic acid (ANS), fibrillar morphology of >200 nm long with average diameter of 10-12 nm and negative minima at 223 nm in Circular Dichroism (CD) spectrum. The SDS- and native PAGE revealed the presence of gliadin and glutenin as the constituent proteins in the isolated protein aggregates. Although, the presence of amyloid-like structures in white bread is evident, further studies would be essential to establish their functional role and health implications.

Discerning Modulation of α-Synuclein Amyloid Assembly by α-Crystallin.

Altered protein folding leading to the formation of structured aggregates such as amyloid fibrils has gained significant attention due to its association with neurodegenerative diseases. α-Synuclein, a small intrinsically disordered protein, gets transformed into amyloid fibrils under unfavorable conditions and contributes to the progression and pathology of Parkinson's disease (PD). Under normal physiological conditions, amyloid formation is controlled by many chaperones and chaperone-like proteins. However, with aging, the protein homeostasis machinery becomes less efficient, causing the loss of proper functioning of chaperones and leading to aberrant protein folding and amyloid formation. Here, we provide in-depth information on the modulation of α-synuclein amyloid assembly by a heterogeneous complex of bovine eye lens protein, α-crystallin, which is known to possess chaperone-like activity. We have used a multiparametric approach to discern the critical events through which α-crystallin abolishes α-synuclein amyloid formation. Our biochemical and biophysical data analysis revealed that α-crystallin, at substoichiometric ratios, alleviates α-synuclein amyloid assembly and drives it into soluble dead-end intermediates. We also demonstrated that α-crystallin was equally efficient in arresting amyloid assembly by some of the PD-related mutants suggesting the significance of chaperone-like activity of α-crystallin under pathological conditions. Finally, we validated our results using human crystallin derived from cataract patients. Based on our findings, we propose that the interaction of α-crystallin directs α-synuclein into a soluble amyloid-incompetent form. Our results suggest that the generic antiamyloid property of chaperone-like proteins, such as α-crystallin, can be harnessed to design protein and peptide-based novel therapeutics for prevention and treatment of deadly neurodegenerative diseases.

Amyloids and Amyloid-like Protein Aggregates in Foods: Challenges and New Perspectives.

Protein misfolding and amyloid formations are associated with many neurodegenerative and systemic diseases. The discovery of Alzheimer's disease and its association with the accumulation of Amyloid-ß (Aß) peptides in the plaques uncovered the pleiotropic nature of peptides/ proteins. As of today, more than 50 proteins/ peptides are reported to form amyloids or amyloid-like protein aggregates under different conditions, establishing that amyloid formation could be a generic property of many proteins. In principle, under certain conditions, all the proteins have this property to form amyloid-like aggregates, which can be toxic or non-toxic. The extensive research in this direction led to an understanding of the ubiquitous nature of amyloids. Mounting evidences suggest that processed foods, particularly protein-rich foods, could be a plethora of amyloids or amyloid-like protein aggregates. Many are reported to be toxic, and their consumption raises health concerns. The assimilation of dietary proteins in the human body largely depends upon their conformational states and the digestive integrity of the gastrointestinal system. Amyloids or amyloid-like protein aggregates are usually protease resistant, and their presence in foods is likely to reduce nutritional value. Several biochemical and biophysical factors, commonly evident in various food processing industries, such as high temperature, the addition of acid, etc., are likely to induce the formation of protease-resistant protein aggregates. Aging significantly alters gastrointestinal health, predisposing aged individuals to be more susceptible to protein aggregation-related diseases. Consumption of foods containing such protein aggregates will lead to a poor supply of essential amino acids and might exaggerate the amyloid-related disease etiology. On the other hand, the gut microbiome plays a crucial role during pathological events leading to the development of Alzheimer's and Parkinson's diseases. The activity of gastrointestinal proteases, pH change, gut microbiome, and intestinal epithelium integrity would largely determine the outcome of consuming foods loaded with such protein aggregates. The current review outlines the recent development in this area and a new perspective for designing safe protein-rich diets for healthy nutrition.>.

Isolation and characterization of amyloid-like protein aggregates from soya beans and the effect of low pH and heat treatment on their stability.

