Synopsis

Cancers frequently exhibit RNA dysregulation, which involves alternative transcript splicing. However, no ‘protein’ dysregulation, such as ribosomal peptide alterations, has ever been identified or put forth as the main factor in the development of cancer. Nobody has ever mentioned subtle peptide modifications like insertion of glycine residue (+ Gly) after peptide elongation in the ribosome, despite the fact that aberrant abnormal RNA transcripts have been found to influence cancer initiation, progression, metastasis, and therapy resistance and are frequently translated into cancer-specific proteins. Cancer may develop if the + Glycine mutation does not occur or does not occur at the proper location in the protein frame. This is because mutations like + Gly give the protein and, consequently, the cell a new function. “A large ‘RNA’ library aids in the discovery of cancer-causing mutations”. Based on our results in the silkworm moth chemosensory protein (CSP) gene family [1,2,3,4,5,6,7,8], we suggest that “a large ‘peptide’ library in the gland and/or other tissues may help discover novel cancer-causing mutations”.

The impact of RNA editing on gene expression and cancer is discussed in the first part. A single gene can produce a variety of isoforms, thanks to RNA editing, but it can also cause some genetic diseases, like cancer, by changing the regulatory mechanisms that control oncogenic gene expression, such as changing the transcriptome scales in messenger RNA (mRNA) editing levels, which, for instance, influence innate immune responses [9]. Therefore, it is widely believed that the connection between the nucleus and the ribosome, genome, genomic DNA, gene, gene expression, and protein synthesis, i.e., the intermediary mRNA strand required for protein formation, is merely the deciding factor for the development of cancer [10]. Apolipoprotein B mRNA editing enzyme (Apobec-1; C-to-U conversion) acting on single-strand DNA as substrate edits RNA molecules, as do adenosine deaminases acting on mRNA (ADAR; A-to-I conversion) or transfer RNA (ADAT; A-to-I conversion) [11,12,13,14,15,16,17]. However, the selection of RNA and/or DNA substrates can always vary depending on the environment, the tissue, and the type of cell [17]. As a result, some APOBEC structural domains can arbitrate C-to-U conversion on RNA [18], but some ADATs from Trypanosoma kinetoplastids can convert C-to-U base on single-strand DNA [19,20,21]. Due to the degeneration of numerous RNA- and DNA-dependent catalytic enzymes, the loss of multi-specific editing enzymes, and variations in transcript profiles found in particular immune response pathways, RNA editing in epithelial and mesenchymal tumors mainly impact the abundance and stability of particular transcripts [22, 23]. Because many cancer genomic studies, in particular, have identified Apobec-1 and C-to-U editing signatures in tumor cell-specific somatic mutations linked to innate immunity, we are constantly looking for a treatment for cancer and the innate immune system that is similar to the treatments for other cancers [24,25,26,27].

In addition, a second part or section discusses the effects of beneficial bacterial systems (bioproducts), a recently developed and expanding field that offers an alternative to pharmaceutical drugs in the treatment of numerous diseases via immune system modulation in diverse animal models (Table 1). Like many other bacterial probiotics, Bacillus/Lactobacillus is an intriguing tool for the prevention and treatment of cancer, atherosclerosis, metabolic disorders, obesity-related dyslipidemia, and cholesterolemia. Bacterial strains that breakdown carcinogens, modify (boost) the immune system, and/or exhibit anti-tumor features such as fat synthesis [28]. The problem of developing a novel natural therapy for the treatment of cancer has made long-chain polyunsaturated fatty acids (PUFAs), specifically omega-3 and omega-6 PUFAs, into very significant prospects [29]. Lactic acid bacteria (Lactobacillus strains), which have been demonstrated to regulate gut flora and, hence, enhance the host’s immune response and overall physiology, thus appear as the top prospects for study on probiotics and cancer [30]. Probiotics that are utilized to treat colorectal cancer, such as Lactobacillus acidophilus and L. fermentum, have been demonstrated to possess potent anticancer effects [31, 32]. Specific chemicals secreted by L. acidophilus cause anti-tumorigenic molecules to assault cancer cells [32]. Similar to this, 5-fluorouracil (Adrucil), a well-known ‘drug’ cancer treatment, was examined, and it was shown to be highly beneficial when used in a specific dual combination with L. acidophilus and L. casei [33]. To regulate lipid metabolism and activate the host immune system, we recently employed L. or Bacillus cocktails in tri-therapy to encourage the proliferation and metabolism of helpful bacterial strains in the gut flora (Table 1) [34,35,36]. Data on the impact of L. cocktail treatment under cyclophosphamide chemotherapy settings are presented [37]. The effect of bioproducts on host DNA could be increased yet further. However, no one has ever investigated the direct effects of L. on host genetics and ribosome peptide mutations. Using natural bacterial tri-therapy (three-L.) to regulate intestinal flora, lipid metabolism and the immune system could be incredibly helpful in developing new strategies to interfere with RNA editing and cancer mutagenesis. Particularly in the cancer bioproduct market growth, peptide mutations are a novel fundamental medical science area that should be investigated.

Table 1 In mammalian models of hyperlipidemia, scour, and cancer, composition of natural medicinal Bacillus/Lactobacillus bioproduct (tri-therapy) for regulation of gut flora, immune system, lipid metabolism, and cell function [34,35,36,37]
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The review’s goal is to interpret newly discovered peptide point mutations in insects as new potential cancer research targets and to propose that RNA + peptide point mutations in bacterial strains be investigated as potential cancer therapies. New medical perspectives or viewpoints are necessary to treat genetic abnormalities of the glands, neurological system, and/or metabolism, including cancers of unknown primary (CUPs). It may be essential to conduct research on the “CSP” protein gene family, which contains RNA + peptide mutations; in this regard, given that, we believe ‘a lack of these mutations is the main cause of CUP’. We started by looking at the various organisms’ enzymes, mutations, editing sites, and DNA/RNA-dependent mechanisms. Then we concentrated on the many peptide mutations found in the sex pheromone gland from the female Bombyx mori silkworm moth. To support a new working hypothesis that certain CSP peptide point mutations, such as + Gly, + Cys, + Phe-Val-Phe, and many others, achieved by the cell ribosome, have functional effects on the protein, we used a modeling structural approach (SWISS-MODEL) on two B. mori CSPs, BmorCSP6 and BmorCSP11. The primary outcome of peptide point mutations, like RNA mutations, is the loss of alpha-helical turns, suggesting that these exceedingly ‘bizarre’ odd changes have a high number and complex diversity and target crucial protein structural domains in a tissue-dependent manner. It is crucial to confirm the regulatory mechanisms behind + Gly in the flank of Cys for cancer therapy utilizing crystallization techniques before conceiving, designing or highlighting them. However, if Linux, NMR, or X-ray results support our hypothesis, it will undoubtedly reveal a new area of ribosome mutational activity. Many cancers still lack a known cellular genesis. We predict that the Bombyx peptide alterations may have a significant impact on cancer CUP therapy and that bacterial peptide editing methods, particularly Lactobacillus combined with CRISPR/Cas, may be utilized to control ribosomes, re-orient the + Gly, + Cys, or + Phe-Val-Phe insertion mutation to the proper site, and treat cancer in humans.

RNA mutations brought on by viruses and bacteria in humans

There are undoubtedly as many editing mechanisms in eukaryotic and prokaryotic cells, as there are malignancies. Infectious diseases that are prone to epidemics and pandemics, like COVID-19, and SARS-CoV-2, with all of its variants, demonstrate the wide range of mutational events that an RNA can undergo. Genetic diseases and malignancies also have a rich diversity of variants. Novel genetic RNA-based therapeutics are needed to address SARS virus mutations because SARS-CoV-2 mutations depend on the host RNA editing system [38]. To survive and spread, RNA viruses (A-rich strands) exploit a variety of genetic alterations or ‘changes’. This genetic proclivity comprises a variety of unusual translational events, such as internal ribosome entry to + G (G base insertion), biases to-U, and stop codon mutations, in addition to high mutation rates, high yields, and short replication times [39, 40]. This is interesting because in several tissues of the silkworm, Bombyx mori, we found a comparable frequency of mutational events in the coding region of RNA strands [see 1–8].

