Bacteriophage-encoded chaperonins stimulate prion protein fibrillation in an ATP-dependent manner.

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

Magnetically Controlled Hyaluronic Acid-Maghemite Nanocomposites with Embedded Doxorubicin.

The controllable delivery of drugs is a key task of pharmacology. For this purpose, a series of polymer composites was synthesized via the cross-linking of hyaluronate and a hyaluronate/polyacrylate mixture with Fe2O3 nanoparticles. The cross-linking imparts magnetic properties to the composites, which are more pronounced for the ternary hyaluronate/polyacrylate/γ-Fe2O3 composites compared with the binary hyaluronate/Fe2O3 composites. When dispersed in water, the composites produce microsized hydrogel particles. Circulation of the ternary microgels in an aqueous solution at a speed of 1.84 cm/s can be stopped using a permanent external magnet with a magnetic flux density of 400 T. The composite hydrogels can absorb the antitumor antibiotic doxorubicin (Dox); the resulting constructs show their cytotoxicity to tumor cells to be comparable to the cytotoxicity of Dox itself. The addition of the hyaluronidase enzyme induces degradation of the binary and ternary microgels down to smaller particles. This study presents prospectives for the preparation of magnetically controlled biodegradable polymer carriers for the encapsulation of bioactive substances.

The role of sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the development of pathologies-from asthenozoospermia to carcinogenesis.

The review considers various aspects of the influence of the glycolytic enzyme, sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) on the energy metabolism of spermatozoa and on the occurrence of several pathologies both in spermatozoa and in other cells. GAPDS is a unique enzyme normally found only in mammalian spermatozoa. GAPDS provides movement of the sperm flagellum through the ATP formation in glycolytic reactions. Oxidation of cysteine residues in GAPDS results in inactivation of the enzyme and decreases sperm motility. In particular, reduced sperm motility in diabetes can be associated with GAPDS oxidation by superoxide anion produced during glycation reactions. Mutations in GAPDS gene lead in the loss of motility, and in some cases, disrupts the formation of the structural elements of the sperm flagellum, in which the enzyme incorporates during spermiogenesis. GAPDS activation can be used to increase the spermatozoa fertility, and inhibitors of this enzyme are being tried as contraceptives. A truncated GAPDS lacking the N-terminal fragment of 72 amino acids that attaches the enzyme to the sperm flagellum was found in melanoma cell lines and then in specimens of melanoma and other tumors. Simultaneous production of the somatic form of GAPDH and sperm-specific GAPDS in cancer cells leads to a reorganization of their energy metabolism, which is accompanied by a change in the efficiency of metastasis of certain forms of cancer. Issues related to the use of GAPDS for the diagnosis of cancer, as well as the possibility of regulating the activity of this enzyme to prevent metastasis, are discussed.

Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

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.

Does the SARS-CoV-2 Spike Receptor-Binding Domain Hamper the Amyloid Transformation of Alpha-Synuclein after All?

Interactions of key amyloidogenic proteins with SARS-CoV-2 proteins may be one of the causes of expanding and delayed post-COVID-19 neurodegenerative processes. Furthermore, such abnormal effects can be caused by proteins and their fragments circulating in the body during vaccination. The aim of our work was to analyze the effect of the receptor-binding domain of the coronavirus S-protein domain (RBD) on alpha-synuclein amyloid aggregation. Molecular modeling showed that the predicted RBD complex with monomeric alpha-synuclein is stable over 100 ns of molecular dynamics. Analysis of the interactions of RBD with the amyloid form of alpha-synuclein showed that during molecular dynamics for 200 ns the number of contacts is markedly higher than that for the monomeric form. The formation of the RBD complex with the alpha-synuclein monomer was confirmed immunochemically by immobilization of RBD on its specific receptor ACE2. Changes in the spectral characteristics of the intrinsic tryptophans of RBD and hydrophobic dye ANS indicate an interaction between the monomeric proteins, but according to the data of circular dichroism spectra, this interaction does not lead to a change in their secondary structure. Data on the kinetics of amyloid fibril formation using several spectral approaches strongly suggest that RBD prevents the amyloid transformation of alpha-synuclein. Moreover, the fibrils obtained in the presence of RBD showed significantly less cytotoxicity on SH-SY5Y neuroblastoma cells.

Sorted Bulls' X-Chromosome-Bearing Spermatozoa Show Increased GAPDHS Activity Correlating with Motility.

Sperm sexing is a technique for spermatozoa sorting into populations enriched with X- or Y-chromosome-bearing cells and is widely used in the dairy industry. Investigation of the characteristics of sorted semen is of practical interest, because it could contribute to the enhancement of sexed semen fertility characteristics, which are currently lower than those of conventional semen. Comparison of a spermatozoa population enriched with X-chromosome-bearing cells to a mixed population is also intriguing in the context of potential differences that drive the mechanisms of primary sex-ratio determination. In this work, sexed (X spermatozoa) and conventional spermatozoa of Holstein bulls were analyzed for the content and enzymatic activity of GAPDHS, a sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase that plays a significant role in the regulation of flagellar activity. No difference in the amount of this glycolysis enzyme per cell was revealed, but, notably, GAPDHS enzymatic activity in the sexed samples was significantly higher. Enzymatic activity among the group of sexed but not conventional sperm samples positively correlated with spermatozoa motility, which indicates the significant role of this enzyme for the sorted cells population.