Purified soya bean proteins (glycinin and conglycinin) are known to form amyloid-like aggregates in vitro at a higher temperature. Soya beans (chunks) are textured proteinaceous vegetables made from defatted soya flour by heating it above 100°C and extruding under high pressure. Therefore, it was assumed that subjecting the soya bean proteins to high temperatures raises the possibility of forming amyloids or amyloid-like protein aggregates. Hence, the present study aimed to examine the presence of amyloid-like protein aggregates in soya beans. The isolated protein aggregates from hydrated soya beans displayed typical characteristics of amyloids, such as the red shift in the absorption maximum (λmax ) of Congo red (CR), high Thioflavin T (ThT), and 8-Anilinonapthalene-1-sulfonate (ANS) binding, and fibrilar morphology. Furthermore, these aggregates were found to be stable against proteolytic hydrolysis, confirming the specific property of amyloids. The presence of amyloid-like structures in soya beans raises concerns about their implications for human nutrition and health. PRACTICAL APPLICATIONS: Protein aggregation has usually been considered detrimental. The traditional food-processing conditions, such as thermal processing, are associated with protein denaturation and aggregation. The formation of ordered protein aggregates with extensive ß-sheet are progressively evident in various protein-rich foods known as amyloid, which expands food safety concerns. Instead, it is also associated with poor nutritional characteristics. The present study concerns the presence of amyloid-like protein aggregates in widely consumed native soya beans, which are manufactured by extensive heat treatment of defatted soy flour. Although there is no indication of their toxicity, these aggregates are found to be proteolytically resistant. The seminal findings in this manuscript suggest that it is time to adapt innovative food processing and supplementation of bioactive molecules that can prevent the formation of such protein aggregates and help maximize the utilization of protein-based nutritional values.

Structural and functional swapping of amyloidogenic and antimicrobial peptides: Redefining the role of amyloidogenic propensity in disease and host defense.

Peptides constitute an essential component of all organisms' protein homeostasis ranging from bacteria, plants, and animals. They have organically been evolved to perform a wide range of essential functions, including their role as neurotransmitters, antimicrobial peptides (AMPs), and hormones. AMPs are short peptides synthesized by almost all organisms, implicated in guarding the host from various microbial infections. Their inherent ability to differentiate the target microbes from the host confers them excellent prospects in fighting against microbial infections and affirming their robust therapeutic potential against numerous drug-resistant microbes. Amyloidogenic peptides (AMYs) represent another class of short peptides armed with inherent aggregation propensity and form fibrillar aggregates rich in cross ß-sheet structure. They are often involved in various degenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and type-2 diabetes. Although these two distinct classes of peptides (i.e., AMPs and AMYs) appear to be functionally divergent, recent studies suggest that they possess a significant degree of structural and functional reciprocity. Consistent with this, many AMPs display amphiphilic nature, and hence, they can facilitate membrane remodeling processes, such as pore formation and fusion, similar to AMYs. The mounting evidence suggests the inherent ability of AMPs to self-assemble to form amyloid-like structures. On the other hand, the demonstration of antimicrobial properties of AMYs in their monomeric conformation provides a hint about the existence of an evolutionary linkage between these two classes of peptides. The congregation of specific amino acids to form aggregation-prone regions in a protein/peptide might have served as an evolutionary reservoir from which AMPs and AMYs were consecutively evolved. The current article reviews the fundamental features of the AMPs, AMYs, and their inter-relatedness and emerging paradigm for their inter-conversion.

Isolation and characterisation of milk-derived amyloid-like protein aggregates (MAPA) from cottage cheese.

Cottage cheese, extensively consumed worldwide, contains coagulated milk protein (casein), produced through boiling and acidification of milk. Casein forms amyloid or amyloid-like structures at high temperatures and low pH. Due to the similarities in the preparation of casein amyloids and cottage cheese, we hypothesized the presence of amyloid or amyloid-like protein aggregates in cottage cheese. To examine this hypothesis, cottage cheese was prepared from cow (Bos indicus) milk and isolated amyloids through a water extraction method. The isolated protein aggregates displayed typical characteristics of amyloids, such as a bathochromic shift in the wavelength of maximum absorption (λmax) of Congo red (CR), high thioflavin T (ThT) binding, increased surface hydrophobicity, and high ß-sheet structure. However, they did not show antibacterial activity and toxic properties against erythrocytes. Our study revealed that the heat-treatment and subsequent acidification during cottage cheese preparation lead to the formation of non-toxic amyloid-like aggregates.