In viruses and the eukaryotes that house them, as well as in archaea, bacteria, and prokaryotes, RNA editing is employed to modify tRNA, ribosomal RNA, messenger RNA, and microRNA. The most prevalent RNA editing change, with the exception of ferns, flowers, and plants, has been described in practically all living things that have been researched: the A-to-I (A-to-G) mutation (see Table 2). Instead A-to-I (A-to-G) mutations, plants change RNA through C-to-U and/or U-to-C transitions [41, 42]. However, base deletion/insertion mutation exclusively impacts the U base in plants [43, 44]. Base deletion/insertion mutation does occur, despite the fact that C-to-U conversion is the most frequent RNA editing in plant mitochondria and chloroplasts (Table 2) [43, 44]. Similar to plant cells, bacterial prokaryotic systems have a high rate of A-to-I and C-to-U mutations, which recode not only tRNA but also protein-coding genes (Table 2) [45, 46]. Accurate profile of RNA editing sites in a variety of bacterial systems, species, or strains may eventually show more varied mutation profiling, including U base deletion/insertion, using deep transcriptome and genome sequencing data. Similar RNA editing factors exist in both plants and bacteria [47], and numerous genes, such as CSPs, and mechanisms, such as RNA + peptide editing, have almost certainly been horizontally transferred from bacteria to plants. This transfer most likely had a big significant impact on how plants evolved and adapted [48,49,50]. Consequently, there is a good chance that RNA editing in plants and other living organisms take root, start, grow and spread from their symbionts. A-to-I, C-to-U, U-to-C, N-to-N, and stop codon nucleotide base mutations are just a few of the RNA mutations that dinoflagellates (algae, protists, marine plankton, inter- and intra-cellular endo-photosymbionts of many marine metazoans and protozoan hosts, such as stony corals and jellyfish) have (Table 2) [51]. Alveolates, flagellates, ciliates, and apicomplexan parasites’ ribosomes have never been subjected to research on peptide mutations. Symbiosis may be essential for mutation exchanges between the bacterial or flagellate symbionts and the coral, cnidarian, gasteropod, or cephalopod host in the marine environment, where cephalopods show particularly high levels of RNA editing (A-to-I and G-to-A; Table 2), [52, 53]. Additionally, there are horizontal gene transfers between bacteria and plants to fungi [54], and the RNA editing profiles of the two are very similar (A-to-I and C-to-U mutations; Table 2) [44, 45, 55, 56]. Therefore, not only CSP but also RNA + peptide editing genes and mechanisms may have been passed from bacteria to plants, fungi (such as mushrooms, mold, and yeast), and the bulk of multicellular eukaryotes, including insects.

Table 2 Prokaryotes and eukaryotes, particularly insects and single-celled dinoflagellates (protists, marine plankton), have a wide range of RNA editing
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A-to-I conversion mutation is the most prevalent RNA editing process in many metazoan eukaryotic animals, such as amphibians, reptiles, rodents, primates, and humans (Table 2). A-to-I mutation (ADAR activity) is crucial for the brain editome and behavior in worms as well [57,58,59]. Depending on the species, organs, and tissues, additional RNA editing mechanisms, such as the C-to-U mutation, are active in mammals [60]. Many-banded krait (Bungarus multicinctus) venom gland tissue has been described as having RNA editing sites [61]. C-to-U has mostly been investigated in mice and other rodents for apolipoprotein B (apoB) in organs that metabolize fat, such as the liver and intestines [62]. Comparing the brain to the heart, kidney, lung, liver, spleen, and thymus, the brain contains the highest amounts of base mutations (A-to-I) [63]. Not only in 3’UTRs but also in microRNA target locations, A-to-I and C-to-U alterations are found in mouse liver, white adipose, and bone tissues, suggesting that A-to-I and C-to-U RNA editing in mammals has distinct regulatory and functional implications [64]. Importantly, G-to-A mutations have also been found in primates, humans, and cephalopods, in addition to A-to-I and C-to-U (Table 2). While G-to-A conversion appears to be necessary for the development of new and more advantageous sites for A-to-I RNA editing events in primates (and squids), G-to-A editing has been associated to chronic social defeat stress in mice [65]. For a certain cell, tissue, or organism to diversify, expand and evolve, G-to-A conversion to enhance A-to-I may be essential [66].

Adenosine-to-inosine conversion by ADAR, which is mainly expressed in the CNS (central nervous system) and is essential for brain function, is the most prevalent type of RNA editing in humans [67, 68]. Human cells can also have other mutations, such as C-to-U, U-to-C, G-to-A, and U-to-A edits (Table 2). Both innate and adaptive immunity, including antibody diversification and numerous antiviral responses, are likely to benefit from the C-to-U editing of protein coding sites by the APOBEC enzyme in metabolic organs, macrophages, and/or hypoxia-induced monocytes [69, 70]. On the other hand, U-to-C, G-to-A, and U-to-A base conversion mutations are thought to be conditionally fatal and have been connected to a number of human illnesses, including cancer and disorders of the brain and neurological system [71].

C-to-U conversion is a relatively infrequent occurrence in humans, and it often seems to be brought on by synonymous mutations, making it non-adaptive [72]. Could these atypical, odd, weird, unusual ‘error’-prone mutations like C-to-U, U-to-C, U-to-A, and G-to-A, be required for the diversity of RNA editing sites for A-to-I in particular cells or tissues of humans in terms of the evolutionary success of G-to-A and A-to-I connections in primates (and squids)? In the face of several varied RNA mutation substitutions as odd as a guanine base altered to an adenine, could it be that protein recoding extends beyond DNA and/or RNA and includes some ribosome-found mechanisms, making it adaptive?

Are there mutations in human and insect cell RNA and peptides?

The growth, development, and evolution of particular cells, tissues, and organisms can all be significantly impacted by a wide variety of mutations and/or mutants with a variety of mutation combinations. Cancer may arise from a dysregulation of the mechanism causing this phenomena of multi-mutations, as opposed to merely an unfavorable U-to-C, C-to-U, U-to-A, or G-to-A conversion (Fig. 1).

Fig. 1
figure 1

RNA and peptide mutations that can affect how a cell functions. A Cellular cytoplasmic and nuclear RNA editing. ADAR: Adenosine deaminase acting on RNA (A-to-I conversion), ADAT: Adenosine deaminase acting on transfer RNA (C-to-U); APOB: apolipoprotein B mRNA editing catalytic polypeptide-like family (APOBEC) enzyme (C-to-U conversion), rAPOB: reverse APOBEC (U-to-C), RNAP: RNA polymerase, RPOL: DNA-directed RNA polymerase subunit L (G-to-A), UDG: uracil-DNA glycosylase (GC-to-TA). An MDVCL motif can convert into several amino acid sequences (IDFVI, VDFAL, and VGFAL), when ADAR, ADAT, APOB, rAPOB, and UDG enzymes are involved, leading to a variety of mutant isoforms. B Ribosome-based editing of peptides in cells. An amino acid motif like MDVCL (Met-Asp-Val-Cys-Leu) can produce additional peptide variants in the ribosome if Phe-Val-Phe or Gly is inserted as a mutation near Cys (1). It is possible to introduce new mutations in the mutant variant pattern VDFAL (Val-Asp-Phe-Ala-Leu), such as Val-Asp being changed to Ala-Cys-Thr-Lys, Cys-Ala-Thr-Lys, Glu-Pro-Gln, Glu-Gln-Pro, Glu-Pro, Pro-Glu, Gln-Glu, or Gln alone (2). When a mutant variant motif, such as VGFAL (Val-Gly-Phe-Ala-Leu), is subjected to an exchange of position between Phe and Ala residues, conversion mutation can result (3). IDFVI (Ile-Asp-Phe-Val-Ile) is a mutant variant motif that can contain one Cys in place of Asp-Phe-Val to produce the novel amino acid motif Ile-Cys-Ile with two Ile residues and a new free Cysteine anchor (4). The size of α-helix 2 can be increased by inserting the Phe-Val-Phe motif (5). Inserting Gly near Cys shortens α2 (6). By providing fresh disulfide linkage S–S, the addition of Ala-Cys and/or Ala-Cys to proteins can aid in the production of new protein dimers (7). The surface of α2 has amino acid pairs Phe-Ala or Ala-Phe that can mediate interactions with various ligands (8). In the N-terminal tail, a + Cys residue can design and construct new peptide building blocks or make new protein-building bonds, which is necessary for new cell activity (9) [see 1]. On B. mori CSPs, these peptide variants were sequenced in SDS-gel–LC–MS–MS analysis [1]. Our theory is that dysregulation in A. and/or B. (1–9) can lead to hereditary genetic disease and malignant cancer disorders (*)

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Some of the potential advantages of U-to-C, C-to-U, U-to-A, and/or G-to-A mutations are shown in Fig. 1. By utilizing several editing enzymes as various as ADAR, ADAT, APOBEC, reverse APOBEC, and uracil-DNA glycosylase (UDG), a gene fragment that codes for Met-Asp-Val-Cys-Leu could express various extra supplemental peptides (Fig. 1A). Multiple polymerases and editing enzymes in the nuclear and cytoplasmic compartments of the cell can be combined to produce more than four novel motifs (Fig. 1A). Ile-Asp-Phe-Val-Ile, Val-Asp-Phe-Ala-Leu, and Val-Gly-Phe-Ala-Leu can substitute for a short pentapeptide motif like Met-Asp-Val-Cys-Leu. Numerous enzymes catalyze nucleic acid changes in various cellular compartments (Fig. 1A). UDG enzyme, which recodes a genomic sequence in the nucleus by switching out a GC base pairing for thymine to adenine, would be necessary for high RNA multi-diversity. This enables the change from methionine to Ile by delivering U bases to particular sites on the RNA strand. When paired with ADAR in the nucleus or cytoplasm, UDG can also assist in shifting Methionine to Val residues, exhibiting amazing flexibility in DNA/RNA strand recoding (Fig. 1A). Even further base editing could take place in the cell nucleus on the same DNA/RNA strand. In addition to ADAT, RNA polymerases and nucleotidyl transferases like RNAP and RPOL that may alter G-to-A on DNA also accept further mutations. By combining these novel mutations, additional A base sites are built, which might act and serve as templates for ADAR enzyme’s A-to-G switch (Fig. 1A). In addition, APOBEC enzyme and/or reverse APOBEC activities mediate C-to-U and/or U-to-C conversion in the cytoplasm, which leads to the synthesis of new extra peptide variants (VCL transition to FVI or FAL; Fig. 1A). At the nucleus and cytoplasmic fraction levels, that is, prior to the ribosome and all protein translation processes, DNA and RNA polymerization appears to be particularly helpful for entirely replacing a protein motif. The crucial point to keep in mind is that, as evidenced by insects, DNA/RNA editing not only contributes to a wide and deep spectrum of transcriptome diversity, but also does so in a tissue-specific manner [see 1–8]. Because of this, we can expect that various editing enzymes will co-occur in particular cells and tissues, resulting in a tremendous diversity of RNA variants that will act and serve as templates for the production of a huge variety of protein isoforms at the ribosome level (Fig. 1A).

