Recombinant Mouse Probable peptidyl-tRNA hydrolase (Ptrh1)

What is Mouse Probable peptidyl-tRNA hydrolase (Ptrh1) and what is its basic function?

Ptrh1 belongs to the PTH family of enzymes that exhibit peptidyl-tRNA hydrolase activity. This activity is crucial for releasing tRNA from premature translation termination products known as peptidyl-tRNA. The enzyme functions by catalyzing the hydrolysis of the ester bond between the peptide and the tRNA, allowing both components to be recycled and reused in protein synthesis. This recycling mechanism is essential for maintaining cellular homeostasis and preventing the accumulation of potentially toxic peptidyl-tRNAs .

Two different enzymes with peptidyl-tRNA hydrolase activity have been identified: Pth, present in bacteria and eukaryotes, and Pth2, found in archaea and eukaryotes. Mouse Ptrh1 shares significant homology with bacterial peptidyl-tRNA hydrolase Pth, suggesting evolutionary conservation of this essential function .

What is the structural organization of Mouse Ptrh1?

Mouse Ptrh1 shares structural similarities with bacterial Pth, which exhibits an α/β fold formed by three layers with a mixed five-strand β-sheet at the core. Critical residues for enzymatic activity include positions homologous to H46 and D121, as mutations at these sites (H46N and D121A substitutions) abolish Ptrh1's hydrolase activity. These residues are highly conserved across species, indicating their fundamental importance to the catalytic mechanism .

The structural characteristics of Ptrh1 enable it to interact with various substrates, including 60S and 80S ribosomal complexes, as well as free peptidyl-tRNAs, demonstrating the enzyme's versatility in different cellular contexts.

How does Ptrh1 differ from other peptidyl-tRNA hydrolases?

While Ptrh1 shares functional similarities with other peptidyl-tRNA hydrolases, it exhibits distinct substrate specificities and cellular localization patterns compared to related enzymes like Ptrh2. Unlike mitochondrial Ptrh2, which belongs to a different peptidyl-hydrolase class, Ptrh1 operates primarily in the cytosol. Despite these differences, both enzymes can release nascent chains (NCs) from 60S/80S ribosomal complexes and exhibit similar dependencies on mRNA length and nascent chain characteristics .

Notably, other putative peptidyl-tRNA hydrolases like PtrhD1 and the mitochondrial factor ICT1 (containing a GGQ motif) do not demonstrate the same activity profile as Ptrh1 in experimental assays, highlighting the functional specialization within this enzyme family .

What are the recommended methods for detecting Mouse Ptrh1 in experimental samples?

Several validated techniques are available for detecting Mouse Ptrh1 in experimental samples:

• Western Blotting: Anti-PTRH1 antibodies (such as clone 3O6 ZooMAb® Rabbit Monoclonal) have been validated for Western blotting at 1:1,000 dilution in various cell lysates including Jurkat, MCF-7, and Raw264.7 cells .

• Immunohistochemistry: Paraffin-embedded tissue sections can be analyzed using anti-PTRH1 antibodies at 1:100 dilution, with validated detection in human adrenal gland tissue sections .

• Immunocytochemistry: Detection of Ptrh1 in intact cells (including MCF7 and Jurkat cells) can be achieved using a 1:100 dilution of specific antibodies .

• ELISA: Quantitative analysis of Mouse Ptrh1 can be performed using sandwich ELISA techniques that employ a combination of capture and detection antibodies specific for Ptrh1 .

How can I quantitatively measure Mouse Ptrh1 levels in tissue or cell samples?

Quantitative assessment of Mouse Ptrh1 levels can be achieved using a sandwich ELISA approach, which follows this methodological workflow:

• Initial capture of Ptrh1 from samples using pre-coated microplates with immobilized anti-Ptrh1 antibodies

• Addition of biotin-conjugated detection antibodies specific for Ptrh1

• Introduction of Streptavidin-conjugated Horseradish Peroxidase (HRP)

• Addition of substrate solution to develop color proportional to Ptrh1 concentration

• Measurement of color intensity after stopping the reaction

This method allows for precise quantification of Ptrh1 levels in various mouse tissue extracts and cell culture samples. Standard curves should be generated using recombinant Ptrh1 of known concentration to ensure accurate quantification.

What is the role of Ptrh1 in ribosome-associated quality control pathways?

Ptrh1 plays a significant role in ribosome-associated quality control (RQC) pathways, specifically in the release of non-ubiquitinated nascent chains (NCs) from ribosomal complexes. Research has revealed that Ptrh1 can release nascent polypeptides from both 60S ribosomal subunits and intact 80S ribosomes under specific conditions .

