Recombinant Human Putative peptidyl-tRNA hydrolase PTRHD1 (PTRHD1)

How does PTRHD1 differ from bacterial peptidyl-tRNA hydrolases?

PTRHD1 demonstrates significant functional divergence from bacterial peptidyl-tRNA hydrolases despite sequence similarities. While bacterial Pth enzymes like E. coli Pth1 actively cleave peptidyl-tRNA, experimental evidence shows PTRHD1 lacks this hydrolytic activity . Specifically, complementation studies using temperature-sensitive Pth1 (Pth1TS) strains demonstrated that while functional Pth enzymes (like S. typhimurium Pth1) can rescue the temperature-sensitive phenotype at 42°C, PTRHD1 fails to complement this function . Additionally, direct biochemical assays confirm PTRHD1's inability to cleave peptidyl-tRNA substrates even at higher concentrations or extended incubation periods . These fundamental differences suggest PTRHD1 evolved distinct functions in eukaryotes compared to its bacterial counterparts in the peptidyl-tRNA hydrolase family.

What are the known molecular characteristics of the PTRHD1 protein?

The PTRHD1 protein exhibits several distinctive molecular characteristics based on experimental characterization. It can be successfully expressed as a soluble recombinant protein in E. coli expression systems and purified to >95% homogeneity using single-step metal chelation chromatography . Although initially classified as a peptidyl-tRNA hydrolase based on sequence analysis, functional studies reveal it binds to multiple nucleic acid types including tRNA, rRNA, and double-stranded DNA, suggesting potential roles in nucleic acid metabolism or regulation . Interestingly, studies of pathogenic variants have identified stable truncated forms of the protein that escape nonsense-mediated mRNA decay, as demonstrated by western blotting and isoelectric focusing analysis of patient-derived cells . This stability of mutant forms might contribute to the complex pathophysiology observed in patients with PTRHD1 mutations, as these truncated proteins may retain partial functions or interfere with normal cellular processes.

What expression systems are effective for recombinant PTRHD1 production?

E. coli expression systems have proven highly effective for recombinant PTRHD1 production, with the protein expressing well as a soluble entity rather than forming inclusion bodies . Experimental data shows successful induction of PTRHD1 expression using 250 μM IPTG in E. coli cultures . This expression approach yields sufficient quantities of soluble protein for subsequent purification and functional studies. The ability to produce PTRHD1 in a bacterial system suggests the protein does not require extensive post-translational modifications or eukaryotic chaperones for proper folding, which simplifies production protocols for research applications. When designing expression constructs, researchers should consider incorporating affinity tags that facilitate downstream purification while minimizing interference with the protein's structure and function.

What purification strategies yield highest purity PTRHD1 for functional studies?

Single-step metal chelation chromatography has been demonstrated as an efficient purification method for recombinant PTRHD1, yielding protein preparations with >95% purity suitable for functional and structural studies . This approach leverages affinity tags (typically histidine tags) engineered into the recombinant protein construct to facilitate selective binding to immobilized metal ions. The high efficiency of this method suggests PTRHD1 expresses with minimal contaminating E. coli proteins that might otherwise complicate purification. For researchers requiring ultra-pure preparations for crystallography or other sensitive applications, this primary purification step could be supplemented with size exclusion chromatography to remove any remaining high or low molecular weight contaminants or protein aggregates. Purified PTRHD1 remains stable and retains its nucleic acid binding capabilities, making it suitable for detailed biochemical characterization studies.

How can researchers verify successful expression and purification of functional PTRHD1?

Verification of successfully expressed and purified functional PTRHD1 requires a multi-faceted approach combining protein analysis and functional assays. SDS-PAGE analysis should demonstrate a band at the expected molecular weight (approximately 15-25 kDa based on published studies), with minimal contaminating bands indicating high purity . Western blotting using PTRHD1-specific antibodies provides additional confirmation of protein identity. For functional verification, nucleic acid binding assays represent the most appropriate approach given PTRHD1's demonstrated ability to bind tRNA, rRNA, and dsDNA . Electrophoretic mobility shift assays (EMSAs) with labeled nucleic acid substrates can assess binding activity. Additionally, researchers should verify the absence of peptidyl-tRNA hydrolase activity using appropriate substrates, as this negative result is consistent with authentic PTRHD1 function . Finally, circular dichroism spectroscopy can confirm proper protein folding by assessing secondary structure elements, ensuring the recombinant protein adopts a native-like conformation.