It is interesting to note that Xuan et al. documented not just a huge number of RNA-level mutation combinations but also an even bigger number of peptide-level mutation combinations [see 1–8]. Here are a few ribosome peptide mutations that were discovered in the female silkworm’s sex pheromone gland. In Fig. 1B, a couple of these mutations are displayed. A Cysteine residue in a specific location on the protein sequence can lead to an insertion mutation, such as the addition of the Phe-Ala-Phe motif or a Gly residue, on its flank. The Met-Asp-Val-Cys-Leu amino acid motif is converted into a Met-Asp-Val-Cys-Phe-Val-Phe-Leu or Met-Asp-Val-Cys-Gly-Leu group sequence at the ribosome level (Fig. 1B). This modification is significant because it affects not only the motif’s composition but also the alpha-helical profiles [see 1–8]. Adding Gly near Cys can alter the alpha-helix structure depending on the protein gene family, according to modeling studies on insect binding proteins [see 8]. Other peptide variations, such as Val-Asp-Phe-Ala-Leu, may contain additional ribosomal peptide alterations. These mutations can replace a pair of amino acids with just one residue, a different pair of residues, or a new motif made up of three or four amino acids, such as Val–Asp being replaced with Ala-Cys-Thr-Lys, Cys-Ala-Thr-Lys, Glu-Pro-Gln, Glu-Gln-Pro, Glu-Pro, Pro-Glu, Gln-Glu, or Gln (Fig. 1B). The ribosome appears to undergo a number of protein rearrangements and mutation combinations in an extraordinarily well-organized manner.

These alterations are not the result of random mutations. All of the peptide-level mutation combinations mentioned here have been sequenced using gel digestion and liquid chromatography with tandem mass spectrometry (LC–MS–MS) [1]. They are not random because they are precise sequenced combinations of Ala–Cys/Cys-Ala or Gln, Glu, and Pro residues [1]. Combinations of mutations adding different types of amino acids have not been sequenced in any of the more than 4000 peptides [1]. Mutations at the peptide level involve the addition of certain elements (Fig. 1B). One example of this is the addition of extra Cysteine residues, which can be exploited to build novel disulfide bridges and supramolecular structural complexes (Fig. 1B). Another illustration is a Phe-Ala switch to an Ala-Phe switch, which is a specific amino acid permutation, a peptide mutation that affects the identity or position of two amino acid residues at a particular spot on the protein structure (Fig. 1B). Given that alpha-helices promote ligand exchange through peptide changes, this may have an impact on how certain alpha-helices interact with one another (Fig. 1B). The Asp-Phe-Val motif was exclusively replaced by Cysteine, changing the main sequence, shortening the peptide and the alpha-helix, building new disulfide bonds, and giving rise to novel functional properties in the protein (Fig. 1B) [see 1–8, 73–75]. Because of this, RNA editing affects the diversity of the transcriptome, but there are a number of additional pathways on the ribosome that lead to an even larger and much more radically diversified proteome. This occurrence of amino acid residues being added to protein structures after ribosome translation may be an adaptation to low genetic diversity and/or genome decay, or it may be the outcome of genome and ribosome convergent evolution, which generates a wide range of functional protein variants [76, 77]. Our investigation in silkworm revealed that “The cell can synthesize a stunning number of isoform products from a single gene due to several nuclear/cytosolic base editing mechanisms on DNA and RNA strands, as well as different multiple ribosomal combinations of peptide stretch mutations” (Fig. 1) [see 1–8].

According to this idea of RNA + peptide mutations in living cells, genetic mutations happen before any changes in the natural environment. Internal cell or tissue mutations can be selected, much as how we would pick a certain cabinet drawer to open, because advantageous, beneficial, benign point mutations that support organismal evolution are not caused by the natural environment. Every cell or group of cells in a tissue has mutations that are already present and spontaneously produced in them for multifunction and/or extension of cellular activities due to high proteome diversity. These mutations contain advantageous, beneficial, positive, real healing mutations.

If mutations in RNA and peptides can produce a wide range of proteins, evolution can proceed without amassing genes or enlarging the genome. In multicellular eukaryotic cells, alternative splicing is thought to be the primary source of RNA and protein variation [78]. RNA + peptide mutations are two additional sources of transcriptome and proteome variety that are far more potent and versatile than alternative splicing for producing an extremely large number of variants in a specific protein gene family [see 1–8, 78–79]. The force of various numerous functional molecules in a cell is considerably increased by splicing + editing + ribosome peptide mutation [79]. This suggests that it may be essential to comprehend how bacterial cells adapt and evolve. In addition, cephalopods with plasticity in their brains, such as cuttle, squid, and octopus, have been demonstrated [80]. It is probable that RNA + peptide mutations are also important and required in avian hearing systems given that the calcium-activated potassium channel slowpoke (cSlo), which has hundreds of mutation variations and each of which is tuned to a distinct sound frequency, is [81]. Combinations of splicing, RNA, and peptide mutations may be useful for ultrasound detection in a number of organisms, including bats, dogs, dolphins, fish, and frogs [82,83,84,85]. For the ability to distinguish between hundreds of sounds and detect high- and low-frequency noises in quiet and complex noisy environment, RNA + peptide mutations may be crucial. To remove a wide range of toxins and/or make a wide range of metabolites that give the human body energy, continuous RNA + peptide mutations may impact not just the auditory system but also immune and metabolic organs like the gut and liver. A few examples of the organs and tissues that could engage in harmful poisonous toxic metabolic processes and develop cancer include the brain, heart, liver, kidneys, skeletal muscle, and adipose tissue [86,87,88,89,90,91]. The propensity for cancerous cells, tumors, metastasis, and RNA + peptide editing activities may, therefore, be connected. Cancer is not brought on by the genes y or x. Cancer may originate from a mutation that alters the complex RNA + peptide editing process that underlies it in the ribosome compartment of the cell. Cancer may develop when one of these countless other peptide motif mutations, such as Glycine insertion in the right flank of Cysteine, does not occur (Fig. 1).

Tissue-specific RNA and peptide mutations in insects

The diversity of Dscam protein isoforms in the central nervous system of insects increases dramatically as a result of a gene like Down syndrome cell adhesion molecule (Dscam), which can exist in the form of more than 38,016 distinct RNA variations [92]. In light of proteome analysis and numerous peptide mutations in the ribosome, it is significant to take note of this. Factually essential for the development of neural connections in the CNS, Dscam is an immunoglobulin that performs as an axon guidance receptor in the fly brain. When the diversity of Dscam variations can help in identifying neuronal cells, it becomes necessary to look for RNA + peptide mutations in this protein gene family. Splicing might provide a means of producing unique cell identities in the CNS [93]. However, RNA + peptide mutations have the potential to significantly increase the number of cell identities in the CNS, neurons, dendritic cells, epithelial cells, stem cells, and elsewhere, across the body [see 1–8]. Similar to this, there are around 20 RNA variations (A-to-I and C-to-U mutations) of the Blattela germanica sodium channel BgNav, each of which correlates to a particular gating characteristic [94]. It is possible that A-to-I and C-to-U conversions in the cytoplasm will be followed by ribosome peptide mutations that will significantly improve the profile of specialized ion channels with distinct status or gating properties. Neuronal ion channels, BgNav, and Dscam immunoglobulin are not the only proteins that are mutating. Numerous even more “versatile” (“prone to mutation and multi function”) protein gene families have also been identified, in addition to the ovarian endocrine system of an insect vector, such as Rhodnius prolixus, the main triatomine vector of the Chagas parasite [95, 96]. Given that these cells and tissues, like the moth sex pheromone gland, have extremely high metabolic rates, the presence of so many editing sites in the ‘CSP’ genes is not surprising [see 1–8, 97–98]. The mechanisms of alternative splicing and RNA editing, which regulate how genes are expressed, and ribosome peptide mutations may be closely related. The potential for tumor cell appearance and cancer progression can then be closely correlated with the full regulatory epigenetic system, which includes alternative splicing + RNA editing + ribosome peptide mutation.