The enzyme's action in releasing non-ubiquitinated NCs may represent an alternative pathway for processing stalled translation products that have not been targeted for proteasomal degradation through the canonical RQC pathway involving NEMF/Listerin-mediated ubiquitination. This suggests a complex regulatory network for managing translation products, with Ptrh1 potentially serving as an important checkpoint in determining the fate of nascent polypeptides .

How does the substrate specificity of Ptrh1 differ from other translation termination mechanisms?

Ptrh1 exhibits a unique substrate specificity profile that contrasts with other translation termination mechanisms, particularly puromycin-mediated peptide release:

These opposing specificities suggest that Ptrh1 targets a distinct population of stalled ribosomes compared to canonical termination mechanisms, potentially addressing translation complexes where the CCA end of peptidyl-tRNA is not properly accommodated in the peptidyl transferase center (PTC) .

What structural determinants influence Ptrh1 activity on ribosomal nascent chain complexes?

Several structural factors have been identified that significantly impact Ptrh1 activity on ribosomal nascent chain complexes:

• mRNA length after the P site: Ptrh1 shows highest activity on complexes with <3 nucleotides downstream of the P site, with activity decreasing as the mRNA length increases beyond this threshold. Complexes with ≥9 nucleotides downstream show negligible Ptrh1 activity .

• Nascent chain length: The length of the nascent polypeptide strongly modulates Ptrh1 activity, with optimal activity observed on longer nascent chains (66-75 amino acids). RNCs containing short (4-6 amino acids) or medium-length (14-55 amino acids) nascent chains are poor substrates for Ptrh1-mediated hydrolysis .

• tRNA identity: Experimental evidence indicates that Ptrh1's activity is not significantly affected by the identity of the tRNA in the P site, as RNCs containing various tRNAs (including tRNA^Ser and tRNA^Cys) remain susceptible to Ptrh1-mediated hydrolysis .

• Protein folding state: The folding state of the nascent polypeptide emerging from the ribosomal exit tunnel appears to have minimal impact on Ptrh1 activity, suggesting that the enzyme primarily recognizes features of the peptidyl-tRNA junction rather than the structural characteristics of the nascent chain itself .

How can I design experiments to assess Ptrh1 activity in vitro?

To effectively evaluate Ptrh1 activity in vitro, researchers should consider the following experimental approach:

• Preparation of substrate RNCs: Generate ribosome-nascent chain complexes by translating appropriate mRNAs (with/without stop codons and varying 3'UTR lengths) in rabbit reticulocyte lysate (RRL) or other translation systems. For optimal Ptrh1 activity, design constructs that produce nascent chains of sufficient length (>65 amino acids) tethered to P-site tRNA .

• Purification of RNCs: Isolate 80S or 60S RNCs using sucrose density gradient (SDG) centrifugation, being mindful that 60S RNC preparations may contain small amounts of contaminating 80S RNCs .

• Activity assay setup:

  • Incubate purified RNCs with recombinant Ptrh1 under varying conditions

  • Include controls with catalytically inactive Ptrh1 mutants (H46N, D121A)

  • Test activity across a range of Mg²⁺ concentrations to determine optimal conditions

• Product analysis: Monitor the release of nascent chains using techniques such as SDS-PAGE and autoradiography (for radiolabeled nascent chains) or western blotting (for specific detection) .

• Comparative analysis: Include parallel experiments with puromycin to distinguish between different mechanisms of nascent chain release and to identify RNC populations that may be specifically targeted by Ptrh1 .

What are the key considerations when comparing Ptrh1 activity across different experimental systems?

When comparing Ptrh1 activity across experimental systems, researchers should account for several factors that may influence results:

• Source of recombinant Ptrh1: Expression systems and purification methods can affect enzyme activity. Ensure consistent protein quality through rigorous quality control testing of recombinant preparations.

• Buffer composition: Ionic strength, pH, and divalent cation concentrations (particularly Mg²⁺) can significantly impact Ptrh1 activity. Standardize buffer conditions across experiments or systematically evaluate the effect of buffer variations .

• RNC preparation methods: Different approaches to generating RNCs (in vitro translation systems, reconstituted systems, etc.) may yield complexes with subtle structural differences that affect Ptrh1 susceptibility.

• Species differences: While the core function of Ptrh1 is conserved, species-specific variations may exist. When comparing mouse Ptrh1 with orthologs from other organisms, consider potential differences in substrate preference or activity profiles .

• Interaction with cofactors: The presence of additional cellular factors might modulate Ptrh1 activity in ways not fully recapitulated in simplified in vitro systems. Consider supplementing reactions with cellular extracts to identify potential cofactors or inhibitors.