If PTRHD1 is not a peptidyl-tRNA hydrolase, what are its proposed alternative functions?

Despite its name suggesting peptidyl-tRNA hydrolase activity, experimental evidence conclusively demonstrates PTRHD1 lacks this enzymatic function . Current research suggests alternative roles based on its nucleic acid binding capabilities. PTRHD1 exhibits binding affinity for multiple nucleic acid types including tRNA, rRNA, and double-stranded DNA , pointing toward potential functions in RNA metabolism, gene expression regulation, or DNA/RNA-dependent cellular processes. The protein's association with intellectual disability, spasticity, and early-onset parkinsonism when mutated suggests critical functions in neuronal development or maintenance. Some researchers hypothesize PTRHD1 may function as an RNA chaperone, facilitating proper RNA folding or RNA-protein interactions essential for neuronal function. Alternatively, it might participate in ribonucleoprotein complexes involved in RNA processing, transport, or localized translation in neurons. These proposed functions would align with both its nucleic acid binding properties and the neurological phenotypes observed in patients with PTRHD1 mutations.

How do frameshift mutations in PTRHD1 affect protein function at the molecular level?

Frameshift mutations in PTRHD1 create truncated protein products with potentially altered function or stability. In one documented case, a homozygous 28-nucleotide frameshift deletion introduced a premature stop codon in PTRHD1 exon 1 . Surprisingly, transcript analysis revealed these mutant mRNAs escaped nonsense-mediated decay (NMD), with real-time PCR demonstrating higher mutant mRNA expression compared to wild-type . Western blotting and isoelectric focusing confirmed the presence of stable truncated PTRHD1 protein in patient cells . These molecular findings suggest a complex pathomechanism where truncated protein products may exert detrimental effects through:

• Loss of critical functional domains required for normal PTRHD1 activity

• Potential dominant-negative effects if truncated proteins interact with binding partners

• Possible gain-of-function effects if truncation exposes normally concealed protein regions

• Altered subcellular localization affecting access to natural substrates or binding partners

The persistence of stable mutant protein rather than complete absence further complicates understanding of disease mechanisms and highlights the importance of characterizing the functional consequences of specific mutations.

What evidence supports PTRHD1's role in nucleic acid binding versus hydrolase activity?

Experimental evidence clearly differentiates PTRHD1's actual function from its initially predicted hydrolase activity. Direct biochemical assays demonstrate PTRHD1 does not cleave peptidyl-tRNA substrates, even at elevated concentrations or extended incubation times, while validated Pth enzymes like E. coli Pth1 efficiently hydrolyze these substrates under identical conditions . Furthermore, complementation studies in temperature-sensitive Pth1 bacterial strains show PTRHD1 cannot rescue the hydrolase-deficient phenotype at non-permissive temperatures (42°C), while known Pth enzymes successfully complement this deficiency . In contrast, follow-up studies reveal PTRHD1 binds multiple nucleic acid types including tRNA, rRNA, and double-stranded DNA , suggesting nucleic acid interaction represents its true biological function. This binding activity aligns with the neurological phenotypes observed in patients with PTRHD1 mutations, as RNA metabolism and regulation play critical roles in neuronal development and function. The disparity between PTRHD1's name and actual function underscores the limitations of functional prediction based solely on sequence homology.

What is the evidence linking PTRHD1 mutations to intellectual disability and parkinsonism?

Multiple independent genetic studies have established PTRHD1 mutations as causative factors for a distinctive neurodevelopmental and neurodegenerative phenotype. Biallelic pathogenic variants in PTRHD1 have been identified in several families with overlapping clinical presentations featuring intellectual disability, spasticity, and early-onset parkinsonism . These findings have been replicated across different ethnic backgrounds, strengthening the genotype-phenotype correlation. In one extensively documented case, a homozygous 28-nucleotide frameshift deletion in PTRHD1 exon 1 was identified in four affected members of a consanguineous family, all presenting with the characteristic triad of symptoms . The genetic evidence is further supported by the absence of other plausible genetic causes in affected individuals and cosegregation of PTRHD1 mutations with disease phenotypes within families. Currently, at least 20 unique public DNA variants have been reported in PTRHD1 , with ongoing research likely to identify additional disease-associated variants. This accumulating evidence firmly establishes PTRHD1 as a disease-causing gene for this specific neurological syndrome.

