1-L Transcription in Prion Diseases.

Understanding the pathogenesis and mechanisms of prion diseases can significantly expand our knowledge in the field of neurodegenerative diseases. Prion biology is increasingly recognized as being relevant to the pathophysiology of Alzheimer's disease and Parkinson's disease, both of which affect millions of people each year. This bioinformatics study used a theoretical protein-RNA recognition code (1-L transcription) to reveal the post-transcriptional regulation of the prion protein (PrPC). The principle for this method is directly elucidated on PrPC, in which an octa-repeat can be 1-L transcribed into a GGA triplet repeat RNA aptamer known to reduce the misfolding of normal PrPC into abnormal PrPSc. The identified genes/proteins are associated with mitochondria, cancer, COVID-19 and ER-stress, and approximately half are directly or indirectly associated with prion diseases. For example, the octa-repeat supports CD44, and regions of the brain with astrocytic prion accumulation also display high levels of CD44.

1-L Transcription of SARS-CoV-2 Spike Protein S1 Subunit.

The COVID-19 pandemic prompted rapid research on SARS-CoV-2 pathogenicity. Consequently, new data can be used to advance the molecular understanding of SARS-CoV-2 infection. The present bioinformatics study discusses the "spikeopathy" at the molecular level and focuses on the possible post-transcriptional regulation of the SARS-CoV-2 spike protein S1 subunit in the host cell/tissue. A theoretical protein-RNA recognition code was used to check the compatibility of the SARS-CoV-2 spike protein S1 subunit with mRNAs in the human transcriptome (1-L transcription). The principle for this method is elucidated on the defined RNA binding protein GEMIN5 (gem nuclear organelle-associated protein 5) and RNU2-1 (U2 spliceosomal RNA). Using the method described here, it was shown that 45% of the genes/proteins identified by 1-L transcription of the SARS-CoV-2 spike protein S1 subunit are directly linked to COVID-19, 39% are indirectly linked to COVID-19, and 16% cannot currently be associated with COVID-19. The identified genes/proteins are associated with stroke, diabetes, and cardiac injury.

Transcription of the Envelope Protein by 1-L Protein-RNA Recognition Code Leads to Genes/Proteins That Are Relevant to the SARS-CoV-2 Life Cycle and Pathogenesis.

The theoretical protein-RNA recognition code was used in this study to research the compatibility of the SARS-CoV-2 envelope protein (E) with mRNAs in the human transcriptome. According to a review of the literature, the spectrum of identified genes showed that the virus post-transcriptionally promotes or represses the genes involved in the SARS-CoV-2 life cycle. The identified genes/proteins are also involved in adaptive immunity, in the function of the cilia and wound healing (EMT and MET) in the pulmonary epithelial tissue, in Alzheimer's and Parkinson's disease and in type 2 diabetes. For example, the E-protein promotes BHLHE40, which switches off the IL-10 inflammatory "brake" and inhibits antiviral THαß cells. In the viral cycle, E supports the COPII-SCAP-SREBP-HSP90α transport complex by the lowering of cholesterol in the ER and by the repression of insulin signaling, which explains the positive effect of HSP90 inhibitors in COVID-19 (geldanamycin), and E also supports importin α/ß-mediated transport to the nucleus, which explains the positive effect of ivermectin, a blocker of importins α/ß. In summary, transcription of the envelope protein by the 1-L protein-RNA recognition code leads to genes/proteins that are relevant to the SARS-CoV-2 life cycle and pathogenesis.

1-L Transcription in Alzheimer's Disease.

Alzheimer's disease is a very complex disease and better explanations and models are needed to understand how neurons are affected and microglia are activated. A new model of Alzheimer's disease is presented here, the ß-amyloid peptide is considered an important RNA recognition/binding peptide. 1-L transcription revealed compatible sequences with AAUAAA (PAS signal) and UUUC (class III ARE rich in U) in the Aß peptide, supporting the peptide-RNA regulatory model. When a hypothetical model of fibril selection with the prionic character of amyloid assemblies is added to the peptide-RNA regulatory model, the downregulation of the PI3K-Akt pathway and the upregulation of the PLC-IP3 pathway are well explained. The model explains why neurons are less protected from inflammation and why microglia are activated; why mitochondria are destabilized; why the autophagic flux is destabilized; and why the post-transcriptional attenuation of the axonal signal "noise" is interrupted. For example, the model suggests that Aß peptide may post-transcriptionally control ELAVL2 (ELAV-like RNA binding protein 2) and DCP2 (decapping mRNA protein 2), which are known to regulate RNA processing, transport, and stability.