Insects, in particular whiteflies, bees, beetles, flies, butterflies, and moths, can act and serve as the initial scientific models for the investigation of RNA + peptide mutations prior to cancer research. The RNA sequence of the whitefly Bemisia tabaci shows localized base substitutions that correspond to biotype (B or Q), possibly in a way that is associated with pesticide resistance [97, 98, 99, 100]. The honeybee brain contains a strikingly high proportion of A-to-I and U-to-C changes [100]. There is a correlation between behavior and this intense RNA editing activity in the bee’s brain [101]. About one-of-a-kind A-to-I RNA editing signatures exist in the insect brain, which are probably crucial for the cell and synaptic range required for a variety of signals and CNS functions [102]. In addition to the integrative function of the brain, genetic RNA + peptide editing can assist repaint blue Morpho or Papilio wings to evade predators and/or find a compatible mate [103, 104]. For molting, warning, and migration (when adults seek out green overwintering grounds), as well as for reproductive behavior (when females seek out brightly shiny, metallic colored blue males), the evolution of various editing mechanisms at a butterfly mimicry or wing coloration locus is significant [105,106,107,108]. It is known that some wing color patterns can emerge in response to environmental stressors including temperature and climate change [109, 110]. It’s interesting to note that certain antennal olfactory protein genes are induced in migrant moths in a population-dependent manner, which denotes that the antennal olfactory protein profile can be physiologically produced as a result of specific flowers, lands, crops, and/or plant odors [111]. This olfactory system adaptation is comparable to pattern adaptation, development, shape, and morphology of the butterfly wings. Insects may regulate (and coordinate) the evolution and adaptation of different tissues in response to their environment, thanks to their high degree of polymorphism in RNA editing, alternative splicing, and protein gene expression [1,2,3,4,5,6,7,8, 100]. Along with the brain, wings, antennae, and leg appendages, the female moth’s sex pheromone gland appears to be a significant site for a variety of RNA and peptide mutations that may be required for pheromonogenesis, or the biosynthesis of sex pheromones. The silkworm moth B. mori shows a number of RNA + peptide mutations at the level of the female sex pheromone gland, particularly in the lipid, fatty acid, and pheromone precursor transporters, or ‘CSP proteins’ [see 1,2,3,4,5,6,7,8, 98, 100, 112].

Pheromone-producing cells, antennae, the epidermis, the cuticle, the gut, and fat body exposed to chemical stress are only a few physiological systems where ‘CSPs’ (or ‘ChemoSensory Proteins’) have been found. Additionally, developing tissues and regenerating legs have been found to express them [8, 112, 113]. This ‘versatile’ family of small soluble protein transporters is also found in bacteria, where it transports xenobiotics, plant natural products, and/or fatty acid lipids. The metabolism, growth tissue repair, growth neural tissue development, insecticide resistance, and sex pheromone production/perception are just a few of the multiple roles that this protein family has been linked to [8, 48, 49, 97,98,99,100, 112,113,114,115]. Despite their billions of years of evolution, eukaryotes and prokaryotes may share the same core RNA + peptide mutation mechanisms that control cells and lives [115]. Understanding CSPs in insects and bacteria may, therefore, be necessary to comprehend how cells, tissues, and organs use RNA editing to multiply, increase, expand, proliferate, and/or acquire a highly specific and specialized function.

Tissue-specific RNA and peptide mutations in moth CSPs

Although the function of these multiple RNA variants and peptide isoforms is unknown, the tissue specificity of so many CSP mutations, including base deletions, insertions, stop codon replacements, and numerous peptide editings in addition of A-to-I, C-to-U, U-to-C, G/C-to-I, and U-to-A/G conversions as found in moths, strongly suggests novel mechanisms governing cell multifunction, or cell multipotency, pluripotency, and totipotency [1,2,3,4,5,6,7,8].

The core of N- and C-helical domains and/or the flank of Cys-Cys disulfide bridges are two critical protein functional location sites where these peptide mutations occur. These location sites are not chosen at random. Since the mutations in the N-terminal region occur in a predetermined order, they are also not random: (1) amino acid inversion mutations (free tail), (2) inversion after intron boundary (α3), (3) Glycine insertion near Cys at position 36, (4) Glycine or Glutamine insertion in the left flank of Cysteine at position 55, and/or (5) insertion of one Glycine residue in the right flank of Cysteine at position 55. The inclusion of Gly, Gln, or a specific three-residue pattern like Phenylalanine-Valine-Phenylalanine also completely eliminates arbitrary, chance or random events in favor of a tight mechanism (Fig. 1B) [see 1–8].

The structural analysis of BmorCSP6 and BmorCSP11, the two BmorCSP proteins in the silkworm sex pheromone gland with the largest isoform variety, eventually serves as a particularly effective illustration of this [1]. The BmorCSP6 and BmorCSP11 model structures’ peptide-level mutation comparison points to the occurrence of tight regulatory events to substantial peptide point mutations in the ribosomal BmorCSP protein sequence (Fig. 2). BmorCSP6 and BmorCSP11 are single intron genes with lengths of 747 and 843 bps, respectively (BABH01021424 and BABH01021434). The nature of the inserted elements (retroposon/BmorCSP6: Bm1; BmorCSP11: BmRTE) differ most notably. BmorCSP6 and BmorCSP11 can be expressed in the adult CNS, head, antennae, legs, epidermis, wings, fat body, and sex pheromone gland in addition to many other tissues. Following insecticide stress, the BmorCSP11 gene is markedly upregulated in the gut [see 113]. Both CSP6 and CSP11 are upregulated in the fat body of the moth after avermectin exposure [113], indicating that they are involved in a number of metabolic processes like lipid synthesis, storage, and release in response to energy needs, hematopoiesis, cytoplasmic fatty acid droplets, and lipid fuel metabolism. CSP6, which is present in Escherichia coli and other enterobacterales, is most likely involved in a wide variety of metabolic activities [8, 49, 114, 115]. In term of amino acid composition, BmorCSP6 and BmorCSP11 share about 37% identity [1]. We sequenced 35 variants of BmorCSP6 and 27 variants of BmorCSP11 in the peptide libraries from the female moth sex pheromone gland; however, neither BmorCSP6 nor BmorCSP11 had splicing forms. Although BmorCSP6 and BmorCSP11 both had four C-tail variations, the N-tail changed the most (Fig. 2) [1]. On the other hand, BmorCSP6 and BmorCSP11 can be easily separated from one another on the number and location of mutations in the central core of the protein. The 67-RKIV–RKYD-90 motif in BmorCSP6 has roughly 10 mutations, compared to BmorCSP11’s 14 mutations in the 28-KCFLDQGPC-36 motif (Fig. 2) [1].

Fig. 2
figure 2

Positions of peptide mutations on the model structures of BmorCSP6 and BmorCSP11. Protein structure modeling is done using Swissmodel.expasy.org. MbraCSPA6 served as the template reference (1kx9.1, X-ray, 1.6 Å, monomer, ligand none) [73]. The moth CSP structure is described by four cysteines in conserved positions (Cys29, Cys36, Cys55, and Cys58), two disulfide bridges (S1: Cys29-Cys36, S2: Cys55-Cys58), and six alpha-helices (α1–α6) [73,74,75]. The moth peptide mutations and locations were reported by Xuan et al. (2014) [1]. A The location of peptide mutations on BmorCSP6. + Cys: Cysteine insertion mutation, + Lys: Lysine insertion mutation. In bold black: genomic DNA-encoded amino acid pattern, in yellow: variant amino acid motif in the flexible loop between α5 and α6 (ADYWEQMKA-to-ADYWEACTK, ADYWECATK, AAYWECATK), in red: variant amino acid motif at the end of the C-tail (FLAGQN to FLAGMK, FLAGQDEK, FLAGGAGK). An area of the N-terminus with a high mutation burden (α1) is designated by the purple asterisk. The locations of the + Glycine insertion mutation are indicated by the two red arrows: (1) on the side of Cys55 to the left of S2, and (2) in the flexible loop between α5 and α6. B The location of peptide mutations on BmorCSP11. + Cys: Cysteine insertion mutation, + Cys-Cys: double Cys insertion mutation motif, X + Cys: motif deletion and insertion of Cysteine. In bold black: genomic DNA-encoded amino acid pattern, in red: variant amino acid motif at the end of the C-terminal tail (TAFINAMD to TAFCTDNK, MDAK, QCSK). The triangle designates the location of the conversion mutation (KED to DEK), which occurred right before the C-terminal tail α6. The N-terminus (α1) and the disulfide bridge S1 (Cys29-Cys36, α2) are where there is a considerable mutation load (mainly + Cys), as indicated by the huge asterisk in bold purple. The three red arrows point to the positions of the + Glycine insertion mutation: (1) to the left of Cys29 (S1), (2) to the left of Cys36, and (3) to the right of Cys36 (S1, α2–α3 loop)

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The protein structures of BmorCSP6 and BmorCSP11 are illustrated in Fig. 2, along with the locations of specific peptide mutations. Modeling of the protein structures is done using Mamestra brassicae chemosensory protein A6 (MbraCSPA6, 1kx9.1, X-ray, 1.6 Å, monomer, ligand none) as a template reference (see methodology in chapter 5), with a focus on α-helix profiling coverage [116,117,118,119]. Alpha-helical profilings are largely conserved across CSPs. Since the positions of α-helices in MbraCSPA6 (X-Ray) and BmorCSP1 (NMR) are so strikingly similar, we chose the X-ray protein crystal for modeling; the only variation between the two 3D structures is the length of α2 [see 73–75]. Insertion mutation (+ Cysteine) occurs at α-helices α1 and α5 of the BmorCSP6 model. The + Cys mutation also occurs after the α-helix α3. After α-helix α1, a + Lys mutation occurs (EN-to-EKN). A particular motif replacement (ADYWEQMKA-to-ADYWEACTK, ADYWECATK, or AAYWECATK) is present in the loop between α-helices α5 and α6, and several mutations are found in the N-terminus (α1). FLAGQN is replaced by FLAGMK, FLAGQDEK, or FLAGGAGK on the C-tail (α6). On the left side of the second disulfide bridge, at position 55, the flank of Cysteine in BmorCSP6 (as well as BmorCSP1, BmorCSP2, BmorCSP3 and BmorCSP14) has a + Glycine residue insertion. Another Glycine is added between Tyr-88 and Asp-89 in the middle of the loop, between positions α5 and α6 (Fig. 2A).