How might PTRHD1 dysfunction contribute to neurodevelopmental and neurodegenerative phenotypes?

The contribution of PTRHD1 dysfunction to both neurodevelopmental and neurodegenerative phenotypes likely stems from its role in neural RNA metabolism or gene expression regulation. Since PTRHD1 demonstrates binding activity with various nucleic acids including tRNA, rRNA, and dsDNA , disruption of these interactions could impair multiple cellular processes critical for neuronal development and maintenance. Potential pathogenic mechanisms include:

• Disrupted localized protein synthesis in neurons, affecting synaptic plasticity essential for learning and memory

• Abnormal processing of specific RNA species critical for neuronal differentiation or survival

• Altered gene expression patterns affecting neurodevelopmental trajectories or neuronal homeostasis

• Impaired stress response mechanisms in neurons, increasing vulnerability to degeneration

• Accumulation of toxic RNA-protein aggregates similar to other neurodegenerative conditions

The combined presentation of intellectual disability (neurodevelopmental) and parkinsonism (neurodegenerative) suggests PTRHD1 functions across neuronal lifespans, with early manifestations reflecting developmental abnormalities and later-onset parkinsonian features indicating progressive neurodegeneration, particularly affecting dopaminergic neurons in the substantia nigra.

What is the spectrum of clinical presentations associated with PTRHD1 mutations?

Patients with biallelic PTRHD1 mutations present with a distinctive clinical syndrome characterized by three cardinal features: intellectual disability, spasticity, and early-onset parkinsonism . The intellectual disability component typically manifests in childhood as developmental delay affecting cognitive functions, with varying severity across patients. Spasticity predominantly affects lower limbs, resulting in abnormal gait and motor difficulties. Parkinsonian symptoms generally emerge earlier than in idiopathic Parkinson's disease, with classical features including bradykinesia, rigidity, resting tremor, and postural instability . Additional reported neurological manifestations may include:

• Speech impairments ranging from dysarthria to more severe communication difficulties

• Behavioral disturbances in some patients

• Variable progression rates of motor symptoms

• Responsiveness to standard dopaminergic therapies for parkinsonian features

This clinical constellation represents a distinct neurogenetic syndrome with both developmental and degenerative components. The recognition of this specific phenotype helps clinicians identify potential PTRHD1 mutation carriers among patients with complex neurological presentations, particularly those with early-onset parkinsonism combined with intellectual disability.

What cellular and animal models best recapitulate PTRHD1-associated pathology?

Developing cellular and animal models that accurately recapitulate PTRHD1-associated pathology requires strategic approaches based on current understanding of the protein's function and disease mechanisms. For cellular models, patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons offer the most physiologically relevant system, maintaining the exact genetic background associated with disease . CRISPR-engineered cell lines carrying specific PTRHD1 mutations in isogenic backgrounds provide complementary controlled systems for mechanistic studies. For animal models, both knockout and knock-in approaches in mice have merit:

Researchers should select models based on specific experimental questions, with combined approaches typically yielding most comprehensive insights into PTRHD1 biology and pathology.

How can researchers overcome challenges in studying PTRHD1 nucleic acid interactions?

Studying PTRHD1's nucleic acid interactions presents several technical challenges that require specialized approaches. To effectively characterize these interactions, researchers should employ multiple complementary techniques:

• RNA Immunoprecipitation followed by sequencing (RIP-seq) can identify endogenous RNA binding partners of PTRHD1 in relevant cell types. This approach requires highly specific antibodies against PTRHD1 or epitope-tagged versions expressed at near-physiological levels .

• Electrophoretic Mobility Shift Assays (EMSAs) using purified recombinant PTRHD1 and labeled nucleic acid substrates provide direct evidence of binding and can determine binding affinities and specificities. Researchers should test various nucleic acid types (tRNA, rRNA, mRNA, dsDNA) with different sequence compositions to establish binding preferences .

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) enable real-time, label-free measurement of binding kinetics and affinities between PTRHD1 and nucleic acids.

• Cross-linking and Immunoprecipitation followed by sequencing (CLIP-seq) techniques provide higher resolution mapping of specific binding sites within RNA molecules.