How does Ptrh1 function interact with the ubiquitin-proteasome system in translation quality control?

Ptrh1 appears to function in parallel with the canonical ubiquitin-proteasome-dependent ribosome-associated quality control (RQC) pathway. In the standard RQC pathway, stalled nascent chains are ubiquitinated by the Listerin E3 ligase in association with NEMF, targeting them for proteasomal degradation. In contrast, Ptrh1 provides an alternative mechanism for processing non-ubiquitinated nascent chains .

Research indicates that association with NEMF/Listerin and subsequent ubiquitination of nascent chains results in peptidyl-tRNA accommodation that renders 60S RNCs resistant to Ptrh1-mediated hydrolysis but susceptible to ANKZF1-induced cleavage. This suggests a regulatory mechanism that directs nascent chains down different processing pathways based on their ubiquitination status .

Another RQC component, TCF25, has been identified as ensuring preferential formation of K48-ubiquitin linkages during Listerin-mediated ubiquitination, further highlighting the complex interplay between different quality control mechanisms .

What evidence exists for Ptrh1 involvement in pathological conditions or disease models?

While direct evidence linking Ptrh1 dysfunction to specific pathological conditions in mouse models remains limited in the provided search results, the fundamental role of peptidyl-tRNA hydrolases in translation and quality control suggests potential implications in various diseases:

• Neurodegenerative disorders: Defects in ribosome-associated quality control have been implicated in several neurodegenerative conditions. Alterations in Ptrh1 function could potentially contribute to proteotoxic stress observed in these disorders.

• Cancer biology: Dysregulation of translation and protein quality control mechanisms represents a hallmark of cancer. The availability of antibodies validated in cancer cell lines (MCF-7, Jurkat) suggests Ptrh1 expression in tumor cells, though its functional significance requires further investigation .

• Developmental disorders: Given the essential nature of translation fidelity for proper development, Ptrh1 dysfunction could potentially impact embryonic or postnatal development.

Further research using genetic models (knockout or conditional Ptrh1-deficient mice) would be valuable to elucidate the physiological and pathological relevance of Ptrh1 in mammalian systems.

What are the emerging techniques for studying Ptrh1 interactions with ribosomal complexes?

Several advanced techniques are emerging as powerful tools for investigating Ptrh1 interactions with ribosomal complexes:

• Cryo-electron microscopy (cryo-EM): High-resolution structural analysis of Ptrh1 bound to ribosomal complexes could provide crucial insights into the molecular mechanism of peptidyl-tRNA hydrolysis and substrate recognition.

• Ribosome profiling: This technique allows genome-wide analysis of ribosome positioning and could help identify endogenous substrates particularly susceptible to Ptrh1 activity in vivo.

• CRISPR-Cas9 genome editing: Generation of Ptrh1-deficient cell lines or animal models using CRISPR-Cas9 technology would facilitate comprehensive functional analysis of this enzyme in physiological contexts.

• Proteomics approaches: Advanced mass spectrometry techniques could help identify Ptrh1 interaction partners and characterize post-translational modifications that might regulate its activity.

• Single-molecule techniques: Methods such as fluorescence resonance energy transfer (FRET) could provide dynamic information about Ptrh1 binding to ribosomal complexes and the conformational changes associated with catalysis.

What are the unexplored aspects of Ptrh1 function that warrant further investigation?

Despite progress in understanding Ptrh1 function, several important questions remain unanswered and represent promising areas for future research:

• Regulation of Ptrh1 activity: The mechanisms controlling Ptrh1 expression, localization, and activity in different cellular contexts remain poorly understood. Investigation of potential post-translational modifications or interaction partners that modulate Ptrh1 function would provide valuable insights.

• Physiological substrates: While in vitro studies have characterized Ptrh1 activity on defined substrates, the identity of its primary physiological targets in vivo requires further exploration. Identifying which endogenous mRNAs or translation contexts most frequently engage Ptrh1 would enhance our understanding of its biological role.

• Tissue-specific functions: The relative importance of Ptrh1 across different tissues and developmental stages remains largely unexplored. Analysis of tissue-specific expression patterns and conditional knockout models could reveal specialized functions in particular cellular contexts.

• Evolutionary adaptations: Comparative analysis of Ptrh1 orthologs across species could uncover evolutionary adaptations and specialized functions that have emerged in different organisms.

• Therapeutic potential: The possibility of modulating Ptrh1 activity for therapeutic purposes in conditions associated with defective translation or protein quality control represents an exciting frontier for translational research.