The role of the protein-RNA recognition code in neurodegeneration.

MicroRNAs are small endogenous RNAs that pair and bind to sites on mRNAs to direct post-transcriptional repression. However, there is a possibility that microRNAs directly influence protein structure and activity, and this influence can be termed post-translational riboregulation. This conceptual review explores the literature on neurodegenerative disorders. Research on the association between neurodegeneration and RNA-repeat toxicity provides data that support a protein-RNA recognition code. For example, this code explains why hnRNP H and SFPQ proteins, which are involved in amyotrophic lateral sclerosis, are sequestered by the (GGGGCC)n repeat sequence. Similarly, it explains why MNBL proteins and (CTG)n repeats in RNA, which are involved in myotonic dystrophy, are sequestered into RNA foci. Using this code, proteins involved in diseases can be identified. A simple protein BLAST search of the human genome for amino acid repeats that correspond to the nucleotide repeats reveals new proteins among already known proteins that are involved in diseases. For example, the (CAG)n repeat sequence, when transcribed into possible peptide sequences, leads to the identification of PTCD3, Rem2, MESP2, SYPL2, WDR33, COL23A1, and others. After confirming this approach on RNA repeats, in the next step, the code was used in the opposite manner. Proteins that are involved in diseases were compared with microRNAs involved in those diseases. For example, a reasonable correspondence of microRNA 9 and 107 with amyloid-ß-peptide (Aß42) was identified. In the last step, a miRBase search for micro-nucleotides, obtained by transcription of a prion amino acid sequence, revealed new microRNAs and microRNAs that have previously been identified as involved in prion diseases. This concept provides a useful key for designing RNA or peptide probes.

Magnetization of active inclusion bodies: comparison with centrifugation in repetitive biotransformations.

BACKGROUND: Physiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle "gelatinous masses" of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field. RESULTS: Magnetically modified inclusion bodies of UDP-glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure. CONCLUSIONS: It is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .

Protein-RNA and protein-glycan recognitions in light of amino acid codes.

BACKGROUND: RNA-binding proteins, in cooperation with non-coding RNAs, play important roles in post-transcriptional regulation. Non-coding micro-RNAs control information flow from the genome to the glycome by interacting with glycan-synthesis enzymes. Glycan-binding proteins read the cell surface and cytoplasmic glycome and transfer signals back to the nucleus. The profiling of the protein-RNA and protein-glycan interactomes is of significant medicinal importance. SCOPE OF REVIEW: This review discusses the state-of-the-art research in the protein-RNA and protein-glycan recognition fields and proposes the application of amino acid codes in profiling and programming the interactomes. MAJOR CONCLUSIONS: The deciphered PUF-RNA and PPR-RNA amino acid recognition codes can be explained by the protein-RNA amino acid recognition hypothesis based on the genetic code. The tripartite amino acid code is also involved in protein-glycan interactions. At present, the results indicate that a system of four codons ("gnc", where n=g - guanine, c - cytosine, u - uracil or a - adenine) and four amino acids (G - glycine, A - alanine, V - valine, D - aspartic acid) could be the original genetic code that imprinted "rules" into both recognition processes. GENERAL SIGNIFICANCE: Amino acid recognition codes have provocative potential in the profiling and programming of the protein-RNA and protein-glycan interactomes. The profiling and even programming of the interactomes will play significant roles in diagnostics and the development of therapeutic procedures against cancer and neurodegenerative, developmental and other diseases.

Protein-RNA recognition: cracking the code.

In early papers, the intent was to find a simple protein-RNA/DNA recognition code. Many people expected a one-to-one correspondence between amino acids and nucleic bases, similar to the code that specifies how one DNA base pairs with another. Despite the lack of such a code, which was evident in the first crystal structures, researchers were indeed unwilling to give up on the idea. Despite the intense interest, a simple one-to-one correspondence has not materialised. The work presented here revisits this theme, and reports a general trend in which four elementary amino acids - G, A, V, and D - have a specific selectivity for four basic nucleotides - g, c, u, and a. During the evolution, as amino acid alphabets increased, new amino acids substituted G, A, V, D amino acids in way to keep hydropathic similarity and the selectivity to minimise errors in established RNA-protein interactions, 1-letter code was created. Additionally, the first nucleotide in codons is used for a 2-letter code. Protein-RNA recognition, visualised by these two code principles, uses a rotation of sensing and anti-sensing sequences in architecture of recognising peptides.

Degradation of polyphosphates by polyphosphate kinases from Ruegeria pomeroyi.