Heat treatment of soluble proteins isolated from human cataract lens leads to the formation of non-fibrillar amyloid-like protein aggregates.

The loss of crystallins solubility with aging and the formation of amyloid-like aggregates is considered the hallmark characteristic of cataract pathology. The present study was carried out to assess the effect of temperature on the soluble lens protein and the formation of protein aggregates with typical amyloid characteristics. The soluble fraction of lens proteins was subjected for heat treatment in the range of 40-60 °C, and the nature of protein aggregates was assessed by using Congo red (CR), thioflavin T (ThT), and 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays, circular dichroism (CD), Fourier-transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The heat-treated protein samples displayed a substantial bathochromic shift (≈15 nm) in the CR's absorption maximum (λmax) and increased ThT and ANS binding. The heat treatment of lens soluble proteins results in the formation of nontoxic, ß-sheet rich, non-fibrillar, protein aggregates similar to the structures evident in the insoluble fraction of proteins isolated from the cataractous lens. The data obtained from the present study suggest that the exposure of soluble lens proteins to elevated temperature leads to the formation of non-fibrillar aggregates, establishing the role of amyloid in the heat-induced augmentation of cataracts pathology.

Aggregation hot spots in the SARS-CoV-2 proteome may constitute potential therapeutic targets for the suppression of the viral replication and multiplication.

The emergence of novel coronavirus SARS-CoV-2 is responsible for causing coronavirus disease-19 (COVID-19) imposing serious threat to global public health. Infection of SARS-CoV-2 to the host cell is characterized by direct translation of positive single stranded (+ ss) RNA to form large polyprotein polymerase 1ab (pp1ab), which acts as precursor for a number of nonstructural and structural proteins that play vital roles in replication of viral genome and biosynthesis of new virus particles. The maintenance of viral protein homeostasis is essential for continuation of viral life cycle in the host cell. To test whether the protein homeostasis of SARS-CoV-2 can be disrupted by inducing specific protein aggregation, we made an effort to examine whether the viral proteome contains any aggregation prone regions (APRs) that can be explored for inducing toxic protein aggregation specifically in viral proteins and without affecting the host cell. This curiosity leads to the identification of several (> 70) potential APRs in SARS-CoV-2 proteome. The length of the APRs ranges from 5 to 25 amino acid residues. Nearly 70% of total APRs investigated are relatively smaller and found to be in the range of 5-10 amino acids. The maximum number of ARPs (> 50) was observed in pp1ab. On the other hand, the structural proteins such as, spike (S), nucleoprotein (N), membrane (M) and envelope (E) proteins also possess APRs in their primary structures which altogether constitute 30% of the total APRs identified. Our findings may provide new windows of opportunities to design specific peptide-based, anti-SARS-CoV-2 therapeutic molecules against COVID-19.

Amyloid cross-sequence interaction between Aß(1-40) and αA(66-80) in relation to the pathogenesis of cataract.

Alzheimer's disease (AD) and cataract represent two common protein misfolding diseases closely associated with aging. Growing evidence suggests that these two diseases may be interrelated with each other through cross-sequence interactions between ß-amyloid (Aß) peptide and the short aggregating peptides derived from proteolytic breakdown of α-crystallin. αΑ(66-80) is one of several peptides produced by the proteolytic breakdown of α-crystallin in aged eye lens. Although it is evident that the Aß(1-40) and αΑ(66-80) coexist in aged eye lenses and both the peptides are known to form macromolecular assemblies, their cross-sequence interaction and the seeding behavior are not known. In this study, the aggregation behavior of αΑ(66-80) has been examined in the presence of Aß(1-40) on using thioflavin T (ThT) based aggregation kinetics. The presence of monomeric Aß(1-40) augmented the aggregation kinetics of αΑ(66-80) and reduced the lag time of αΑ(66-80) aggregation. However, the addition of Aß(1-40) or αΑ(66-80) fibrils (seeds) didn't result in any change in the rate of αΑ(66-80) aggregation. In this in vitro study, we could show that the presence Aß(1-40) has substantial effect on the aggregation of αΑ(66-80), which suggests a possible interaction between AD and cataract pathologies.

Trehalose Inhibits the Heat-Induced Formation of the Amyloid-Like Structure of Soluble Proteins Isolated from Human Cataract Lens.