Modulation of TRPV1 and TRPA1 Channels Function by Sea Anemones' Peptides Enhances the Viability of SH-SY5Y Cell Model of Parkinson's Disease.

Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.

Polyelectrolytes for Enzyme Immobilization and the Regulation of Their Properties.

In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems (self-regulated enzyme-polyelectrolyte complexes, artificial chaperones, polyelectrolyte brushes, layer-by-layer immobilization and others). These works represent the field of "smart polymers", whilst the trivial use of charged polymers as carriers for adsorption or covalent immobilization of proteins is beyond the scope of this short review. In addition, we have included a section on the molecular modeling of interactions between proteins and polyelectrolytes, as modeling the binding of proteins with a strictly defined, and already known, spatial structure, to disordered polymeric molecules has its own unique characteristics.

Hydroxycinnamic Acid Derivatives from Coffee Extracts Prevent Amyloid Transformation of Alpha-Synuclein.

Earlier we showed that derivatives of hydroxycinnamic acids prevent amyloid transformation of alpha-synuclein and prion protein. The aim of this work was to determine the content of 3-hydroxycinnamic acid derivatives in coffee extracts and to evaluate their activity in relation to alpha-synuclein amyloid aggregation. Hydroxycinnamic acid derivatives were identified in aqueous and ethanol extracts of coffee beans by quantitative mass spectrometric analysis. Only 3,4-dimethoxycinnamic acid (13-53 µg/mL) was detected in significant amounts in the coffee extracts, while ferulic acid was present in trace amounts. In addition, 3-methoxy-4-acetamidoxycinnamic acid (0.4-0.8 µg/mL) was detected in the roasted coffee extracts. The half-maximum inhibitory concentrations of alpha-synuclein fibrillization reaction in the presence of coffee extracts, as well as inhibitory constants, were determined using thioflavin T assay. The inhibitory effect of black and green coffee extracts on alpha-synuclein fibrillization is dose-dependent, and in a pairwise comparison, the constants of half-maximal inhibition of fibrillization for green coffee extracts are comparable to or greater than those for black coffee. Thus, coffee extracts prevent pathological transformation of alpha-synuclein in vitro, probably due to the presence of 3,4-dimethoxycinnamic acid in them. Consequently, coffee drinks and coffee extracts can be used for the prevention of synucleinopathies including Parkinson's disease.

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.

Effect of bacteriophage-encoded chaperonins on amyloid transformation of α-synuclein.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

Potential Effect of Post-Transcriptional Substitutions of Tyrosine for Cysteine Residues on Transformation of Amyloidogenic Proteins.

The review considers the reasons and consequences of post-transcriptional tyrosine substitutions for cysteine residues. Main attention is paid to the Tyr/Cys substitutions that arise during gene expression in bacterial systems at the stage of protein translation as a result of misrecognition of the similar mRNA codons. Notably, translation errors generally occur relatively rarely - from 10-4 to 10-3 errors per codon for E. coli cells, but in some cases the error rate increases significantly. For example, this is typical for certain pairs of codons, when the culture conditions change or in the presence of antibiotics. Thus, with overproduction of the recombinant human alpha-synuclein in E. coli cells, the content of the mutant form with the replacement of Tyr136 (UAC codon) with a cysteine residue (UGC codon) can reach 50%. Possible reasons for the increased production of alpha-synuclein with the Tyr136Cys substitution are considered, as well as consequences of the presence of mutant forms in preparations of amyloidogenic proteins when studying their pathological transformation in vitro. A separate section is devoted to the Tyr/Cys substitutions occurring due to mRNA editing by adenosine deaminases, which is typical for eukaryotic organisms, and the possible role of this process in the amyloid transformation of proteins associated with neurodegenerative diseases.

Synthetic Sulfated Polymers Control Amyloid Aggregation of Ovine Prion Protein and Decrease Its Toxicity.

Amyloid aggregation, including aggregation and propagation of prion protein, is a key factor in numerous human diseases, so-called amyloidosis, with a very poor ability for treatment or prevention. The present work describes the effect of sulfated or sulfonated polymers (sodium dextran sulfate, polystyrene sulfonate, polyanethole sulfonate, and polyvinyl sulfate) on different stages of amyloidogenic conversion and aggregation of the prion protein, which is associated with prionopathies in humans and animals. All tested polymers turned out to induce amyloid conversion of the ovine prion protein. As suggested from molecular dynamics simulations, this effect probably arises from destabilization of the native prion protein structure by the polymers. Short polymers enhanced its further aggregation, whereas addition of high-molecular poly(styrene sulfonate) inhibited amyloid fibrils formation. According to the seeding experiments, the protein-polymer complexes formed after incubation with poly(styrene sulfonate) exhibited significantly lower amyloidogenic capacity compared with the control fibrils of the free prion protein. The cytotoxicity of soluble oligomers was completely inhibited by treatment with poly(styrene sulfonate). To summarize, sulfonated polymers are a promising platform for the formulation of a new class of anti-prion and anti-amyloidosis therapeutics.