• For structural characterization of PTRHD1-nucleic acid complexes, researchers might employ X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy depending on complex stability and size.

When interpreting results, researchers should consider whether binding occurs at physiologically relevant affinities and whether interactions demonstrate sequence or structure specificity that could indicate biological function.

What experimental approaches can identify PTRHD1 binding partners and signaling pathways?

Identifying PTRHD1's protein binding partners and associated signaling pathways requires systematic application of complementary approaches to build a comprehensive interaction network. The following methodologies offer particular advantages for PTRHD1 research:

• Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling: By fusing PTRHD1 to a biotin ligase or peroxidase, researchers can identify proteins in close proximity to PTRHD1 in living cells. This approach captures both stable and transient interactions in their native cellular context .

• Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS): This technique identifies stable protein complexes containing PTRHD1. Comparing interactomes of wild-type versus mutant PTRHD1 can reveal interactions disrupted by disease-causing mutations.

• Yeast two-hybrid screening: While this approach may yield false positives, it can identify direct binary interactions and serves as a complementary method to validate findings from other techniques.

• Phosphoproteomic analysis in cells with PTRHD1 knockout or overexpression: This approach can reveal signaling pathways affected by PTRHD1 perturbation, even if PTRHD1 does not directly participate in these pathways.

• Transcriptomic and ribosome profiling analyses: Given PTRHD1's nucleic acid binding properties, these approaches can identify genes and pathways regulated at the transcriptional or translational level by PTRHD1.

Researchers should prioritize validation of identified interactions in multiple experimental systems and correlate findings with disease phenotypes to establish biological and pathological relevance.

What therapeutic strategies might address PTRHD1-associated neurological disorders?

Developing therapeutic strategies for PTRHD1-associated neurological disorders requires consideration of the complex pathophysiology involving both neurodevelopmental and neurodegenerative processes. Several approaches warrant investigation:

Research at present should focus on identifying the most critical molecular consequences of PTRHD1 dysfunction to guide rational therapeutic development.

How might structural biology approaches advance understanding of PTRHD1 function?

Structural biology approaches offer transformative potential for understanding PTRHD1 function and developing targeted therapeutics. High-resolution structural data would elucidate:

• The molecular basis for PTRHD1's nucleic acid binding capabilities, including specific binding motifs and interaction surfaces that mediate binding to tRNA, rRNA, and dsDNA .

• Conformational changes that may occur upon nucleic acid binding, potentially revealing allosteric regulation mechanisms.

• How disease-causing mutations alter protein structure, stability, or interaction surfaces, providing direct insight into pathomechanisms .

• Potential sites for small molecule intervention that could modulate PTRHD1 function therapeutically.

Researchers should pursue multi-faceted structural approaches including:

Integrating computational approaches with experimental structural biology would accelerate progress in understanding PTRHD1 structure-function relationships.

What emerging technologies show promise for advancing PTRHD1 research?

Several cutting-edge technologies show particular promise for transforming PTRHD1 research and overcoming current limitations in understanding its functions and disease associations:

• Spatial transcriptomics combined with single-cell RNA sequencing can reveal cell type-specific expression patterns of PTRHD1 and its potential binding partners in human brain tissue, providing critical context for understanding its neurological functions and disease associations .

• Advanced genome editing approaches including base editing and prime editing offer precise methods to introduce or correct specific PTRHD1 mutations in cellular and animal models without double-strand breaks, enabling more accurate disease modeling .

• AlphaFold2 and other AI-driven protein structure prediction tools can generate hypothetical structural models of PTRHD1 and its complexes with nucleic acids, guiding experimental approaches even before crystal structures are available .

• Optical control of protein function through optogenetic approaches could enable temporally precise manipulation of PTRHD1 activity in specific neuronal populations, helping dissect its roles in different developmental stages and brain regions.

• Massively parallel reporter assays could systematically test how PTRHD1 affects expression of thousands of genetic elements simultaneously, potentially revealing global regulatory patterns.

• Organoid models derived from patient iPSCs represent promising systems for studying PTRHD1 function in three-dimensional neural tissues that recapitulate aspects of human brain development and organization.

These emerging technologies, particularly when applied in combination, have the potential to rapidly advance understanding of PTRHD1 biology and pathology, potentially accelerating therapeutic development.