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.

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.

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.

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.

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.

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.

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) physiologically aggregated within inclusion bodies (IBs) retaining their biological activity as immobilized biocatalysts.

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.

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.

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.

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.

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.

Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3).

Active inclusion bodies of polyphosphate kinase 3 and cytidine 5'-monophosphate kinase were combined with whole cells that co-express sialic acid aldolase and CMP-sialic acid synthetase. The biocatalytic mixture was used for the synthesis of CMP-sialic acid, which was then converted to 3'-sialyllactose by whole cells.

Bioenergy beads: a tool for regeneration of ATP/NTP in biocatalytic synthesis.

Active inclusion bodies of recombinant polyphosphate kinase were obtained by simple washing of Escherichia coli cells with nonionic detergent and then they were immobilized in agar/TiO2 beads. Bioenergy beads obtained are charged by polyphosphate to act as rechargeable supply of adenosine/nucleoside triphosphates (ATP/NTP), a practical tool for synthesis of artificial receptors.

Production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) using enzymes or whole cells entrapped in calcium pectate-silica-gel beads.

The present study focuses on the application of immobilization technology to enzymic sugar syntheses. The paper describes an improved silica-alginate matrix established for entrapment and encapsulation. The replacement of alginate with pectate provided enhanced chemical resistance of the matrix, which allows the use of 1% (w/v) polyphosphate in reaction mixtures. Polylysine, a reagent for silica condensation, was replaced by a much cheaper alternative, namely polyethyleneimine. The proposed design was applied in the production of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid (CMP-sialic acid) by immobilized recombinant enzymes or Escherichia coli cells containing overexpressed enzymes. A comparison between these two strategies was made. On the basis of the results we conceptualized a system to synthesize sialyloligosaccharides by using a biocatalyst entrapped in calcium pectate-silica gel beads.