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Modification of Glyceraldehyde-3-Phosphate Dehydrogenase with Nitric Oxide: Role in Signal Transduction and Development of Apoptosis.

This review focuses on the consequences of GAPDH S-nitrosylation at the catalytic cysteine residue. The widespread hypothesis according to which S-nitrosylation causes a change in GAPDH structure and its subsequent binding to the Siah1 protein is considered in detail. It is assumed that the GAPDH complex with Siah1 is transported to the nucleus by carrier proteins, interacts with nuclear proteins, and induces apoptosis. However, there are several conflicting and unproven elements in this hypothesis. In particular, there is no direct confirmation of the interaction between the tetrameric GAPDH and Siah1 caused by S-nitrosylation of GAPDH. The question remains as to whether the translocation of GAPDH into the nucleus is caused by S-nitrosylation or by some other modification of the catalytic cysteine residue. The hypothesis of the induction of apoptosis by oxidation of GAPDH is considered. This oxidation leads to a release of the coenzyme NAD+ from the active center of GAPDH, followed by the dissociation of the tetramer into subunits, which move to the nucleus due to passive transport and induce apoptosis. In conclusion, the main tasks are summarized, the solutions to which will make it possible to more definitively establish the role of nitric oxide in the induction of apoptosis.

Structural and Computational Study of the GroEL-Prion Protein Complex.

The molecular chaperone GroEL is designed to promote protein folding and prevent aggregation. However, the interaction between GroEL and the prion protein, PrPC, could lead to pathogenic transformation of the latter to the aggregation-prone PrPSc form. Here, the molecular basis of the interactions in the GroEL-PrP complex is studied with cryo-EM and molecular dynamics approaches. The obtained cryo-EM structure shows PrP to be bound to several subunits of GroEL at the level of their apical domains. According to MD simulations, the disordered N-domain of PrP forms much more intermolecular contacts with GroEL. Upon binding to the GroEL, the N-domain of PrP begins to form short helices, while the C-domain of PrP exhibits a tendency to unfold its α2-helix. In the absence of the nucleotides in the system, these processes are manifested at the hundred nanoseconds to microsecond timescale.

Infection of Human Cells by SARS-CoV-2 and Molecular Overview of Gastrointestinal, Neurological, and Hepatic Problems in COVID-19 Patients.

The gastrointestinal tract is the body's largest interface between the host and the external environment. People infected with SARS-CoV-2 are at higher risk of microbiome alterations and severe diseases. Recent evidence has suggested that the pathophysiological and molecular mechanisms associated with gastrointestinal complicity in SARS-CoV-2 infection could be explained by the role of angiotensin-converting enzyme-2 (ACE2) cell receptors. These receptors are overexpressed in the gut lining, leading to a high intestinal permeability to foreign pathogens. It is believed that SARS-CoV-2 has a lesser likelihood of causing liver infection because of the diminished expression of ACE2 in liver cells. Interestingly, an interconnection between the lungs, brain, and gastrointestinal tract during severe COVID-19 has been mentioned. We hope that this review on the molecular mechanisms related to the gastrointestinal disorders as well as neurological and hepatic manifestations experienced by COVID-19 patients will help scientists to find a convenient solution for this and other pandemic events.

Thermocontrolled Reversible Enzyme Complexation-Inactivation-Protection by Poly(N-acryloyl glycinamide).

A prospective technology for reversible enzyme complexation accompanied with its inactivation and protection followed by reactivation after a fast thermocontrolled release has been demonstrated. A thermoresponsive polymer with upper critical solution temperature, poly(N-acryloyl glycinamide) (PNAGA), which is soluble in water at elevated temperatures but phase separates at low temperatures, has been shown to bind lysozyme, chosen as a model enzyme, at a low temperature (10 °C and lower) but not at room temperature (around 25 °C). The cooling of the mixture of PNAGA and lysozyme solutions from room temperature resulted in the capturing of the protein and the formation of stable complexes; heating it back up was accompanied by dissolving the complexes and the release of the bound lysozyme. Captured by the polymer, lysozyme was inactive, but a temperature-mediated release from the complexes was accompanied by its reactivation. Complexation also partially protected lysozyme from proteolytic degradation by proteinase K, which is useful for biotechnological applications. The obtained results are relevant for important medicinal tasks associated with drug delivery such as the delivery and controlled release of enzyme-based drugs.

Novel cryo-EM structure of an ADP-bound GroEL-GroES complex.

The GroEL-GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL-GroES1 complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.