Recombinant Pig 40S ribosomal protein S23 (RPS23)

What is the basic structure of recombinant pig RPS23 protein?

Recombinant pig RPS23 is a 142 amino acid protein (spanning residues 2-143) with a molecular weight of approximately 16 kDa, typically expressed with a His-tag for purification purposes. The protein can be recombinantly produced in yeast expression systems with purification yields of >90% purity . The amino acid sequence (GKCRGLRTARKLRSHRRDH KWHDKQYKKAHLGTALKANPFGGASHAKGIVLEKVGVEAKQPNSAIRKCVRVQLIKNGKKITAFVPNDGCLNFIEENDEVLVAGFGRKGHAVGDIPGVRFKVVKVANVSLLALYKGKKERPRS) is highly conserved across species, reflecting its essential role in ribosome function . The protein contains distinct domains, including an evolutionarily divergent N-terminal extension (residues 1-46) and a more conserved C-terminal globular domain (residues 47-143), with the N-terminal region being particularly important for protein-protein interactions including with hydroxylases .

Where is RPS23 located within the ribosome, and what is its primary function?

RPS23 is positioned at the heart of the ribosomal decoding center within the 40S ribosomal subunit, making it critically positioned to influence translational accuracy. Crystal structure analyses have confirmed RPS23 as a core component of the "accuracy center" that directly influences how ribosomes interpret the genetic code . Within assembled ribosomes, certain regions of RPS23 (particularly the N-terminal extension) become buried and inaccessible to modifying enzymes, suggesting that post-translational modifications must occur before ribosome assembly . The protein's strategic position allows it to affect both elongation accuracy and termination efficiency during protein synthesis. Mutations in RPS23 and its bacterial homolog (S12) have been historically linked to alterations in translational accuracy, first discovered through seminal work with streptomycin-resistant E. coli strains . The functional importance of RPS23 is underscored by its high evolutionary conservation from bacteria through mammals, indicating strong selection pressure to maintain its critical role in protein synthesis.

How is recombinant pig RPS23 typically expressed and purified for research purposes?

Recombinant pig RPS23 is predominantly expressed in yeast expression systems rather than bacterial systems, which allows for proper folding and some post-translational modifications. The standard procedure involves cloning the pig RPS23 coding sequence (amino acids 2-143) into a yeast expression vector with an N-terminal His-tag for affinity purification . After expression induction, cells are lysed under native conditions, and the protein is typically purified using nickel affinity chromatography, with purification yields reaching >90% purity as confirmed by SDS-PAGE analysis . The purified protein can be used for various applications including ELISA-based interaction studies and functional assays. For researchers requiring alternative expression systems, it's important to note that expression in E. coli, mammalian cells, or via baculovirus infection may result in different protein characteristics, purity levels, and lead times . When studying hydroxylation states of RPS23, researchers should be aware that expression conditions, particularly oxygen levels, can significantly affect the pattern and extent of post-translational modifications.

What post-translational modifications occur on RPS23, and how do they differ across species?

RPS23 undergoes oxygen-dependent prolyl hydroxylation at specific proline residues, with remarkable species-specific patterns. In lower eukaryotes like Saccharomyces cerevisiae, Schizosaccharomyces pombe, and green algae, RPS23 undergoes an unprecedented dihydroxylation modification on proline residue Pro-64 (in yeast), catalyzed by prolyl dihydroxylases like Tpa1p . In contrast, higher eukaryotes including humans exhibit only monohydroxylation of RPS23, specifically prolyl trans-3-hydroxylation of Pro-62 (the human equivalent position), catalyzed by the human Tpa1p homolog OGFOD1 . This evolutionary shift from dihydroxylation to monohydroxylation represents a fascinating example of how post-translational modification systems have evolved across eukaryotic lineages. The hydroxylation occurs on unassembled RPS23 before its incorporation into ribosomes, as the modification site becomes inaccessible in fully assembled 40S ribosomal subunits . These modifications have been definitively characterized using mass spectrometric analyses, which have proven essential for distinguishing between the different hydroxylation states.

Which enzymes are responsible for RPS23 hydroxylation, and how are they regulated?

RPS23 hydroxylation is catalyzed by a highly conserved subfamily of Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases whose catalytic domains are closely related to transcription factor prolyl trans-4-hydroxylases that function as oxygen sensors in the hypoxic response in animals . In S. cerevisiae, the enzyme Tpa1p catalyzes dihydroxylation, while in S. pombe, the prolyl-3,4-dihydroxylase Ofd1 performs this function . In humans, the homologous enzyme OGFOD1 catalyzes prolyl trans-3-hydroxylation . These enzymes require molecular oxygen, Fe(II), and 2-oxoglutarate as co-substrates, making their activity inherently oxygen-sensitive. Under hypoxic conditions, the reduced availability of oxygen inhibits the hydroxylase activity, creating a direct link between oxygen sensing and translational regulation . In S. pombe, the Ofd1-mediated hydroxylation occurs while RPS23 is in complex with the nuclear import adaptor Nro1, which subsequently imports RPS23 into the nucleus for assembly into 40S ribosomes . This system creates an elegant regulatory mechanism where oxygen levels directly influence both RPS23 modification and ribosome assembly.