The age-dependent loss of solubility and aggregation of crystallins constitute the pathological hallmarks of cataract. Several biochemical and biophysical factors are responsible for the reduction of crystallins' solubility and formation of irreversible protein aggregates, which display amyloid-like characteristics. The present study reports the heat-induced aggregation of soluble proteins isolated from human cataract lenses and the formation of amyloid-like structures. Exposure of protein at 55 °C for 4 h resulted in extensive (≈ 60%) protein aggregation. The heat-induced protein aggregates displayed substantial (≈ 20 nm) redshift in the wavelength of maximum absorption (λmax) of Congo red (CR) and increase in Thioflavin T (ThT) fluorescence emission intensity, indicating the presence of amyloid-like structures in the heat-induced protein aggregates. Subsequently, the addition of trehalose resulted in substantial inhibition of heat-induced aggregation and the formation of amyloid-like structure. The ability of trehalose to inhibit the heat-induced aggregation was found to be linearly dependent upon its concentration used. The optimum effect was observed in the presence of 30-40% (w/v) trehalose where the aggregated was found to be reduced from 60 to 30%. The present study demonstrated the ability to trehalose to inhibit the protein aggregation and interfere with the formation of amyloid-like structures.

Computational investigation for modeling the protein-protein interaction of TasA(28-261)-TapA(33-253): a decisive process in biofilm formation by Bacillus subtilis.

Biofilms have a significant role in microbial persistence, antibiotic resistance, and chronic infections; consequently, there is a pressing need for development of novel "anti-biofilm strategies." One of the fundamental mechanisms involved in biofilm formation is protein-protein interactions of "amyloid-like proteins" (ALPs) in the extracellular matrix. Such interactions could be potential targets for development of novel anti-biofilm strategies; therefore, assessing the structural features of these interactions could be of great scientific value. Characterization of structural features the of protein-protein interaction with conventional structure biology tools including X-ray diffraction and nuclear magnetic resonance is technically challenging, expensive, and time-consuming. In contrast, modeling such interactions is time-efficient and economical, and might provide deeper understanding of structural basis of interactions. Although it is often acknowledged that molecular modeling methods have varying accuracy, their careful implementation with supplementary verification methods can provide valuable insight and directions for future studies. With this reasoning, during the present study, the protein-protein interaction of TasA(28-261)-TapA(33-253) (which is a decisive process for biofilm formation by Bacillus subtilis) was modeled using in silico approaches, viz., molecular modeling, protein-protein docking, and molecular dynamics simulations. Results obtained here identified amino acid residues present within intrinsically disordered regions of both proteins to be critical for interaction. These results were further supported with principal component analyses (PCA) and free energy landscape (FEL) analyses. Results presented here represent novel finding, and we hypothesize that amino acid residues identified during the present study could be targeted for inhibition of biofilm formation by B. subtilis.

Predicted aggregation-prone region (APR) in ßB1-crystallin forms the amyloid-like structure and induces aggregation of soluble proteins isolated from human cataractous eye lens.

The aggregation of ß-crystallins in the human eye lens constitutes a critical step during the development of cataract. We anticipated that the presence of Aggregation-Prone Regions (APRs) in their primary structure, which might be responsible for conformational change required for the self-assembly. To examine the presence of APRs, we systematically analyzed the primary structures of ß-crystallins. Out of seven subtypes, the ßB1-crystallin found to possess the highest aggregation score with 9 APRs in its primary structure. To confirm the amyloidogenic nature of these newly identified APRs, we further studied the aggregation behavior of one of the APRs spanning from 174 to 180 residues (174LWVYGFS180) of ßB1-crystallin, which is referred as ßB1(174-180). Under in vitro conditions, the synthetic analogue of ßB1(174-180) peptide formed visible aggregates and displayed high Congo red (CR) bathochromic shift, Thioflavin T (ThT) binding and fibrilar morphology under transmission electron microscopy, which are the typical characteristics of amyloids. Further, the aggregated ßB1(174-180) was found to induce aggregation of the soluble fraction of proteins isolated from the human cataractous lens. This observation suggests that the presence of APRs in ßB1-crystallin might be serving as one of the intrinsic supplementary factors responsible for constitutive aggregation behavior of ßB1-crystallin and development of cataract.

Impact of semen-derived amyloid (SEVI) on sperm viability and motility: its implication in male reproductive fitness.