The profile of Glycine insertion mutations in BmorCSP11 protein is rather very distinct. In BmorCSP11 (as well as BmorCSP4, BmorCSP7, BmorCSP9, BmorCSP13, and BmorCSP17), we could not find any + Gly residue insertion mutation at Cys55 [see 1, 8]. At the disulfide bridge S1’s boundary, adjacent to Cys29 and Cys36, BmorCSP11 protein has a + Gly mutation (Fig. 2B) [see 1]. Glycine is added near to Cysteine on both sides of the S1 disulfide bridge, i.e., on the left side of Cys29 and the right side of Cys36. Consequently, it appears that the moth CSP peptide mutations are definitely not random. Depending on the CSP, they show up in a very specific position. BmorCSP6, BmorCSP1, BmorCSP2, BmorCSP3, BmorCSP8, BmorCSP12, BmorCSP14, and BmorCSP15 all have a specific Gly mutation at Cys55. Gly can be inserted into CSP1 and CSP2 on either the left or right side of Cys55. It is exclusively inserted on the right side in CSP8, CSP12, and CSP15. CSP11, like CSP1, CSP3, CSP13, and CSP15, has + Glycine at the Cys29 level (DGC, FGC, KGC, and NGC). Among the Bombyx CSPs, only BmorCSP11 (and BmorCSP2) exhibit + Glycine mutation at Cys36 (S1, Fig. 2B) [see 1]. In BmorCSP2, Gly binds to Cys36 on the right side (-CGT-), but in BmorCSP11, it can bind to both sides (-PGC- and -PCG-) [see 1]. This strongly implies that the CSPs’ peptide mutations are under the ribosome’s control.

Phe-Val-Phe motif is mutated in BmorCSP7 in place of Glycine on the side of Cys36. This emphasizes how selective amino acid insertion mutations adjacent to Cysteine residue can be [see 1, 7–8]. BmorCSP11 variant has more than simply Gly-Cys; it also exhibits a variety of additional peptide mutations. Peptide sequencing of one variation of the BmorCSP11 protein revealed an insertion mutation (insertion of Cys-Ser-Glu-Cys motif: + Cysteine) at α1 [1]. Another kind of mutation or peptide rearrangement can result in the complete deletion and replacement of the QYDFDV motif by Cysteine alone at the N-terminus of BmorCSP11 [1] (Fig. 2B). Pro-Gly-Cys, Cys-Pro-Asp, Pro-Cys-Gly, and Cys-Gly motifs can accumulate near α-helix α2 [1]. Another + Cys mutation occurs in the C-terminal region (position α6). In the loop between α-helices α5 and α6, there are no mutations other than the KED > DEK conversion [see 1]. The C-tail of BmorCSP11 (α6) changes from FINAMD to FCTDNK, FMDAK, or FQCSK, providing an opportunity to anchor a new disulfide bridge Cys-Cys (Fig. 2B) [1].

The 3D model structures of BmorCSP6 and BmorCSP11 proteins were compared, and the comparison (see Fig. 2) strongly suggests a specific profiling of peptide mutations. In particular, the + Cys, + Gly, + Lys, + Cys-Cys, motif deletion and insertion of Cysteine, conversion mutations, and complete rearrangement of amino acid motif, as found in the C-terminal region (α6) and/or in the central loop between α5 and α6 α-helices strongly suggest not a random but a very specific profiling of peptide mutations. This could result in the development of a brand-new function (neofunctionalization) and/or an enhancement of an already existing function, depending on the cell, tissue, organ, and/or specific physiological situation. For instance, lysine residue insertion, can assist the protein acquire basic polar charges, enabling it to form hydrogen bonds with aldehyde carbonyl functional groups and enhancing binding affinity with fatty aldehyde molecules like hexadecenal and/or RNA ligands [120, 121]. Proteins that play a role in tissue formation, chromatin repair, cell–cell communication, cell cycle, signaling, splicing, and nuclear transport, among other activities, are driven by lysine mutations [122].

The function of a protein can change as a result of peptide mutations

There would be no association between the peptide mutations observed in Bombyx and cancer or genetic disease if the functional structure of the protein was unaffected. To see whether peptide mutations affect protein function, we looked at the mutation’s position and how it affected specific structural elements of the protein as a preliminary investigation for additional CSP-cancer research (Fig. 3). For modeling investigations utilizing SWISS-MODEL and QMEAN [123, 124], the X-ray structure of MbraCSPA6 shows a 30–44% match or sequence identity with BmorCSP6 (Seq Identity 44.23%, GMQE 0.67, QMEAN -0.9, QMEANDisco Global 0.68 ± 0.08) and BmorCSP11 (Seq Identity 29.52%, GMQE 0.62, QMEAN -0.20, QMEANDisco Global 0.66 ± 0.08; Figs. 2 & 3 and Table 3). Particularly in α-helical profiling, the coverage is really near to 100%. SWISS-MODEL-Expasy used BmorCSP sequence and CSP chimera, or chimeric sequences built from peptide mutation motifs, as templates. This was used in ClustalW for sequence alignment to test the working hypothesis that peptide mutations can change a protein’s function.

Fig. 3
figure 3

Effects of peptide mutations on the structure of the CSP protein (SWISSMODEL analysis). Variant protein structures are modeled using Swissmodel.expasy.org. MbraCSPA6 is served as the template reference (1kx9.1, X-ray, 1.6 Å, monomer, ligand none) [73]. The moth CSP structure is described by four Cysteines in conserved positions (Cys29, Cys36, Cys55, and Cys58), two disulfide bridges (S1: Cys29-Cys36, S2: Cys55-Cys58), and six alpha-helices (α1–α6) [73,74,75]. The moth peptide mutations and locations were reported by Xuan et al. (2014) [1]. Using ClustalW alignment, chimeric sequences resulting from peptide mutation and either BmorCSP6 or BmorCSP11, were built and then subjected to Swissmodel.expasy.org. The moth CSP mutations are characterized by a lack of alpha-helical turns (see above). A Effects of peptide mutations on the structural model of BmorCSP6. BmorCSP6: BmorCSP6-no mutation, BmorCSP6-Cys: BmorCSP6-KDGMQT to KDCTQT mutation, BmorCSP6-Gly: TACAKC to TAGCAKC, BmorCSP6-Cterm1: FLAGQN to FLAGMK, BmorCSP6-Cterm2: FLAGQN to FLAGQDEK, BmorCSP6-Cterm3: FLAGQN to FLAGGAGK, BmorCSP6-mut1: WEQMKAKY to WEACTKKY, BmorCSP6-mut2: WEQMKAKY to WECATKKY, BmorCSP6-mut3: BmorCSP6-ADYWEQMKAKY to AAYWECATKKY mutation model protein structure. B Effects of peptide mutations on the structural model of BmorCSP11. BmorCSP11: BmorCSP11-no mutation, BmorCSP11-Gly1: BmorCSP11-NYAKCFLD to NYAKGCFLD mutation, BmorCSP11-Gly2: DQGPCTAE to DQGPGCTAE, BmorCSP11-Gly3: DQGPCTAE to DQGPCGTAE, BmorCSP11-Gly4: NYAKCFLDQGPCTAE to NYAKGCFLDQGPGCGTAE, BmorCSP11-Cterm1: TAFINAMD to TAFCTDNK, BmorCSP11-Cterm2: TAFINAMD to TAFMDAK, BmorCSP11-Cterm3: BmorCSP11-TAFINAMD to TAFQCSK mutation model protein structure. The red arrows indicate specific structural changes, primarily in α1 and α6 (BmorCSP6) and α2 and α6 (BmorCSP11)