How does hydroxylation of RPS23 affect translational accuracy and termination efficiency?

RPS23 hydroxylation has profound effects on translational accuracy, particularly on stop codon readthrough, with impacts varying up to ~10-fold depending on the specific mRNA sequence context . Unlike most previously characterized translational accuracy modulators (such as antibiotics or the prion state of the S. cerevisiae translation termination factor eRF3), RPS23 hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner . This dual functionality allows for fine-tuned regulation of translation termination in response to oxygen availability. The mechanism appears to involve structural changes in the ribosomal decoding center that affect how efficiently stop codons are recognized by release factors versus suppressor tRNAs. Under certain conditions, the hydroxylation status of RPS23 can determine cell viability as a consequence of nonsense codon suppression, highlighting the physiological significance of this modification . The sequence-specific nature of these effects suggests that RPS23 hydroxylation may preferentially regulate the expression of specific subsets of genes, particularly those containing premature termination codons or programmed readthrough sequences.

What extra-ribosomal functions does RPS23 perform in cellular oxygen sensing?

Beyond its canonical role in ribosomes, RPS23 functions as a key component in oxygen homeostasis, particularly in fission yeast where it participates in a sophisticated oxygen-sensing system. In S. pombe, unassembled RPS23 forms a regulatory complex with the prolyl dihydroxylase Ofd1 and the nuclear import adaptor Nro1, which collectively control the hypoxic response by modulating the activity of the sterol regulatory element-binding protein (SREBP) transcription factor Sre1 . Under low oxygen conditions, Ofd1 hydroxylase activity is inhibited, stabilizing the Ofd1-RPS23-Nro1 complex and sequestering Ofd1 from binding to Sre1 . This sequestration allows Sre1 to activate hypoxic gene expression, creating a direct link between oxygen availability, RPS23 modification state, and transcriptional responses. The binding between Ofd1 and RPS23 occurs specifically at the N-terminal extension domain (amino acids 1-23) of RPS23, a region that becomes inaccessible once RPS23 is incorporated into ribosomes . This system demonstrates how unassembled ribosomal proteins can serve regulatory functions completely distinct from their roles in translation.

How does the RPS23 gene family relate to neurodegenerative disease mechanisms?

The RPS23 gene has given rise to a family of retroposed genes (RPS23rg) that appear to play significant roles in neurodegenerative disease pathways. The mouse gene Rps23 retroposed gene 1 (Rps23rg1) regulates β-amyloid (Aβ) levels and tau phosphorylation, two major pathological hallmarks of Alzheimer's disease (AD) . Rps23rg1 originated through retroposition of the mouse ribosomal protein S23 (Rps23) mRNA, and a similar process generated another functionally expressed gene, Rps23rg2, in mice . Both Rps23rg1 and Rps23rg2 are reversely transcribed relative to the parental Rps23 gene and encode transmembrane proteins . Functional studies have shown that these proteins interact with adenylate cyclases and upregulate cAMP levels, activating protein kinase A (PKA) and thereby inhibiting glycogen synthase kinase-3 (GSK-3) activity, tau phosphorylation, and Aβ generation . The RPS23RG protein family members function similarly to reduce AD-like pathologies, though RPS23RG2 shows weaker effects than RPS23RG1 . Interestingly, while retroposition of Rps23 mRNA occurred multiple times in different species, humans may not possess functional Rps23rg homologs, suggesting species-specific differences in how RPS23-derived genes may influence neurodegeneration.

How can researchers effectively analyze RPS23 hydroxylation states and their functional impacts?

Analysis of RPS23 hydroxylation requires a multi-faceted approach combining mass spectrometry, biochemical assays, and functional readouts. Mass spectrometric analyses have been the gold standard for identifying and characterizing the hydroxylation modifications of RPS23, capable of distinguishing between mono- and dihydroxylation states . To study the enzymes responsible for these modifications, in vitro hydroxylation assays using purified enzymes (such as Tpa1p, Ofd1, or OGFOD1) with recombinant RPS23 as substrate can be performed, monitoring the reaction by mass spectrometry or using oxygen consumption assays . For functional studies, translational readthrough assays using reporter constructs with premature stop codons can quantify how RPS23 hydroxylation affects termination efficiency in different sequence contexts . Researchers can modulate hydroxylation using genetic approaches (deletion or mutation of hydroxylase genes) or small-molecule inhibitors of Fe(II) and 2-oxoglutarate dependent oxygenases . Protein-protein interaction studies, such as GST pull-down assays, have been valuable for mapping binding interfaces between RPS23 and its hydroxylases, revealing that Ofd1 directly binds to the N-terminal extension domain (amino acids 1-23) of RPS23 .

What approaches can be used to study the extra-ribosomal functions of RPS23?