Human semen contains a large number of macromolecules, including proteins/enzymes and carbohydrates, regulating and protecting sperm cells. Proteomic analysis of human seminal fluid led to the discovery of semen amyloids derived from short peptide fragments of the proteins prostatic acid phosphatase (PAP) and semenogelin (SG) which are known to play a crucial role in enhancing HIV infection. However, the relevance of their existence in human semen and role in maintaining sperm behavior remains unclear. Distinct physiological, biochemical, and biophysical attributes might cause these amyloids to influence sperm behavior positively or negatively, affecting fertilization or other reproductive processes. We assessed the direct effect of amyloids derived from a PAP248-286 fragment, on sperm motility and viability, which are crucial parameters for assessment of sperm quality in semen. Co-incubation of human sperm with PAP248-286 amyloids at normal physiological concentrations formed in buffer led to significant reduction in sperm viability, though approximately a 10× higher concentration was needed to show a similar effect with amyloid formed in seminal fluid. Both forms of PAP248-286 amyloid also had a significant impact on sperm motility at physiological levels, in agreement with a previous report. Our study suggests that PAP248-286 amyloids can directly influence sperm motility and viability in a concentration-dependent manner. We hypothesise that the direct toxic effect of PAP248-286 amyloid is normally mitigated by other seminal fluid ingredients, but that in pathological conditions, where PAP248-286 concentrations are elevated and it plays a role in determining sperm health and viability, with relevance for male fertility as well as sterility.

Pheromone peptide cOB1 from native Enterococcus faecalis forms amyloid-like structures: A new paradigm for peptide pheromones.

Pheromone peptides are an important component of bacterial quorum-sensing system. The pheromone peptide cOB1 (VAVLVLGA) of native commensal Enterococcus faecalis has also been identified as an antimicrobial peptide (AMP) and reported to kill the prototype clinical isolate strain of E. faecalis V583. In this study, the pheromone peptide cOB1 has shown to form amyloid-like structures, a characteristic which is never reported for a pheromone peptide so far. With in silico analysis, the peptide was predicted to be highly amyloidogenic. Further, under experimental conditions, cOB1 formed aggregates displaying characteristics of amyloid structures such as bathochromic shift in Congo red absorbance, enhancement in thioflavin T fluorescence, and fibrillar morphology under transmission electron microscopy. This novel property of pheromone peptide cOB1 may have some direct effects on the binding of the pheromone to the receptor cells and subsequent conjugative transfer, making this observation more important for the therapeutics, dealing with the generation of virulent and multidrug-resistant pathogenic strains.

Thrombin stimulates gene expression and secretion of IL-11 via protease-activated receptor-1 and regulates extravillous trophoblast cell migration.

Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times. Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.

The mechanism of phosphatidylcholine-induced interference of PAP (248-286) aggregation.

Seminal amyloids are well known for their role in enhancing HIV infection. Among all the amyloidogenic peptides identified in human semen, PAP248-286 was found to be the most active and was termed as semen-derived enhancer of viral infection (SEVI). Although amyloidogenic nature of the peptide is mainly linked with enhancement of the viral infection, the most active physiological conformation of the aggregated peptide remains inconclusive. Lipids are known to modulate aggregation pathway of a variety of proteins and peptides and constitute one of the most abundant biomolecules in human semen. PAP248-286 significantly differs from the other known amyloidogenic peptides, including Aß and IAPP, in terms of critical concentration, surface charge, fibril morphology, and structural transition during aggregation. Hence, in the present study, we aimed to assess the effect of a lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), on PAP248-286 aggregation and the consequent conformational outcomes. Our initial observation suggested that the presence of the lipid considerably influenced the aggregation of PAP248-286 . Further, ZDOCK and MD simulation studies of peptide multimerization have suggested that the hydrophobic residues at C-terminus are crucial for PAP248-286 aggregation and are anticipated to be major DOPC-interacting partners. Therefore, we further assessed the aggregation behaviour of C-terminal (PAP273-286 ) fragment of PAP248-286 and observed that DOPC possesses the ability to interfere with the aggregation behaviour of both the peptides used in the current study. Mechanistically, we propose that the presence of DOPC causes considerable inhibition of the peptide aggregation by interfering with the peptide's disordered state to ß-sheet transition.

Mammalian antimicrobial peptide protegrin-4 self assembles and forms amyloid-like aggregates: Assessment of its functional relevance.