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Table 3 Model results of mutations on BmorCSP6 and BmorCSP11 structure compared to template 1kx9.1 chemosensory protein A6 (X-ray, 1.6 Å, monomer, ligand none) in swissmodel.expasy.org. (order by GMQE, global model quality estimation) [123]
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Modeling the structural elements built by peptide mutations sequenced in the female moth sex pheromone gland reveals that mutations like Cysteine addition, Glycine insertion, and entire C-tail rearrangement can induce particular functional modifications in the protein structure (Fig. 3 and Table 3). Table 3 lists the model results of mutations on BmorCSP6 and BmorCSP11 compared to the template 1kx9.1 chemosensory protein A6 (X-ray, 1.6 Å, monomer, ligand none). The results are sorted in swissmodel.expasy.org. by GMQE and QMEAN. QMEAN significantly changed in the + Glycine mutation at position Cys55 and the + Cysteine mutation in 77–86 domain (BmorCSP6; Table 3). Cys insertion can provide additional sites for the development of the disulfide Cys-Cys and assist in the formation of new fundamental intermolecular building blocks within the same cell. The overall structure of BmorCSP6 is unaffected by the addition of Cysteine to its N-terminus. It is believed that the insertion of Gly (near Cys) will have a much bigger impact, especially in the loop between α3 and α4 (Fig. 3A). The folding of α1–α2 and α4–α5 alpha-helices on the BmorCSP6-Gly protein model (insertion of Gly on the left flank of Cys55) may show how this mutation promotes prism ‘gating’, in which ‘CSP opens in response to a specific stimulus' (Fig. 3A). Additionally, in BmorCSP6 protein models (mut2 and mut3), insertion of a CATK motif in the loop between α5 and α6 has a very significant impact on protein folding but insertion of an ACTK motif in this place had little effect on protein folding (Fig. 3A). AAYWECATK replaces ADYWEQMKA, respectively adding an alpha-helical turn to α5 and removing one from α6, respectively (mut3; Fig. 3A). Even α1 appears to have undergone significant alteration in mut3 (loss of alpha-helical turn; Fig. 3A). Similar to ACTK and + Cys mutations, C-terminal mutations like FLAGQN switch to FLAGMK, FLAGQDEK, or FLAGGAGK had no effect on BmorCSP6’s overall folding (Figs. 2 and 3A). However, as seen with FLAGQDEK and FLAGGAGK, it is very possible that these C-tail mutations modify how a ligand molecule or protein partner interacts with it when the last turn of α6 is altered (the last turn shuts; Fig. 3A). The carboxyl terminus (C-tail) is where the majority of interactions between receptors and protein chaperones take place [125]. As with cytoglobin and salivary D7 OBP protein, it is connected to ligand-binding characteristics [126, 127]. It has been demonstrated that salivary D7 OBP protein’s C-terminal domain undergoes significant conformational changes upon binding hormone noradrenaline neurotransmitter, which serve to stabilize the bound ligand and expand the structural diversity of mediators that can be accommodated at the ligand-binding site [127]. The size, pliability, and composition of the C-terminal α-tail can be three important aspects of the ligand binding and releasing mechanisms that may change as a result of peptide mutations in the odorant binding protein (‘OBP’) family, whose C-terminal alpha-helix (α7) inserts into the binding pocket to expulse the ligand [128,129,130,131].

Modeling the varied protein structure caused by BmorCSP11-peptide mutations sequenced in the sex pheromone gland of the silkworm moth B. mori reveals notable variations in functional regions like the C-terminal tail (α-helix α6) and different turns of α-helix α2 (Fig. 3B and Table 3). QMEAN was changed for + Gly near Cys36 on BmorCSP11 (Table 3). When + Gly occurs on both sides of Gly36, this mutation eliminates the last turn of α-helix α2 (Fig. 3B). The structure of BmorCSP11 is unaffected when + Gly only appears on one side of Cys, but Glycine insertion near Cys29 breaks the central turn of α2 (Fig. 3B). Similar observations were made with BmorCSP1 and BmorCSP2, respectively [1, 2, 8]. Therefore, despite the fact that various mechanisms appear to change the structure of CSPs, loss of α-helical turns appears to be a frequent process for this protein gene family (Fig. 3B). It seems that the replacement of TAFINAMD with TAFCTDNK in the C-tail regulates the alpha-helix α6 (Fig. 3B). The last turn of α6 is shortened when TAFMDAK or TAFQCSK are used as substitute peptide mutations (Fig. 3B). Loss of α-helical turns in CSPs appears to be influenced by the protein group and α-helix position. BmorCSP11 and many other CSPs lose α-helical turns typically at the N- and C-termini, but BmorCSP6 gets a α-helical turn on position α5 (Fig. 3) [2, 8]. The fact that the α-helix at the bottom of the prism (α3), which represents the bolster base side of the CSP structure, is unaltered in BmorCSP1, BmorCSP2, and both the BmorCSP6 and BmorCSP11 protein models (Fig. 3) [1, 2, 8], is an essential element of this BmorCSP mutation analysis. These observations show that the ribosome exerts strict control over the nature and location of these peptide changes, strongly suggesting that they are not random.

This may suggest that critical structural components of a protein, particularly those at the N- and C-termini, can adapt and change as a result of peptide mutations, even though more complex structural analyses of the protein structure (AlphaFold, Linux, Mass Spectrometry, NMR spectroscopy, and X-ray) are required to make definite conclusions about a potential change in protein function. The two ends of the protein, which can take part in a variety of protein–protein interactions or have specific ligand-binding properties, may be significantly targeted by peptide mutations and protein recoding for multifunction.

It is crucial to consider this notion of changing protein partner or ligand stimulation through ribosome peptide mutations in new views, approaches, and tactics for cancer, CUP, and future therapeutics.

In fact, we might speculate that the majority of multifunctional molecules, like CSPs, that can influence a high variety of biological processes might have a regulatory mechanism that modifies peptide mutation in addition to frameshift, editing, and splicing. Specifically, a protein recoding caused by + Gly or + Cys mutations may be essential for cellular processes. More research is required to determine how + Gly near Cys or + Cys mutations in CSPs affect their functional outcomes. This could aid in our understanding of the molecular processes that lead to the synthesis of α-helices and/or the regulation of α-helical and α-helix–loop–α-helix patterns, which are essential for the design, positioning, and maintenance of protein multifunctionality. Protein multifunctionality is one way through which a cell develops more and executes more intricate functions while consuming fewer or no materials. It is likely that suppressing peptide mutations causes cancers like CUPs by preventing the multifunctionality of the protein.

"No matter how carefully the external conditions are applied, it appears that every cell is unique and has its own attitude, as is the case in biology. Cancer is brought on by a dysregulation and failure in protein recoding in the ribosome, not by aberrant splicing or single nucleotide base changes at the DNA/RNA level."

"The mutations +Gly and +Cys mutations are not lethal. Because these peptide changes are necessary for cell multifunction, cancer disorders develop, worsen, and spread if they occur in the incorrect side or do not occur at all."

Therefore, peptide mutations like those observed in the Bombyx moth for sex pheromone gland may enhance the production of stem cells, cell proliferation, neurogenesis, new dendritic spines or pyramidal neurons, or new connections in the brain for motor memory and/or sensory capacities [1,2,3,4,5,6,7,8, 100, 101]. Peptide mutations may be particularly important for a metabolic organ like the liver, gut, or kidney, which must carry out thousands of chemical metabolic interactions. When these interactions are highly dependent on peptide mutations like + Gly or + Cys, controlling protein recoding at the ribosome level may represent a new research direction for preventing all types of CUP malignancies.

The mechanism of RNA editing/peptide mutation for bacterial tumor treatment

With a better understanding of CSPs for CUPs, we would be able to not only comprehend some fundamental mechanisms underlying RNA editing and ribosome peptide mutations, but also to imagine some novel therapeutic approaches. The growth and development of genetic medicine and innovative gene therapies for cancers and CUP malignant tumors will require a thorough understanding of the fundamental mechanisms driving peptide mutations in the ribosome of insect cells. Studies using RNA and peptide mutations in bacterial 70S ribosomes may be even more significant. This would be extremely beneficial for the field of translational medicine in the treatment of cancer, CUP, and other hereditary genetic disorders (Fig. 4).

Fig. 4
figure 4

CRISPR editing bacteria as a basis for RNA + peptide mutation cancer treatment. A DNA- and RNA-dependent base mutations in malignant cells’ nuclei and/or cytoplasm mediated by editing bacteria. DNA > RNA: DNA-dependent base mutation (nucleus), RNA > RNAv: RNA-dependent base mutation (cytoplasm), RNAv: RNA variant, ADAR: Adenosine deaminase acting on RNA (A-to-I), ADAT: Adenosine deaminase acting on transfer RNA (C-to-U); APOB: apolipoprotein B mRNA editing catalytic polypeptide-like family (APOBEC) enzyme (C-to-U), rAPOB: reverse APOBEC (U-to-C), RNAP: RNA polymerase, RPOL: DNA-directed RNA polymerase subunit L (G-to-A), UDG: uracil-DNA glycosylase (GC-to-TA). Using bacteria with specialized editing capabilities, base mutations that affect DNA and/or RNA can be controlled, leading to mutant DNA/RNA variants that are compatible with healthy status. UBac: universal bacteria (controls DNA- and RNA-dependent base mutation, editing of all mutant variants), Bac1: bacterial type 1 (controls A-to-G), Bac2: bacterial type 2 (C-to-U in the nucleus), Bac3: bacterial type 3 (C-to-U in the cytoplasm), Bac4: bacterial type 4 (U-to-C), Bac5: bacterial type 5 (G-to-A, RNAP enzymatic activity), Bac6: bacterial type 6 (G-to-A, RPOL enzymatic activity), Bac7: bacterial type 7 (DNA-dependent polymerization, GC-to-TA, uracil-DNA glycosylase activity). B Peptide mutations mediated by editing bacteria in the ribosome of cancerous cells. UBac: universal bacterial (controls all peptide mutations and permits editing of all different kinds of peptide variations), + : insertion, > : motif replacement, red cross: conversion mutation, X: deletion, X + Cys: motif deletion and insertion of Cysteine. Bac1: bacterial type 1 (control over the Phe–Val–Phe insertion and regulation of α-helix profiling), Bac2: type 2 (control over the Glycine insertion mutation and the shortening of the α-helix), Bac3: type 3 (control over the Cysteine insertion mutation and the formation of a new disulfide bridge), Bac4: type 4 (control over the insertion of Glycine and Cysteine, the shortening of α-helix and the formation of new disulfide bridges), Bac5: type 5 (mutation to Ala-Cys-Thr-Lys), Bac6: type 6 (mutation to Cys-Ala-Thr-Lys, formation of new intramolecular disulfide bridges), Bac7–Bac12: types 7–12 (change in amino acid composition in α-helix), Bac13: type 13 (conversion mutation, switch of amino acid motif), Bac14: type 14 (deletion mutation), Bac15: type 15 (control of deletion mutation and insertion of Cysteine, new intermolecular S–S formation). UBac can be used to manage numerous peptide variants of all shapes and sizes. Bac3 and Bac14 bacteria can be combined to help control deletion mutations, + Cys, and the development of new disulfide bridges. Any + Gly near Cysteine at position 58 is removed in Bac-CRISPR. Lys-Glu-Asp motif can be changed to Asp-Glu-Lys using the endogenous type I CRISPR-Cas system established in bacteria: (1) CRISPR chops the peptide chain and marks the cutting edges, 2) the peptide amino acid cassette identifies the cutting points and insert the entire correct sequence motif. Bac-CRISPR: control over the peptide or amino acid motif’s composition and mutation conversion