Investigating the extra-ribosomal functions of RPS23 requires techniques that can distinguish between its ribosome-bound and free forms, as well as methods to track its interactions with non-ribosomal partners. Ribosome profiling combined with RPS23-specific antibodies can help quantify the distribution of RPS23 between ribosomal and non-ribosomal pools under various conditions . For studying RPS23's role in oxygen sensing, researchers can employ co-immunoprecipitation and GST pull-down assays to capture and analyze the Ofd1-RPS23-Nro1 complex and its dynamics under different oxygen concentrations . Mutational analysis targeting the N-terminal region of RPS23 (amino acids 1-23) can help dissect the specific residues important for interaction with oxygen-sensing machinery while preserving ribosomal functions . The hypoxic response can be monitored through reporter assays for Sre1 transcriptional activity or by measuring expression of hypoxia-responsive genes . To study retroposed RPS23 genes (Rps23rg), researchers can use overexpression and knockdown approaches combined with assays measuring cAMP levels, PKA activity, and downstream effects on tau phosphorylation and Aβ generation . These approaches help delineate how RPS23 and its derived genes function beyond the confines of the ribosome.

What are the key differences between RPS23 in lower versus higher eukaryotes?

While the core structure and ribosomal function of RPS23 remain conserved, several key differences distinguish RPS23 in lower versus higher eukaryotes. The most prominent difference lies in the hydroxylation pattern: lower eukaryotes like S. cerevisiae, S. pombe, and green algae feature dihydroxylation of RPS23 proline residues (specifically Pro-64 in yeast), catalyzed by enzymes like Tpa1p and Ofd1 . In contrast, higher eukaryotes including humans exhibit monohydroxylation, specifically prolyl trans-3-hydroxylation of Pro-62, catalyzed by the enzyme OGFOD1 . This modification difference likely reflects evolutionary adaptations in translational control mechanisms. The regulatory pathways involving RPS23 also show species-specific features. In S. pombe, unassembled RPS23 participates in a specialized oxygen-sensing system through interactions with Ofd1 and Nro1, directly affecting the hypoxic transcription factor Sre1 . Higher eukaryotes may utilize different regulatory pathways involving RPS23, though the connections to oxygen sensing are likely conserved given the oxygen-dependent nature of the hydroxylation. Another significant difference appears in the RPS23 gene family: rodents have generated functional retroposed genes (Rps23rg1 and Rps23rg2) with roles in neurodegeneration pathways, while humans may lack functional RPS23rg homologs despite having undergone RPS23 retroposition events .

How might targeting RPS23 hydroxylation be therapeutically relevant for genetic diseases?

Modulating RPS23 hydroxylation represents a promising therapeutic approach for genetic diseases caused by nonsense mutations, which introduce premature stop codons resulting in truncated, non-functional proteins. Research has demonstrated that RPS23 hydroxylation status significantly affects stop codon readthrough in a sequence-specific manner, with alterations of up to ~10-fold in termination efficiency . Small-molecule inhibitors targeting the RPS23 hydroxylases (the Fe(II) and 2-oxoglutarate dependent oxygenases) have been shown to increase production of full-length proteins from sequences containing clinically relevant nonsense mutations . This approach could potentially benefit patients with genetic diseases like cystic fibrosis, Duchenne muscular dystrophy, or certain cancers where nonsense mutations are prevalent. Unlike broad-spectrum translational readthrough drugs that may induce global translational errors, targeting RPS23 hydroxylation may offer more nuanced control over readthrough in specific sequence contexts . The context-dependent nature of RPS23 hydroxylation effects on translational accuracy suggests that careful optimization would be required to maximize therapeutic benefit while minimizing off-target effects. Additionally, since oxygen levels influence hydroxylase activity, combining hydroxylase inhibitors with controlled oxygen conditions might further enhance therapeutic efficacy in certain clinical settings.

What role might RPS23 and its retroposed genes play in neurodegenerative disease mechanisms?

The discovery that RPS23-derived retroposed genes regulate key pathways in neurodegeneration opens new avenues for understanding and potentially treating conditions like Alzheimer's disease. The mouse genes Rps23rg1 and Rps23rg2 have been shown to reduce AD-like pathologies by regulating β-amyloid levels and tau phosphorylation through interaction with adenylate cyclases . These interactions upregulate cAMP levels, activate protein kinase A (PKA), and subsequently inhibit glycogen synthase kinase-3 (GSK-3) activity, effectively reducing the two major pathological hallmarks of Alzheimer's disease . Understanding these mechanisms could inform new therapeutic strategies targeting adenylate cyclase activation or PKA/GSK-3 signaling pathways. Interestingly, while these retroposed genes are present and functional in mice, humans may not possess functional RPS23rg homologs, suggesting potential species differences in neurodegeneration mechanisms that researchers should consider when translating findings from mouse models to human applications . Further research exploring whether the hydroxylation state of human RPS23 itself influences neurodegenerative pathways could reveal additional therapeutic targets. The role of RPS23 in cellular oxygen sensing may also have implications for understanding how hypoxic stress contributes to neurodegeneration, potentially linking translational regulation to disease progression in conditions where oxygen delivery to neural tissues is compromised.