Polyphosphate kinases 2 (PPK2) are key enzymes for polyphosphate utilisation in bacteria. The genome of Ruegeria pomeroyi, a marine α-proteobacterium, includes three Pseudomonas aeruginosa PPK2 homologs. We expressed these homologs in Escherichia coli as soluble proteins, purified the protein products and compared their metal, pH and nucleotide preferences. The optimal pH was 8.0 for SPO1727 and 9.0 for SPO1256. The SPO0224 gene product had two pH optima at eight and ten. The SPO0224 protein showed little dependence on metal presence, while SPO1256 required Mg(2+). SPO1727 required Mg(2+) but accepted other ions as well.

Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release.

BACKGROUND: Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. RESULTS: In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. CONCLUSIONS: The tailored inclusion bodies are promising "nanopills" for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release.

1-L Transcription in Parkinson's Disease.

BACKGROUND: As a chronic degenerative disorder of the central nervous system that affects both motor and non-motor systems, Parkinson's disease (PD) is very complex, and explanations and models are needed to better understand how dopaminergic neurons are affected and microglia are activated. METHODS: A theoretical protein-RNA recognition code that assumes that the second letter in codons is compatible with specific amino acids involved in protein-RNA recognition was used to search for compatibility of human α-synuclein (α-syn) with mRNAs in the human transcriptome (1-L transcription). RESULTS: The 1-L transcription revealed compatible amino acid sequences with the ATTTA ARE (class I), PAS and polyA in α-syn, supporting a protein-RNA regulatory model. In PD, inflammatory microglia reactions, cognitive decline and motor circuit disturbances are observed. The model theoretically explains why α-syn producing neurons are less protected from inflammation and why microglia are activated. Consistent with knowledge of PD, the identified genes showed how the PI3K-AKT pathway is downregulated, how reactive oxygen species (ROS) production and sensitivity are increased, how mitochondria are destabilized, why autophagy is impaired, and why neuronal depigmentation is observed. CONCLUSIONS: 1-L transcription of α-syn leads to genes/proteins relevant to PD. When α-syn is accepted as a small RNA recognition protein involved in the post-transcriptional regulations, some identified genes indicate that its function is an important regulatory factor associated with intracellular and extracellular transport of RNA vesicles. These vesicles are extremely important in cellular communication. In addition, the spectrum of identified genes strongly indicates that α-syn produced by neuronal cells is required for proper regulation of inflammatory and immune responses.

Physiologically Aggregated LacZ Applied in Trehalose Galactosylation in a Recycled Batch Mode.

Galactooligosaccharides obtained via ß-galactosidase transgalactosylation have health-promoting properties and are widely recognized as effective prebiotics. Trehalose-based galactooligosaccharides could be introduced into food and pharmaceutical industries similarly to trehalose. In light of this, new technological approaches are needed. Recently, in vivo enzyme immobilizations for recombinant proteins have been introduced, and physiological aggregation into active inclusion bodies (aIBs) has emerged as one such method of in vivo immobilization. To prepare LacZ ß-galactosidase in the form of aIBs, we used a short 10 amino acid aggregation-prone tag. These native protein particles were simply washed from the cell lysate and applied in trehalose galactosylation in a recycled batch mode. In this study, aIBs entrapped in alginate beads, encapsulated in alginate/cellulose sulfate/poly(methylene-co-guanidine) capsules and magnetized were compared with free aIBs. Alginate/cellulose sulfate/PMCG capsules showed more suitable properties and applicability for biotransformation of trehalose at its high concentration (25%, w/v) and elevated temperature (50 °C).

Glycocodon theory--the first table of glycocodons.

Hydrophobic cellular membranes separate cells from an environment that is generally based on water. Therefore, it is not surprising that hydrophilic glycans and glycoproteins are exposed on the lipidic surface of membranes and that the glycocalyx has evolved in all basic cell types. During the evolution of multicellular life, the surface exposed protein-glycan interactions were taken as the origin of the language of cell-cell communication. The bioinformatics analysis presented here reveals that the amino acid triplets, the glycocodons, can be deduced for each glycan letter (monosaccharide). This theory proposes to distinguish between the "sugar code" (the sugar sequence) and the "glycocode" (evolutionary selected amino acids recognising the mono-sugar). Similarly to genetic code, original glycocodons are related to G, A, V, and D amino acids. Modern glycocodons can be deduced from GAVD-glycocodons using hydropathic similarity. In general, the amino acid triplets can be assembled from one dipeptide that is specific to a monosaccharide plus a polar amino acid. This theory may shed a different light on the reason for WWD conservation in the active sites of oligosaccharyltransferases and for GGQ in the active sites of ribosomes.

Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase, UDP-glucose pyrophosphorylase) physiologically aggregated within inclusion bodies retaining their biological activity as immobilized biocatalysts.

Pull-Down Into Active Inclusion Bodies and Their Application in the Detection of (Poly)-Phosphates and Metal-Ions.

Inclusion bodies are typically ignored as they are considered unwanted protein waste generated by prokaryotic host cells during recombinant protein production or harmful protein inclusions in human cell biology. However, these protein particles may have applications for in vivo immobilization in industrial biocatalysis or as cell-tolerable protein materials for the pharmaceuticals industry and clinical development. Thus, there is a need to in vivo "pull-down" (insolubilize) soluble enzymes and proteins into inclusion bodies. Accordingly, in this study, sequences from the short-chain polyphosphatase ygiF were used to design pull-down tags capable of detecting (poly)-phosphates and metal ions. These tags were compared with the entire CHAD domain from Escherichia coli ygiF and SACS2 CHAD from Saccharolobus solfataricus. The results demonstrated that highly soluble green fluorescent protein variants could be pulled down into the inclusion bodies and could have modified sensitivity to metals and di-/tri-inorganic phosphates.

Polyphosphate--an ancient energy source and active metabolic regulator.

There are a several molecules on Earth that effectively store energy within their covalent bonds, and one of these energy-rich molecules is polyphosphate. In microbial cells, polyphosphate granules are synthesised for both energy and phosphate storage and are degraded to produce nucleotide triphosphate or phosphate. Energy released from these energetic carriers is used by the cell for production of all vital molecules such as amino acids, nucleobases, sugars and lipids. Polyphosphate chains directly regulate some processes in the cell and are used as phosphate donors in gene regulation. These two processes, energetic metabolism and regulation, are orchestrated by polyphosphate kinases. Polyphosphate kinases (PPKs) can currently be categorized into three groups (PPK1, PPK2 and PPK3) according their functionality; they can also be divided into three groups according their homology (EcPPK1, PaPPK2 and ScVTC). This review discusses historical information, similarities and differences, biochemical characteristics, roles in stress response regulation and possible applications in the biotechnology industry of these enzymes. At the end of the review, a hypothesis is discussed in view of synthetic biology applications that states polyphosphate and calcium-rich organelles have endosymbiotic origins from ancient protocells that metabolized polyphosphate.

High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

Theoretical Analysis of S, M and N Structural Proteins by the Protein-RNA Recognition Code Leads to Genes/proteins that Are Relevant to the SARS-CoV-2 Life Cycle and Pathogenesis.

In this conceptual review, based on the protein-RNA recognition code, some theoretical sequences were detected in the spike (S), membrane (M) and capsid (N) proteins that may post-transcriptionally regulate the host genes/proteins in immune homeostasis, pulmonary epithelial tissue homeostasis, and lipid homeostasis. According to the review of literature, the spectrum of identified genes/proteins shows that the virus promotes IL1α/ß-IL1R1 signaling (type 1 immunity) and immunity defense against helminths and venoms (type 2 immunity). In the alteration of homeostasis in the pulmonary epithelial tissue, the virus blocks the function of cilia and the molecular programs that are involved in wound healing (EMT and MET). Additionally, the protein-RNA recognition method described here identifies compatible sequences in the S1A-domain for the post-transcriptional promotion of PIKFYVE, which is one of the critical factors for SARS-CoV-2 entry to the host cell, and for the post-transcriptional repression of xylulokinase XYLB. A decrease in XYLB product (Xu5P) in plasma was proposed as one of the potential metabolomics biomarkers of COVID-19. In summary, the protein-RNA recognition code leads to protein genes relevant to the SARS-CoV-2 life cycle and pathogenesis.

Prebiotic Peptides Based on the Glycocodon Theory Analyzed with FRET.

In modern protein-carbohydrate interactions, carbohydrate-aromatic contact with CH-π interactions are used. Currently, they are considered driving forces of this complexation. In these contacts, tryptophan, tyrosine, and histidine are preferred. In this study, we focus on primary prebiotic chemistry when only glycine, alanine, aspartic acid, and valine are available in polypeptides. In this situation, when the aromatic acids are not available, hydrogen-bonding aspartic acid must be used for monosaccharide complexation. It is shown here that (DAA)n polypeptides play important roles in primary "protein"-glucose recognition, that (DGG)n plays an important role in "protein"-ribose recognition, and that (DGA)n plays an important role in "protein"-galactose recognition. Glucose oxidase from Aspergillus niger, which still has some ancient prebiotic sequences, is chosen here as an example for discussion.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst.

BACKGROUND: Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. RESULTS: As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. CONCLUSIONS: Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.