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Currently, it is rather unclear how the ribosome controls the high and widespread trafficking of amino acids, pairs of amino acids, entire motifs, a wide range of motif types, and peptide modifications. The results of CSP expression in prokaryotes [see 8, 49, 100, 114, 115] suggest that insects and microorganisms should both be carefully searched for CSP peptide mutations. It is possible that all eukaryotic and prokaryotic cells have a common pathway for the biosynthetic machinery of proteins as revealed by the mechanisms of RNA + peptide mutation in insect and microbial species [132, 133].

With ribosome peptide mutations, we propose a novel area of research for prokaryotes. Prokaryotes’ RNA editing has garnered a lot of interest (Table 2). TadA was the first bacterial RNA editing enzyme to be discovered [134]. Then a genetic structure that is somewhat similar to that described in insects was found in the bacterial kingdom, including a variety of RNA-dependent enzymes, RNA-dependent RNA polymerases (RdRps), ADAT, ADAR, and variations (see Table 2) [135]. The development of bacterial strains that can restore particular base mutations depends on these enzymes, which may be extremely ancient RNA editing systems that mediate A-to-I and C-to-U on pre-mRNA in the nucleus and/or the cytoplasmic cellular compartment (Fig. 4A). Some bacterial species, such as E. coli, are known to express a variety of DNA- and RNA-dependent polymerases [136]. Some universal bacteria (UBac), such as E. coli, may be particularly effective at correcting specific RNA editing dysfunctions since A-to-I and C-to-U abnormalities can be treated at the DNA, RNA, and/or RNA variant levels (Fig. 4A). Additionally, we speculate that E. coli may be as useful to address peptide mutation dysfunction given the similarity between genetic mutations in bacteria and insects. Controlling peptide mutations in E. coli may have the dual benefits of decreasing the strain’s infectious toxicity and using the bacterium as a potential cancer treatment.

A universal bacterial (UBac) model that can handle any DNA/RNA change is proposed in response to multiple deaminases and extra mutational differences that have been described in various bacteria, including APOBEC, reverse APOBEC, U-to-C mutations, and UDG-like enzymes (Fig. 4A). It has been shown that bacterial cells possess AID/APOBEC3s, which may be the last remnants of a very ancient innate system [137]. Gut bacterial species like E. coli and methylotrophic yeasts like Pichia pastoris have become crucial in the production and isolation of mutagenesis enzymes involved in cancer and immunity. This strongly implies that specific microbial strains—like E. coli and P. pastoris—must be used in genetic editing biotechnology and a variety of cancer therapeutic applications (Fig. 4A) [138]. Even while each APOBEC enzyme in mammals has the identical structure, their editing functions (DNA or RNA targets) and tissue-specific expression are rather incredibly different [139]. This could pave the way for the development of a specific bacterial strain expressing APOBEC for the purpose of treating a very particular cancer or tumor.

Similar to this, bacterial UDG repair activity (such as the cold-active UDG from Bacillus, “cUDG-like”, the heat-labile UDG activity from the marine psychrophilic bacterial strain BMTU3346, or other extremozymes) offers a wide range of functions in relation to cancer and tumor suppression, depending on tissue specificities, temperature spikes, and physiological conditions [140,141,142]. These UDGs, which are structurally more flexible than other editing enzymes, provide significant biotechnological potential for tissue adaptation and cancer gene therapy. To regain full control of epigenetic regulation and editing processes at the level of DNA, RNA, and/or RNA variant mutations in the various cellular compartments, integrating Bac1, Bac3, Bac5, and Bac7 may be very beneficial (Fig. 4A). With the aid of specialized bacterial strains and/or DNA/RNA editing enzymes, it is possible to address the tissue specificity of cancer genes (Fig. 4A). This is a really compelling argument, especially in light of the fact that it has been shown that certain tissues can cause various editing enzymes to function incorrectly. APOBEC has been linked to mutations in the bladder, head, neck, breast, and lungs [143]. An A-to-I ADAR enzyme deficit is usually related to cancers of the breast, liver, and lungs [144]. In addition to brain nerve leukodystrophy, the prostate, the lungs, and a number of hematologic cancers have all been linked to RNAP/RNApol [145,146,147,148]. A specialized bacterial approach to cancer treatment may also be used to address chemotherapy resistance and/or some specific epigenetic changes directly linked to cancer drug chemical resistance [149]. As a result, many bacterial prokaryotic systems with a high frequency of A-to-I, C-to-U, and GC-to-TA base conversion mutations may have a distinct advantage not only in recoding DNA, tRNA, microRNA, and protein coding genes in human cells, but also in mediating or maintaining the curative positive effects of anticancer agents to tumor tissues and minimizing toxic lethal side effects in chemotherapy conditions [150, 151].

In addition to all types of RNA editing, bacteria may be useful for all forms of peptide mutations. Increased protein variety and a cell’s capacity to respond to any harmful hazardous environmental or physiological changes are probably determined by the sequence and position of peptide mutations within the ribosome [see 1–8, 97–98, 113, Figs. 1,2,3,4]. The origin of the first cell or protocell, which took place 3.8 billion years ago, may be traced back to this basic cellular adaptation phenomena, which is now quite prevalent [see 8, 115]. These unique ribosomal mechanisms defy the biological consensus that a single gene codes for a single protein; in addition to alternative splicing and RNA editing, we can also ascribe these mutations to peptide editing pathways that produce a remarkable diversity of proteins. Bacterial, insect, and human cells’ ribosomes may have a large number of peptide editing enzymes (perhaps in the ubiquitome) absolutely required for the cell to acquire and develop a new gene function (Fig. 4B). These peptide variations might not be harmful or lethal mutations, and they also could not cause ache, discomfort, illness, pain, problems, symptoms, or other negative effects. There is a very apparent structure to these tissue-specific + Phe-Val-Phe, + Cys, + Gly, + Gln, or X + Cys mutations [see 1–8, Figs. 2 & 3 in this work]. They might be necessary to address intra-cellular heterogeneity and drug therapy resistance at the genetic, epigenetic, and ‘epiproteomic’ levels [see 115, 150–151].

Therefore, it is possible that the discovered changes in the insect ribosome peptide could serve as a diagnostic indicator for CUP. There are many different types of cancer, yet many of them lack a clear etiology. Currently, if it is not feasible or reliable to determine the type of cancer cells, the diagnosis is “CUP” (“Cancer of Unknown Primary”). The prevalence, tissue specificity, abundance, functional impact, impact on protein structure, and high diversity of mutations, including + Phe-Val-Phe, + Cys, + Gly, + Gln, > Ala-Cys-Thr-Lys, > Cys-Ala-Thr-Lys, > Glu-Pro-Gln, > Glu-Gln-Pro, > Glu-Pro, > Pro-Glu, > Gln-Glu, > Gln, amino acid inversion, and deletion + Cysteine insertion, all point to this type of peptide changes in the ribosome as the primary cause of CUPs, even if the regulatory mechanisms causing these mutations have not yet been identified. This suggests that the insect or bacterial cell that is prone to peptide mutations may be crucial for the therapy of CUP (Fig. 4B). Peptide recoding enzymes can differ depending on the cell type, tissue, or bacterial species, similar to ADAR, APOBEC, RNAP/RNApol, and other RNA editing enzymes. So, it is conceivable to think about using specific bacteria and peptide mutations to treat and cure cancers and malignancies like CUPs, which, in the end, could be referred to as “Cancers of Peptide Primary”.