Protegrin-4 (PG-4) is a member of the porcine leukocyte protegrins family of cysteine-rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the ß sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG-4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin-T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non-toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide-based antimicrobials with enhanced stability.

Structure, function and antagonism of semen amyloids.

Amyloid fibrils are linear polypeptide aggregates with a cross-ß structure. These fibrils are best known for their association with neurodegenerative diseases, such as Alzheimer's or Parkinson's, but they may also be used by living organisms as functional units, e.g. in the synthesis of melanin or in the formation of bacterial biofilms. About a decade ago, in a search for semen factors that modulate infection by HIV-1 (a sexually transmitted virus and the causative agent of the acquired immune deficiency syndrome (AIDS)), it was demonstrated that semen harbors amyloid fibrils capable of markedly increasing HIV infection rates. This discovery not only created novel opportunities to prevent sexual HIV-1 transmission but also stimulated research to unravel the natural role of these factors. We discuss here the identification of these intriguing structures, their molecular properties, and their effects on both sexually transmitted diseases and reproductive health. Moreover, we review strategies to antagonize semen amyloid to prevent sexual transmission of viruses.

Effect of Green Tea Polyphenol Epigallocatechin-3-gallate on the Aggregation of αA(66-80) Peptide, a Major Fragment of αA-crystallin Involved in Cataract Development.

PURPOSE: Crystallin is a major protein present in eye lens. Peptide fragment αA(66-80) derived from αA-crystallin possesses high aggregation propensity and forms amyloid-like structures. αA(66-80) aggregates are known to interact with soluble crystallins and destabilize native structures that subsequently undergo aggregation. Crystallin aggregation in eye lens leads to reduction in lens opacity, the condition generally referred to as a cataract. Thus, αA(66-80) aggregation appears to be an important event during cataract development, and therefore, inhibition of αA(66-80) aggregation may be an attractive strategy to intervene in cataract development. MATERIALS AND METHODS: αA(66-80) peptide derived from αA-crystallin possesses high aggregation potential and has a crucial role in cataract development. In order to inhibit the aggregation of αA(66-80) peptide, epigallocatechin-3-gallate (EGCG), a major active constituent of green tea, was employed. The inhibitory effect was assessed by Congo Red (CR) spectral shift assay, Thioflavin-T binding assay, transmission electron microscopy and fluorescence microscopy. RESULTS: The inhibitory potential of EGCG toward αA-crystallin was clearly observed as in the presence of EGCG, the αA(66-80) aggregation was considerably inhibited and the pre-formed fibrillary aggregates of αA(66-80) were found to be disassembled. CONCLUSION: In the present study, we are able to successfully demonstrate that EGCG efficiently blocks the aggregation of αA(66-80) peptide in a concentration-dependent manner. Furthermore, it is also evident that EGCG is able to disaggregate pre-formed αA(66-80) aggregates. The study suggests that EGCG can be a potential molecule that can prevent the initiation of cataract as well as be helpful in the disease reversal.

Assessment of the effect of macromolecular crowding on aggregation behaviour of a model amyloidogenic Peptide.

Accumulation of ordered protein aggregates (or amyloids) represents a hallmark of many diseases (e.g., Alzheimer's disease, type II diabetes, Parkinson's diseases etc.), results from intermolecular association of partially unfolded proteins/ peptides. Such associations usually take place in highly crowded conditions. The aggregates, which are formed under in vitro and in vivo conditions exhibit substantial variations in their structure and function. Such heterogeneities in amyloids might arise due to macromolecular crowding that is usually omitted under in vitro conditions. The current study is an attempt to assess the effects of macromolecular crowding on amyloid formation using a model amyloidogenic peptide. The sequence of the peptide was derived from C-terminal region (RATQIPSYKKLIMY) of PAP(248-286), which naturally occurs in human semen as amyloid aggregates and is known for boosting HIV infectivity. This model peptide forms sedimentable and fibrillar aggregates in aqueous buffer and shows the characteristic features of amyloids. In the presence of macromolecular crowders the morphological features of the amyloids are significantly altered and resulted in the formation of shorter amyloid aggregates. The current study assumes the hypothesis that macromolecular crowding in the biological system favours formation of heterogeneous classes of aggregates and each of them might differ in their biophysical and biological properties.