For these peptide mutation-related primary cancers, the glandular system and stem cells might be of most concern. For CUP, gland and stem cell-specific cancer therapies, choosing a particular microbial strain or peptide mutation may be highly beneficial (Fig. 4B). Protein recombinations based on those observed in Bombyx CSPs may be one of the most critical mechanisms for multipotent/totipotent stem cells in a tissue or organism to differentiate into a specific cell type [see 1–8]. Stem cells, like CSP proteins, play a crucial role in many different physiological systems, including tissue regeneration, hemocyte phagocytosis, innate and adaptive immunity, neurogenesis, brain repair, and neuroplasticity [152,153,154,155,156]. Particularly, there is a strong connection between stems cells and the endocrine glandular system [157, 158]. It is interesting to note that diversity of peptide mutations, according to the female silkworm moth sex pheromone gland, tends to occur preferentially in the glandular system [see 1]. Consequently, a glandular tissue could be targeted specifically using a novel gene therapy based on bacterial strains and peptide mutations. Given that peptide mutations are relatively prevalent in insect glands, it stands to reason that peptide mutations in the cell ribosome may control a range of fatty acid and hormone biosynthetic pathways, as well as specific hormonal activities, targets, or modes of action. Similar to thyroid hormone transporters and pathway genes, thyroid hormone receptors come in a variety of sizes and shapes. Their ratio and volume are highly tissue specific [159, 160]. It follows that it is most likely that some modifications to the ribosome’s peptide mutation machinery are responsible for cancers of the adipose tissue, adrenal gland, breast, hypothalamus, kidney, ovary, pancreas, parathyroid gland, pineal gland, and prostate, among other types of cancer. A specialized bacterium for ribosome peptide mutation correction may aid in the growth, development, and evolution of stem cell or gland-specific cancer medicines (see Fig. 4B) [161, 162].

We found peptide editing in at least two families of pleiotropic transporter proteins, CSPs and OBPs, by examining the sex pheromone gland of the female moth [see 1, 8].

Pleiotropic transporter proteins with peptide mutations, such as CSPs and OBPs, imply that peptide mutation processes exist in a variety of different living organisms besides insects, such as bacteria, fungi, cephalopods, protists, and plants. Each of these species possesses a variety of editing mechanisms that have been demonstrated to change the genomic sequence in the cell nucleus (see Table 2). Although G-to-A mutations have been described in rodents, primates, and cephalopods (Table 2), there has not been any research toward using peptide mutations and plasticity in cell ribosomes to drive evolution in specific supraorganisms. It is rather critical to modify the size and arrangement of α-helices along a transporter sequence in humans utilizing some ribosome peptide mutations because it is possible that these two types of ‘strange’ odd mutations (G-to-A conversion and peptide mutation) enhance the brain’s internal rewiring. For example, Bac2 or Bac4 bacteria may be able to regulate peptide mutations that shorten the α-helices, such as those found in the silkworm [1, 8] (+ Gly, + Gln, + Phe-Val-Phe, etc.; see Fig. 4B). Various bacteria, especially those with + Cys, may help restore the tissue’s capacity to form new intra- and/or inter-disulfide bonds (Fig. 4B). Additional ‘bacterial’ peptide mutations may alter the composition of amino acid residues in secondary structures such as α-helices and the ligand property potentials in the α-helical turn in various families of transporter proteins (Fig. 4B). Based on these structural alterations and the significant number of ribosome peptide mutations sequenced in the moth sex pheromone gland, we propose that we look into peptide editing in humans and determine the mechanism underlying + Gly, + Cys, or + Phe-Val-Phe mutations in some transporter protein families in normal versus cancer patients. Then based on bacterial bioproducts and ribosomal peptide mutations, we might be able to determine the root cause of CUP and, ultimately, develop a novel cancer therapy strategy.

Our novel strategy to treating cancer may involve modified Lactobacillus built with the help of CRISPR/Cas biotechnology, according to the most recent cutting-edge research [163,164,165,166,167,168,169]. Our approach, however, stands out from all others since we are focusing on the ribosome rather than the genome, which will ultimately result in protein structure rearrangement (Fig. 4B). Based on the CRISPR system’s ability to correct precise point mutation, such as single base deletion and/or insertion at the genome, we intend to build a CRISPR bacterial system that aims to delete, add, and/or replace a specific amino acid residue or amino acid pattern at the level of peptides. We have no interest in adding a gene or plasmid cassette. In the end, we intend to use CRISPR to express a single guide RNA or peptide cassette with the ribosome as the primary target rather than the nucleus. In-frame deletions and insertions by one-step transformation, in the manner indicated for locating genomic loci for particular chromosomal alterations [163,164,165,166,167,168,169], may not be possible in our CRISPR approach for ribosome editing. While it has been demonstrated that CRISPR–Cas may successfully delete a gene or a nucleotide base pair, we here propose a new challenge: employing CRISPR-based technology for amino acid inversion. This new approach most likely requires a mechanism like CRISPR–Cas that can eliminate one or two amino acid residues, but also another mechanism that brings the correct mutation at a specific position, such as Glu-Pro, Pro-Glu, Gln-Glu, Gln, Lys-Glu-Asp, or Asp-Glu-Lys motif in the free C-terminal tail (Fig. 4B). This could involve the ability of the enzyme, the spliceosome, the ubiquitome, the amino acid transporter cassette, or any combination of these to detect a particular amino acid pattern, such as the pattern at Cysteine at positions 29, 36 or 55. The removal of + Gly near Cys58 might need CRISPR cutting. In the vicinity of Cys58, no Gly should be inserted [see 1]. Although CRISPR-based technology should be enhanced to enable molecular peptide editing for + Cys or + Gly in the target cell or tissue sample, it may be useful to delete an erroneously improperly incorrected inserted amino acid residue. We probably first need a genotyping sensor to identify the detrimental faulty mutation in a cell or tissue sample [99]. The endogenous type I CRISPR–Cas system may then need to be used in two phases: 1) cut the peptide chain at a specific point, removing the wrong, incorrect or misplaced amino acid to mark the cutting edges; and 2) replace the incorrect faulty mutation with the right proper amino acid residue, pair of amino acids, or whole sequence motif [170] (Fig. 4B). In a manner similar to a ‘reverted’ directed-mutagenesis (we do not produce a mutation, we correct it) and/or genome editing by CRISPR–Cas as already developed for gene editing in cancer, as well as in assisted biotechnological recombineering of Lactobacillus bacterial species, such as L. casei, L. crispatus, L. reuteri, and L. plantarum [170,171,172,173,174], the amino acid transporter cassette is oriented by the markers at the fissure to bring and deliver the repaired mutation to the proper site (Fig. 4B). Based on the study of L. for precision engineering and CRISPR editing, as well as our new knowledge of mutation and CRISPR, we may be able to build a novel approach for cancer biology and therapy that will incorporate ribosome peptide editing, particularly in lactic acid bacteria.

Perspectives and future research

We may examine not only RNA but also peptide mutations in humans and prokaryotic cells, which are known as beneficial bacteria in many conditions such as obesity and cancer, to bring the RNA mutation landscape of cancer to the peptide mutation landscape of cancer. Future strains that have been coupled with specific peptide mutations, and CRISPR editing may be very useful for targeted gene therapy and personalized medicine. This comprises changing the immune system and gastrointestinal tract with commensal bacteria while concentrating on ribosome peptide mutation dysregulation as a significant contributor to CUP.

This approach requires first identifying the relevant biological target or peptide mutation in the ribosome, followed by an understanding of how it leads to a disease condition like CUP in the gland or nervous system. After insects and bacteria, a wide range of species, from worms to mammals, must be studied to determine the significance of peptide changes for the best optimal behavioral response. This is necessary to find out if other organisms have comparable mechanisms for transporter protein recoding and their effects on certain cells or tissues. It is also essential to build a new evolutionary perspective on cell adaptability based on multiple peptide mutations. The theories of evolution and natural selection based on peptide mutation principles typically explain how moths can change the color of their wings, smell, and/or detect billions or hundreds of thousands of sensory signals, and/or deal with hundreds of toxic chemical compounds, how insects can deal with a variety of chemicals found in plants, and/or how coleoid cephalopods (squids) have evolved such a large, flexible, and complex brain, as well as such a diverse and adaptable body. The basis of genomic and personalized translational (precision) medicine in humans may then shift to concentrating on specific ribosomal dysfunctions, i.e., composition- and position-specific modifications of peptide mutations in protein recoding, after highlighting peptide mutations in insects, mollusks, and squids. Mutations in particular protein gene families, including the human solute transporters in the neurosignaling, neurons, and endocrine systems, can result in a variety of tissue cancers depending on their structure and location. Using bacterial genetic tools, cells with particular combinations of peptide mutations (ribosomal editing mechanisms) for protein biosynthesis repair are notable particularly in cases of altered protein gene expression, such as in CUP, gut, lung, or white blood cell cancer. Even more challenging but noteworthy and promising applications of new human tools in genetic medicine include the delivery of engineered cells, patient-derived cells with specific editing mechanisms or CRISPR strategies to match all of common biological processes, particularly protein biosynthesis in the ribosome, as well as stem cells for tissue regeneration and cancer transplantation. Bacteria, insects, cell glands, and stem cells should be employed as model studies for ribosome peptide mutations to discover novel mechanisms, to regulate the production of functional protein isoforms, and to foresee new cancer treatments based on mutations as ‘strange’ as + Glycine residue close to Cysteine position. In this context, mutations like + Glycine adjacent to a Cysteine residue are seen as cellular adaptations, or the evolutionary process for neofunctionalization in cells, or tissues, rather than conditions that can result in disease. By focusing on this particular mechanism, a solution to the problem of human health and cancer, especially CUP, could be developed.